RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

PubMed

Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

2015-08-07

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans

PubMed Central

van Rijnberk, Lotte M.; van der Horst, Suzanne E. M.; van den Heuvel, Sander; Ruijtenberg, Suzan

2017-01-01

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit. PMID:28158315

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells.

PubMed

Fu, Yujie; Kadioglu, Onat; Wiench, Benjamin; Wei, Zuofu; Gao, Chang; Luo, Meng; Gu, Chengbo; Zu, Yuangang; Efferth, Thomas

2015-04-15

The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet. We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis. CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways. CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Molecular Effects of I3C/DIM in Prostate Cancer Cells

DTIC Science & Technology

2005-04-01

DNA fragmentation, resulting in apoptosis (40,41). To (phorbol myristate acetate, etc.), stress (pH, hypoxia, heavy further explore the mechanism of...cytosol into the ling cell proliferation, differentiation, apoptosis, inflammation, mitochondria in both cell lines treated with 13C. However, stress ...of cell prolifer- the control of cell growth, apoptosis, inflammation, ation, cell cycle, apoptosis, signal transduction, onco- stress response, and

DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

PubMed Central

Yedjou, Clement G.; Tchounwou, Hervey M.; Tchounwou, Paul B.

2015-01-01

In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO3)2] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO3)2 for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO3)2-treated cells, indicative of membrane rupture by Pb(NO3)2 compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO3)2 exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO3)2 exposure caused cell cycle arrest at the G0/G1 checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO3)2 inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G0/G1 checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO3)2 exposure and its associated adverse health effects. PMID:26703663

DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

PubMed

Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

2015-12-22

In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

Cell cycle-related metabolism and mitochondrial dynamics in a replication-competent pancreatic beta-cell line.

PubMed

Montemurro, Chiara; Vadrevu, Suryakiran; Gurlo, Tatyana; Butler, Alexandra E; Vongbunyong, Kenny E; Petcherski, Anton; Shirihai, Orian S; Satin, Leslie S; Braas, Daniel; Butler, Peter C; Tudzarova, Slavica

2017-01-01

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca 2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca 2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca 2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca 2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.

Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

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

Vethakanraj, Helen Shiphrah; Babu, Thabraz Ahmed; Sudarsanan, Ganesh Babu

2015-08-28

The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observedmore » in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER{sup +} and ER{sup −} breast cancer cell lines. - Highlights: • Acid Ceramidase inhibitor, Ceranib 2 induced apoptosis in Breast cancer cell lines (MCF 7 and MDA MB 231 cell lines). • Apoptosis is mediated by DNA fragmentation and cell cycle arrest. • Ceranib 2 upregulated the expression of pro-apoptotic genes and down regulated anti-apoptotic gene expression. • More potent compared to the standard drug Tamoxifen.« less

A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

PubMed

Weisskopf, M; Schaffner, W; Jundt, G; Sulser, T; Wyler, S; Tullberg-Reinert, H

2005-10-01

Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

Leptin stimulation of cell cycle and inhibition of apoptosis gene and protein expression in OVCAR-3 ovarian cancer cells.

PubMed

Ptak, Anna; Kolaczkowska, Elzbieta; Gregoraszczuk, Ewa L

2013-04-01

The OVCAR-3 cell line expressing the long (ObRb) and short (ObRt) isoforms of leptin receptor mRNA was used to analyze the effect of leptin on the expression of selected genes and proteins involved in the cell cycle and apoptosis. OVCAR-3 cells were exposed to 2, 20, 40, and 100 ng/ml of leptin. Cell proliferation was determined using the alamarBlue cell viability test and flow cytometry. Apoptosis was measured using a cellular DNA fragmentation ELISA kit. The expression of selected cell cycle and apoptosis genes was evaluated by real-time PCR and confirmed by western blot. The stimulatory action of leptin on cell proliferation was observed as an increase in cells in the S and G2/M phases. Up-regulation of genes responsible for inducing cell proliferation and suppression of genes responsible for inhibition of proliferation were noted. Western blots revealed increased expression of cyclins D and A and inhibition of p21WAF1/CIP1 protein expression by leptin. Inhibition of DNA fragmentation was observed under all leptin doses. Suppression of genes involved in the extrinsic and intrinsic apoptotic pathway was observed. Western blots illustrated decreased Bad, TNFR1, and caspase 6 protein expression in response to leptin treatment. Leptin promotes ovarian cancer cell line growth by up-regulating genes and proteins responsible for inducing cell proliferation as well as down-regulating pro-apoptotic genes and proteins in apoptotic pathways. Results of this study warrant examining the relationship between the risk of ovarian cancer and elevated leptin levels in obese women.

The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

PubMed

Mizejewski, G J

2016-09-01

The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration.

PubMed

Hong, Jing-Fang; Song, Ying-Fang; Liu, Zheng; Zheng, Zhao-Cong; Chen, Hong-Jie; Wang, Shou-Sen

2016-06-01

The aim of the present study was to investigate the in vitro and in vivo anticancer and apoptotic effects of taraxerol acetate in U87 human glioblastoma cells. The effects on cell cycle phase distribution, cell cycle-associated proteins, autophagy, DNA fragmentation and cell migration were assessed. Cell viability was determined using the MTT assay, and phase contrast and fluorescence microscopy was utilized to determine the viability and apoptotic morphological features of the U87 cells. Flow cytometry using propidium iodide and Annexin V-fluorescein isothiocyanate demonstrated the effect of taraxerol acetate on the cell cycle phase distribution and apoptosis induction. Western blot analysis was performed to investigate the effect of the taraxerol acetate on cell cycle‑associated proteins and autophagy‑linked LC3B‑II proteins. The results demonstrated that taraxerol acetate induced dose‑ and time‑dependent cytotoxic effects in the U87 cells. Apoptotic induction following taraxerol acetate treatment was observed and the percentage of apoptotic cells increased from 7.3% in the control cells, to 16.1, 44.1 and 76.7% in the 10, 50 and 150 µM taraxerol acetate‑treated cells, respectively. Furthermore, taraxerol acetate treatment led to sub‑G1 cell cycle arrest with a corresponding decrease in the number of S‑phase cells. DNA fragments were observed as a result of the gel electrophoresis experiment following taraxerol acetate treatment. To investigate the inhibitory effects of taraxerol acetate on the migration of U87 cell, a wound healing assay was conducted. The number of cells that migrated to the scratched area decreased significantly following treatment with taraxerol acetate. In addition, taraxerol acetate inhibited tumor growth in a mouse xenograft model. Administration of 0.25 and 0.75 µg/g taraxerol acetate reduced the tumor weight from 1.2 g in the phosphate‑buffered saline (PBS)‑treated group (control) to 0.81 and 0.42 g, respectively. Similarly, 0.25 and 0.75 µg/g taraxerol acetate injection reduced the tumor volume from 1.3 cm3 in the PBS-treated group (control) to 0.67 and 0.25 cm3, respectively.

Curcumin inhibits growth potential by G1 cell cycle arrest and induces apoptosis in p53-mutated COLO 320DM human colon adenocarcinoma cells.

PubMed

Dasiram, Jade Dhananjay; Ganesan, Ramamoorthi; Kannan, Janani; Kotteeswaran, Venkatesan; Sivalingam, Nageswaran

2017-02-01

Curcumin, a natural polyphenolic compound and it is isolated from the rhizome of Curcuma longa, have been reported to possess anticancer effect against stage I and II colon cancer. However, the effect of curcumin on colon cancer at Dukes' type C metastatic stage III remains still unclear. In the present study, we have investigated the anticancer effects of curcumin on p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. The cellular viability and proliferation were assessed by trypan blue exclusion assay and MTT assay, respectively. The cytotoxicity effect was examined by lactate dehydrogenase (LDH) cytotoxicity assay. Apoptosis was analyzed by DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis. Cell cycle distribution was performed by flow cytometry analysis. Here we have observed that curcumin treatment significantly inhibited the cellular viability and proliferation potential of p53 mutated COLO 320DM cells in a dose- and time-dependent manner. In addition, curcumin treatment showed no cytotoxic effects to the COLO 320DM cells. DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis revealed that curcumin treatment induced apoptosis in COLO 320DM cells. Furthermore, curcumin caused cell cycle arrest at the G1 phase, decreased the cell population in the S phase and induced apoptosis in COLO 320DM colon adenocarcinoma cells. Together, these data suggest that curcumin exerts anticancer effects and induces apoptosis in p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network.

PubMed

Shen, Chengpin; Yu, Yanyan; Li, Hong; Yan, Guoquan; Liu, Mingqi; Shen, Huali; Yang, Pengyuan

2012-06-01

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[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.

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

PubMed

Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

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

Dey, Sumit K.; Saha, Shekhar; Das, Provas

2014-08-01

3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reducedmore » in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.« less

Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos.

PubMed

Miki, Hiromi; Inoue, Kimiko; Ogonuki, Narumi; Mochida, Keiji; Nagashima, Hiroshi; Baba, Tadashi; Ogura, Atsuo

2004-12-01

Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.

Fragment-based drug design and identification of HJC0123, a novel orally bioavailable STAT3 inhibitor for cancer therapy

PubMed Central

Chen, Haijun; Yang, Zhengduo; Ding, Chunyong; Chu, Lili; Zhang, Yusong; Terry, Kristin; Liu, Huiling; Shen, Qiang; Zhou, Jia

2013-01-01

Fragment-based drug design (FBDD) is a promising approach for the generation of lead molecules with enhanced activity and especially drug-like properties against therapeutic targets. Herein, we report the fragment-based drug design, systematic chemical synthesis and pharmacological evaluation of novel scaffolds as potent anticancer agents by utilizing six privileged fragments from known STAT3 inhibitors. Several new molecules such as compounds 5, 12, and 19 that may act as advanced chemical leads have been identified. The most potent compound 5 (HJC0123) has demonstrated to inhibit STAT3 promoter activity, downregulate phosphorylation of STAT3, increase the expression of cleaved caspase-3, inhibit cell cycle progression and promote apoptosis in breast and pancreatic cancer cells with low micromolar to nanomolar IC50 values. Furthermore, compound 5 significantly suppressed estrogen receptor (ER)-negative breast cancer MDA-MB-231 xenograft tumor growth in vivo (p.o.), indicating its great potential as an efficacious and orally bioavailable drug candidate for human cancer therapy. PMID:23416191

The ETD-like fragmentation for small molecules

USDA-ARS?s Scientific Manuscript database

Introduction (137) Flavonoids are a class of plant and fungus secondary metabolites (three-ring structure C6-C3-C6 with various substitutions) involved in many functions such as pigmentation, UV filtration, symbiotic nitrogen fixation, cell cycle inhibitors, and defense mechanisms. These polyphenoli...

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

PubMed Central

2013-01-01

Background Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. Results In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. Conclusions The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells PMID:23506616

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells.

PubMed

Chen, C; Yang, R L

2013-08-01

MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

Antiproliferative action of Xylopia aethiopica fruit extract on human cervical cancer cells.

PubMed

Adaramoye, Oluwatosin A; Sarkar, Jayanta; Singh, Neetu; Meena, Sanjeev; Changkija, Bendangla; Yadav, Prem P; Kanojiya, Sanjeev; Sinha, Sudhir

2011-10-01

The anticancer potential of Xylopia aethiopica fruit extract (XAFE), and the mechanism of cell death it elicits, was investigated in various cell lines. Treatment with XAFE led to a dose-dependent growth inhibition in most cell lines, with selective cytotoxicity towards cancer cells and particularly the human cervical cancer cell line C-33A. In this study, apoptosis was confirmed by nuclear fragmentation and sub-G(0)/G(1) phase accumulation. The cell cycle was arrested at the G(2)/M phase with a decreased G(0)/G(1) population. A semi-quantitative gene expression study revealed dose-dependent up-regulation of p53 and p21 genes, and an increase in the Bax/Bcl-2 ratio. These results indicate that XAFE could be a potential therapeutic agent against cancer since it inhibits cell proliferation, and induces apoptosis and cell cycle arrest in C-33A cells. Copyright © 2011 John Wiley & Sons, Ltd.

The Myxococcus xanthus Spore Cuticula Protein C Is a Fragment of FibA, an Extracellular Metalloprotease Produced Exclusively in Aggregated Cells

PubMed Central

Lee, Bongsoo; Mann, Petra; Grover, Vidhi; Treuner-Lange, Anke; Kahnt, Jörg; Higgs, Penelope I.

2011-01-01

Myxococcus xanthus is a soil bacterium with a complex life cycle involving distinct cell fates, including production of environmentally resistant spores to withstand periods of nutrient limitation. Spores are surrounded by an apparently self-assembling cuticula containing at least Proteins S and C; the gene encoding Protein C is unknown. During analyses of cell heterogeneity in M. xanthus, we observed that Protein C accumulated exclusively in cells found in aggregates. Using mass spectrometry analysis of Protein C either isolated from spore cuticula or immunoprecipitated from aggregated cells, we demonstrate that Protein C is actually a proteolytic fragment of the previously identified but functionally elusive zinc metalloprotease, FibA. Subpopulation specific FibA accumulation is not due to transcriptional regulation suggesting post-transcriptional regulation mechanisms mediate its heterogeneous accumulation patterns. PMID:22174937

The coordination between mechanical and chemical subsystems initiates locomotion of Physarum plasmodial fragments

NASA Astrophysics Data System (ADS)

Zhang, Shun; Guy, Robert; Del Alamo, Juan Carlos

2017-11-01

Physarum polycephalum is a multinucleated slime mold whose endoplasm flows periodically driven by the contraction of its ectoplasm, a dense shell of F-actin cross-linked by myosin molecular motors and attached to the cell membrane. We find that physarum fragments smaller than 100 microns remain round and stay in place. However, larger fragments break symmetry leading to sustained forward locomotion, in process that is reminiscent of an interfacial instability that seems to settle around two different limit cycles (traveling waves and standing waves). We use both theory and experiments to study how coordination emerges between the different mechanical and chemical subsystems of the fragment to initiate locomotion. The role of many involved factors, such as fragment size, substratum adhesiveness, rheological properties, actin polymerization and traction stresses are investigated, and we find they agree well with our predictive model.

[CHROMATIN ORGANIZATION IN CELL CYCLE OF AMOEBA PROTEUS ACCORDING TO OPTICAL TOMOGRAPHY DATA].

PubMed

Demin, S Yu; Berdieva, M A; Podlipaeva, Yu I; Yudin, A L; Goodkov, A V

2015-01-01

For the first time the nuclear cycle of large freshwater amoeba Amoeba proteus was studied by the method of optical tomography. The nuclei were fixed in situ in the cells of synchronized culture, stained by DAPI and examined by confocal laser scanning microscope. 3D-images of intranuclear chromatin were studied in details at different stages of nuclear cycle. The obtained data, together with literary ones allow represent the dynamics of structural organization of the nucleus in Amoeba proteus cell cycle in a new fashion. It was concluded that in this species the two-stage interphase takes place, as well as mitosis of peculiar type which does not correspond to any known type of mitosis according to classification existing now. It is presumed that in the course of nuclear cycle the chromosomes and/or their fragments are amplified, this presumption being in a good correspondence with the data about nuclear DNA hyperreplication in the cell cycle of A. proteus. As a result of chromosomes amplification their number may vary at different stages of cell cycle, and it allows to explain the contradictory data concerning the exact number of chromosomes in this species. The elimination of extra-DNA occurs mainly at the stage between prophase and prometaphase. We presume the majority of chromosomes, or may be even all of them to be referred to cholocentric type according to their behaviour during the mitosis.

The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation

PubMed Central

Shoji, Shisako; Muto, Yutaka; Ikeda, Mariko; He, Fahu; Tsuda, Kengo; Ohsawa, Noboru; Akasaka, Ryogo; Terada, Takaho; Wakiyama, Motoaki; Shirouzu, Mikako; Yokoyama, Shigeyuki

2014-01-01

Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain. PMID:25161877

Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes.

PubMed

Johnson, Mary Kathrine; Cooksey, Amanda M; Wise, Dwayne A

2008-11-01

CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented. (c) 2008 Wiley-Liss, Inc.

Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

PubMed

Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

2015-01-01

Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

PubMed Central

Chen, C.; Yang, R.L.

2013-01-01

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer. PMID:23903687

Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber

NASA Technical Reports Server (NTRS)

Rydberg, B.; Holley, W. R.; Mian, I. S.; Chatterjee, A.

1998-01-01

A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell. Copyright 1998 Academic Press.

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

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

Fujii, Seiko; Division of Maxillofacial Surgery, Kyushu Dental University; Okinaga, Toshinori

2013-05-10

Highlights: •Novel histone deacetylase inhibitor Ky-2, remarkably inhibits myeloma cell growth. •Ky-2 demonstrates no cytotoxicity against normal lymphocytic cells. •Ky-2 induces cell cycle arrest through the cell cycle-associated proteins. •Ky-2 induces Bcl-2-inhibitable apoptosis through a caspase-dependent cascade. -- Abstract: Objectives: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. Materials and methods: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viabilitymore » was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst’s staining was used to detect apoptotic cells. Results: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. Conclusion: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.« less

The dual effects of polar methanolic extract of Hypericum perforatum L. in bladder cancer cells

NASA Astrophysics Data System (ADS)

Nseyo, U. O.; Nseyo, O. U.; Shiverick, K. T.; Medrano, T.; Mejia, M.; Stavropoulos, N.; Tsimaris, I.; Skalkos, D.

2007-02-01

Introduction and background: We have reported on the polar methanolic fraction (PMF) of Hypericum Perforatum L as a novel photosensitizing agent for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). PMF has been tested in human leukemic cells, HL-60 cells, cord blood hemopoietic progenitor cells, bladder cancers derived from metastatic lymph node (T-24) and primary papillary bladder lesion (RT-4). However, the mechanisms of the effects of PMF on these human cell lines have not been elucidated. We have investigated mechanisms of PMF + light versus PMF-alone (dark experiment) in T-24 human bladder cancer cells. Methods: PMF was prepared from an aerial herb of HPL which was brewed in methanol and extracted with ether and methanol. Stock solutions of PMF were made in DSMO and stored in dark conditions. PMF contains 0.57% hypericin and 2.52% hyperforin. The T24 cell line was obtained from American Type Culture Collection (ATCC). In PDT treatment, PMF (60μg/ml) was incubated with cells, which were excited with laser light (630nm) 24 hours later. Apoptosis was determined by DNA fragmentation/laddering assay. DNA isolation was performed according to the manufacture's instructions with the Kit (Oncogene Kit#AM41). Isolated DNA samples were separated by electrophoresis in 1.5% in agarose gels and bands were visualized by ethidium bromide labeling. The initial cell cycle analysis and phase distribution was by flow cytometry. DNA synthesis was measured by [3H] thymidine incorporation, and cell cycle regulatory proteins were assayed by Western immunoblot. Results: The results of the flow cytometry showed PMF +light induced significant (40%) apoptosis in T24 cells, whereas Light or PMF alone produced little apoptosis. The percentage of cells in G 0/G I phase was decreased by 25% and in G2/M phase by 38%. The main impact was observed on the S phase which was blocked by 78% from the specific photocytotoxic process. DNA laddering analysis showed that PMF (60μg/ml) + light at 630nm induced DNA fragmentation in a light dose-dependent manner; in contrast, PMF or light alone did not induce DNA fragmentation. In separate experiments, PMF alone treatment produced a dose-dependent DNA synthesis with a 90% inhibition at a concentration of 25μg/ml (IC90 = 25μg/ml). Expression of p53 and p27 cell cycle regulatory proteins was not altered by PMF alone, however, a dose-dependent increase in p21 expression was observed that correlates with PMF concentrations. Cyclin A and cyclin B protein levels showed a clear decrease inverse to the concentration of PMF. In the absence of light treatment, flow cytometry analysis showed that PMF alone results in G 0/G I cell cycle arrest, with a 2-fold increase in G 0/G I cells concomitant with 50% decrease in cells in both S and G II/M phases. However, flow cytometry on PMF alone-treated cells did not show sub G 0/G I peak, further evidence of the lack of apoptosis as a mechanism of effect of PMF in the dark. Conclusions: With respect to light treatment, apoptosis appears to play a vital role in PDT-induced cytotoxicity. The flow cytometry and DNA laddering results revealed that T24 cells demonstrated apoptotic responses in PMF-mediated PDT. Experiments conducted with PMF alone showed a dose-dependent inhibition of DNA synthesis associated with G 0/G I cell cycle arrest and the extract is able to coordinate changes in key cell cycle regulatory proteins in human bladder cancer cells. Both experimental conditions suggest PMF as a potent and effect anti-proliferative agent in cancer chemoprevention and therapy of human urothelial carcinoma cells.

Plasmodium falciparum exhibits markers of regulated cell death at high population density in vitro.

PubMed

Engelbrecht, Dewaldt; Coetzer, Thérèsa Louise

2016-12-01

The asexual erythrocytic cycle of the protozoan parasite Plasmodium falciparum is responsible for the pathogenesis of malaria and causes the overwhelming majority of malaria deaths. Rapidly increasing parasitaemia during this 48hour cycle threatens the survival of the human host and the parasite prior to transmission of the slow-maturing sexual stages to the mosquito host. The parasite may utilise regulated cell death (RCD) to control the burden of infection on the host and thus aid its own survival and transmission. The occurrence of RCD in P. falciparum remains a controversial topic. We provide strong evidence for the occurrence of an apoptosis-like phenotype of RCD in P. falciparum under conditions of high parasite density. P. falciparum was maintained in vitro and stressed by allowing growth to an unrestricted peak parasitaemia. Cell death markers, including morphological changes, DNA fragmentation, mitochondrial polarisation and phosphatidylserine externalisation were used to characterise parasite death at the time of peak parasitaemia and 24h later. At peak parasitaemia, mitochondrial depolarisation was observed, together with phosphatidylserine externalisation in both parasitised- and neighbouring non-infected erythrocytes. DNA fragmentation coincided with a decline in parasitaemia. Fewer merozoites were observed in mature schizonts at peak parasitaemia. Growth recovery to near-peak parasitaemia was noted within two intraerythrocytic cycles. The combination and chronological order of the biochemical markers of cell death suggest the occurrence of an apoptosis-like phenotype. The identification of a RCD pathway in P. falciparum may provide novel drug targets, particularly if the pathway differs from the host machinery. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Curcumin induces apoptosis and cell cycle arrest via the activation of reactive oxygen species-independent mitochondrial apoptotic pathway in Smad4 and p53 mutated colon adenocarcinoma HT29 cells.

PubMed

Agarwal, Ayushi; Kasinathan, Akiladdevi; Ganesan, Ramamoorthi; Balasubramanian, Akhila; Bhaskaran, Jahnavi; Suresh, Samyuktha; Srinivasan, Revanth; Aravind, K B; Sivalingam, Nageswaran

2018-03-01

Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53. Copyright © 2018. Published by Elsevier Inc.

[Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

PubMed

Solary, E; Dubrez, L; Eymin, B; Bertrand, R; Pommier, Y

1996-03-01

Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

Viscum articulatum Burm. f. aqueous extract exerts antiproliferative effect and induces cell cycle arrest and apoptosis in leukemia cells.

PubMed

Mishra, Ruchi; Sharma, Saurabh; Sharma, Radhey Shyam; Singh, Savita; Sardesai, Milind Madhav; Sharma, Sadhna; Mishra, Vandana

2018-06-12

Viscum articulatum Burm. f. (leafless mistletoe) has been used in traditional system of medicines in India, China, Taiwan, Cambodia, Laos, and Vietnam, to treat blood-related diseases and various inflammatory and degenerative diseases including cancer. Anticancer activities of some phytomolecules purified from Viscum articulatum Burm. f. have been tested. However scientific evidence for the anticancerous potential of aqueous extract of V. articularum (VAQE) used in traditional medicine is lacking. To study the antiproliferative and apoptotic effect of VAQE on Jurkat E6.1 and THP1 leukemia cells. The aqueous extract of the whole plant of Viscum articulatum Burm. f. was prepared in phosphate buffer saline. In VAQE, total soluble protein was estimated using Bradford's dye-binding assay; flavonoid content was determined using aluminum chloride colorimetric assay; and phenolic content was estimated following Folin-Ciocalteu colorimetric assay. XTT cell viability assay was used to test VAQE induced cytotoxicity in Jurkat E6.1 and THP1 leukemia cells and peripheral blood mononuclear cells (PBMC). The effect of VAQE on cell cycle progression was analyzed by PI staining using flow cytometry. Annexin-V-FITC/PI differential staining method was used for detecting the onset of apoptosis in leukemia cells. Rhodamine 123 dye was used to detect the change in mitochondrial membrane potential (MMP) using flow cytometry. DCF-DA fluorescence dye was used to estimate the level of reactive oxygen species (ROS). The ROS inhibitors were used to evaluate the role of ROS in mediating DNA degradation in VAQE-treated leukemia cells. The molecular mechanisms underlying VAQE induced apoptosis induction was studied by analyzing the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins, caspase-8 and caspase-3 enzymes using western blot. Diphenylamine (DPA) assay was used to determine the DNA fragmentation and conclusion of apoptosis. VAQE triggered cytotoxic effect on Jurkat E6.1 (IC 50 -2.4 µg/ml; 24 h) and THP1 (IC 50 -1.0 µg/ml; 24 h) cells in a dose- and time-dependent manner. The apoptosis induction and G2/M arrest of the cell cycle are the cause of VAQE-induced cytotoxicity in leukemia cells. The apoptosis in VAQE-treated Jurkat E6.1 and THP1 cells was mediated via a reduction in MMP, elevation of intracellular ROS, decreased expression of the anti-apoptotic (Bcl-2) and increased expression of the pro-apoptotic (Bax) protein, activation of caspase-8 and caspase-3 and DNA fragmentation. VAQE has a high efficacy to exert a cytotoxic effect in Jurkat E6.1 and THP1 cells and to induce apoptosis and G2/M cell cycle arrest. VAQE induces extrinsic pathway of apoptosis in both the leukemia cell lines via disruption of MMP, intracellular ROS imbalance, increased ratio of Bax/Bcl-2, activation of caspase-8, caspase-3 and ROS-mediated DNA fragmentation. The knowledge gained from the outcomes of the study may encourage the identification of novel chemotherapeutic agent from Viscum articulatum Burm. f. to treat leukemia. Copyright © 2018 Elsevier B.V. All rights reserved.

The More the Merrier?. Entropy and Statistics of Asexual Reproduction in Freshwater Planarians

NASA Astrophysics Data System (ADS)

Quinodoz, Sofia; Thomas, Michael A.; Dunkel, Jörn; Schötz, Eva-Maria

2011-04-01

The trade-off between traits in life-history strategies has been widely studied for sexual and parthenogenetic organisms, but relatively little is known about the reproduction strategies of asexual animals. Here, we investigate clonal reproduction in the freshwater planarian Schmidtea mediterranea, an important model organism for regeneration and stem cell research. We find that these flatworms adopt a randomized reproduction strategy that comprises both asymmetric binary fission and fragmentation (generation of multiple offspring during a reproduction cycle). Fragmentation in planarians has primarily been regarded as an abnormal behavior in the past; using a large-scale experimental approach, we now show that about one third of the reproduction events in S. mediterranea are fragmentations, implying that fragmentation is part of their normal reproductive behavior. Our analysis further suggests that certain characteristic aspects of the reproduction statistics can be explained in terms of a maximum relative entropy principle.

Modeling extracellular matrix degradation balance with proteinase/transglutaminase cycle.

PubMed

Larreta-Garde, Veronique; Berry, Hugues

2002-07-07

Extracellular matrix mass balance is implied in many physiological and pathological events, such as metastasis dissemination. Widely studied, its destructive part is mainly catalysed by extracellular proteinases. Conversely, the properties of the constructive part are less obvious, cellular neo-synthesis being usually considered as its only element. In this paper, we introduce the action of transglutaminase in a mathematical model for extracellular matrix remodeling. This extracellular enzyme, catalysing intermolecular protein cross-linking, is considered here as a reverse proteinase as far as the extracellular matrix physical state is concerned. The model is based on a proteinase/transglutaminase cycle interconverting insoluble matrix and soluble proteolysis fragments, with regulation of cellular proteinase expression by the fragments. Under "closed" (batch) conditions, i.e. neglecting matrix influx and fragment efflux from the system, the model is bistable, with reversible hysteresis. Extracellular matrix proteins concentration abruptly switches from low to high levels when transglutaminase activity exceeds a threshold value. Proteinase concentration usually follows the reverse complementary kinetics, but can become apparently uncoupled from extracellular matrix concentration for some parameter values. When matrix production by the cells and fragment degradation are taken into account, the dynamics change to sustained oscillations because of the emergence of a stable limit cycle. Transitions out of and into oscillation areas are controlled by the model parameters. Biological interpretation indicates that these oscillations could represent the normal homeostatic situation, whereas the other exhibited dynamics can be related to pathologies such as tumor invasion or fibrosis. These results allow to discuss the insights that the model could contribute to the comprehension of these complex biological events.

Size Matters!. Birth Size and a Size-Independent Stochastic Term Determine Asexual Reproduction Dynamics in Freshwater Planarians

NASA Astrophysics Data System (ADS)

Thomas, Michael A.; Quinodoz, Sofia; Schötz, Eva-Maria

2012-09-01

Asexual reproduction by division in higher organisms is rare, because a prerequisite is the ability to regenerate an entire organism from a piece of the original body. Freshwater planarians are one of the few animals that can reproduce this way, but little is known about the regulation of their reproduction cycles or strategies. We have previously shown that a planarian's reproduction strategy is randomized to include fragmentations, producing multiple offspring, as well as binary fissions, and can be partially explained by a maximum relative entropy principle. In this study we attempt to decompose the factors controlling their reproduction cycle. Based on recent studies on the cell cycle of budding yeast, which suggest that molecular noise in gene expression and cell size at birth together control cell cycle variability, we investigated whether the variability in planarian reproduction waiting times could be similarly regulated. We find that such a model can indeed explain the observed distribution of waiting times between birth and next reproductive event, suggesting that birth size and a stochastic noise term govern the reproduction dynamics of asexual planarians.

Synthesis of Apoptotic New Quinazolinone-Based Compound and Identification of its Underlying Mitochondrial Signalling Pathway in Breast Cancer Cells.

PubMed

Zahedifard, Maryam; Faraj, Fadhil Lafta; Paydar, Mohammadjavad; Looi, Chung Yeng; Hasandarvish, Pouya; Hajrezaie, Maryam; Kamalidehghan, Behnam; Majid, Nazia Abdul; Khalifa, Shaden A M; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen; El-Seedi, Hesham R

2015-01-01

The anti-carcinogenic effect of the new quinazolinone compound, named MMD, was tested on MCF-7 human breast cancer cell line. The synthesis of quinazolinone-based compounds attracted strong attention over the past few decades as an alternative mean to produce analogues of natural products. Quinazolinone compounds sharing the main principal core structures are currently introduced in the clinical trials and pharmaceutical markets as anti-cancer agents. Thus, it is of high clinical interest to identify a new drug that could be used to control the growth and expansion of cancer cells. Quinazolinone is a metabolite derivative resulting from the conjugation of 2-aminobenzoyhydrazide and 5-methoxy-2- hydroxybenzaldehyde based on condensation reactions. In the present study, we analysed the influence of MMD on breast cancer adenoma cell morphology, cell cycle arrest, DNA fragmentation, cytochrome c release and caspases activity. MCF-7 is a type of cell line representing the breast cancer adenoma cells that can be expanded and differentiated in culture. Using different in vitro strategies and specific antibodies, we demonstrate a novel role for MMD in the inhibition of cell proliferation and initiation of the programmed cell death. MMD was found to increase cytochrome c release from the mitochondria to the cytosol and this effect was enhanced over time with effective IC50 value of 5.85 ± 0.71 μg/mL detected in a 72-hours treatment. Additionally, MMD induced cell cycle arrest at G0/G1 phase and caused DNA fragmentation with obvious activation of caspase-9 and caspases-3/7. Our results demonstrate a novel role of MMD as an anti-proliferative agent and imply the involvement of mitochondrial intrinsic pathway in the observed apoptosis.

Urtica dioica dichloromethane extract induce apoptosis from intrinsic pathway on human prostate cancer cells (PC3).

PubMed

Mohammadi, A; Mansoori, B; Aghapour, M; Baradaran, B

2016-03-31

Prostate cancer is considered as the major cause of death among men around the world. There are a number of medicinal plants triggering apoptosis response in cancer cells, thus have a therapeutic potential. Therefore, further studies to characterize beneficial properties of these plants in order to introduce novel anti-cancer drugs are the interest of recent researches on the alternative medicine. On the other hand, due to traditional uses and availability of Urtica dioica extract, we decided to evaluate the efficacy of this medicinal herb on pc3 prostate cancer cell line. In the present study the cytotoxic effects of Urtica dioica extract were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and trypan blue viability dye. Then, DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were exploited to measure cell death and apoptosis stage. The expression levels of caspase 3, caspase 9 and Bcl-2 genes were quantified by Real-Time PCR. Finally, Cell cycle was analyzed by flow cytometry. MTT assay showed that dichloromethanolic extract of Urtica dioica significantly inhibited the cell growth. According to the DNA fragmentation and TUNEL assay results, the herbal extract was able to induce apoptosis in prostate cancer cells. Our findings also demonstrated that the plant extract substantially increases the caspase 3 and 9 mRNA expression, while decreases Bcl-2. Cell cycle arrest was occurred in G2 stage, due to the results of flow cytometry. These results indicate that dichloromethanolic extract of Urtica dioica can successfully induce apoptosis in PC3 cells. Therefore, it could be used as a novel therapeutic candidate for prostate tumor treatment.

Electronic waste leachate-mediated DNA fragmentation and cell death by apoptosis in mouse fibroblast (NIH/3T3) cell line.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Filippin-Monteiro, Fabíola B; Sierra, Jelver A; Creczynski-Pasa, Tânia B

2013-08-01

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N-acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N-acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction.

PubMed

Buddhan, Rajamanickam; Manoharan, Shanmugam

2017-01-01

Aim of the present study was to evaluate the in vitro cytotoxic potential of the diosmin in A431 skin cancer cells. The cytotoxic (anti-cell proliferative) potential of diosmin in A431 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell viability), dual staining (apoptotic induction), dichloro-dihydro-fluorescein diacetate assay (reactive oxygen species [ROS] generation), DNA fragmentation study, Western blotting analysis (apoptotic markers expression) and flow cytometry (cell cycle arrest). Diosmin reduced the cell viability of A431 cells in a dose-dependent fashion and the inhibitory concentration 50% value was attained at 45 μg/ml using MTT assay. Diosmin at a concentration of 45 μg/ml generated excessive ROS in A431 cells, as compared to untreated cells. Diosmin treated A431 cells also revealed multiple DNA fragments than the untreated cells. Diosmin upregulated the expression of p53, caspases 3 and 9 and downregulated the expression of Bcl-2, matrix metalloproteinases-2 and 9 in A431 cells. The cytotoxic or anti-cell proliferative potential of diosmin is due to its ROS-mediated apoptotic induction potential, as well as due to its role in the inhibition of invasion in the A431 cells.

Types of rotor failure and characteristics of fragments

NASA Technical Reports Server (NTRS)

Mccarthy, D.

1977-01-01

Noncontained rotor failures in U.K. engines resulting from low cycle fatigue, low cycle fatigue with superimposed high cycle fatigue, and overheating and-or overspeeding were analyzed. The size, shape, weight, velocity, energy, and direction of the fragments released from turbines and compressors were studied and are presented in graph.

Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression

PubMed Central

Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

2015-01-01

Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent. PMID:25792804

Exploration of cell cycle regulation and modulation of the DNA methylation mechanism of pelargonidin: Insights from the molecular modeling approach.

PubMed

Karthi, Natesan; Karthiga, Arumugasamy; Kalaiyarasu, Thangaraj; Stalin, Antony; Manju, Vaiyapuri; Singh, Sanjeev Kumar; Cyril, Ravi; Lee, Sang-Myeong

2017-10-01

Pelargonidin is an anthocyanidin isolated from plant resources. It shows strong cytotoxicity toward various cancer cell lines, even though the carcinogenesis-modulating pathway of pelargonidin is not yet known. One of our previous reports showed that pelargonidin arrests the cell cycle and induces apoptosis in HT29 cells. Flowcytometry and immunoblot analysis confirmed that pelargonidin specifically inhibits the activation of CDK1 and blocks the G2-M transition of the cell cycle. In addition, DNA fragmentation was observed along with induction of cytochrome c release-mediated apoptosis. Hence, the aim of the present study was to investigate the molecular mechanism of pelargonidin's action on cell cycle regulators CDK1, CDK4, and CDK6 as well as the substrate-binding domain of DNMT1 and DNMT3A, which regulate the epigenetic signals related to DNA methylation. The results of docking analysis, binding free energy calculation, and molecular dynamics simulation correlated with the experimental results, and pelargonidin showed a specific interaction with CDK1. In this context, pelargonidin may also inhibit the recognition of DNA and catalytic binding by DNMT1 and DNMT3A. The HOMO-LUMO analysis mapped the functional groups of pelargonidin. Prediction of pharmacological descriptors suggested that pelargonidin can serve as a multitarget inhibitor for cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

National collection of embryo morphology data into Society for Assisted Reproductive Technology Clinic Outcomes Reporting System: associations among day 3 cell number, fragmentation and blastomere asymmetry, and live birth rate.

PubMed

Racowsky, Catherine; Stern, Judy E; Gibbons, William E; Behr, Barry; Pomeroy, Kimball O; Biggers, John D

2011-05-01

To evaluate the validity of collecting day 3 embryo morphology variables into the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS). Retrospective. National database-SART CORS. Fresh autologous assisted reproductive technology (ART) cycles from 2006-2007 in which embryos were transferred singly (n=1,020) or in pairs (n=6,508) and embryo morphology was collected. None. Relationship between live birth, maternal age, and morphology of transferred day 3 embryos as defined by cell number, fragmentation, and blastomere symmetry. Logistic multiple regressions and receiver operating characteristic curve analyses were applied to determine specificity and sensitivity for correctly classifying embryos as either failures or successes. Live birth rate was positively associated with increasing cell number up to eight cells (<6 cells: 2.9%; 6 cells: 9.6%; 7 cells: 15.5%; 8 cells: 24.3%; and >8 cells: 16.2%), but was negatively associated with maternal age, increasing fragmentation, and asymmetry scores. An area under the receiver operating curve of 0.753 (95% confidence interval 0.740-0.766) was derived, with a sensitivity of 45.0%, a specificity of 83.2%, and 76.4% of embryos being correctly classified with a cutoff probability of 0.3. This analysis provides support for the validity of collecting morphology fields for day 3 embryos into SART CORS. Standardization of morphology collections will assist in controlling for embryo quality in future database analyses. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Productive infection of Epstein-Barr virus (EBV) in EBV-genome-positive epithelial cell lines (GT38 and GT39) derived from gastric tissues.

PubMed

Takasaka, N; Tajima, M; Okinaga, K; Satoh, Y; Hoshikawa, Y; Katsumoto, T; Kurata, T; Sairenji, T

1998-08-01

We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G{sub 2}/M arrest, and triggers apoptosis in human leukemia HL-60 cells

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

Magalhães, Hemerson I.F.; Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, Paraíba; Wilke, Diego V.

2013-10-01

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC{sub 50} values in the nanomolar range. Cell cycle arrest in G{sub 2}/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation,more » loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G{sub 2}/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G{sub 2}/M phase of the cell cycle. • PHT induces caspase-dependent apoptosis.« less

Capturing a flavivirus pre-fusion intermediate.

PubMed

Kaufmann, Bärbel; Chipman, Paul R; Holdaway, Heather A; Johnson, Syd; Fremont, Daved H; Kuhn, Richard J; Diamond, Michael S; Rossmann, Michael G

2009-11-01

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting approximately 60 A-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements.

Fungal 7-epi-10-deacetyltaxol produced by an endophytic Pestalotiopsis microspora induces apoptosis in human hepatocellular carcinoma cell line (HepG2).

PubMed

Subban, Kamalraj; Singh, Satpal; Subramani, Ramesh; Johnpaul, Muthumary; Chelliah, Jayabaskaran

2017-11-28

Paclitaxel (taxol) is a potent anticancer drug that is used in the treatment of a wide variety of cancerous. In the present study, we identified a taxol derivative named 7-epi-10-deacetyltaxol (EDT) from the culture of an endophytic fungus Pestalotiopsis microspora isolated from the bark of Taxodium mucronatum. This study was carried out to investigate the effects of fungal EDT on cell proliferation, the induction of apoptosis and the molecular mechanisms of apoptosis in human hepatoma HepG2 cells in vitro. The endophytic fungus was identified by traditional and molecular taxonomical characterization and the fungal EDT was purified using column chromatography and confirmed by various spectroscopic and chromatographic comparisons with authentic paclitaxel. We studied the in vitro effects of EDT on HepG2 cells for parameters such as cell cycle distribution, DNA fragmentation, reactive oxygen species (ROS) generation and nuclear morphology. Further, western blot analysis was used to evaluate Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), p38-mitogen activated protein kinase (MAPK) and poly [ADP-ribose] polymerase (PARP) expression. We demonstrate that the fungal EDT exhibited significant in vitro cytotoxicity in HepG2 cells. We investigated cytotoxicity mechanism of EDT in HepG2 cells. The results showed nuclear condensation and DNA fragmentation were observed in cells treated with fungal EDT. Besides, the fungal EDT arrested HepG2 cells at G2/M phase of cell cycle. Furthermore, fungal EDT induced apoptosis in HepG2 cells in a dose-dependent manner associated with ROS generation and increased Bax/Bcl-2 ratio, p38 MAPKs and PARP cleavage. Our data show that EDT induced apoptotic cell death in HepG2 cells occurs through intrinsic pathway by generation of ROS mediated and activation of MAPK pathway. This is the first report for 7-epi-10-deacetyltaxol (EDT) isolated from a microbial source.

Anti-Cancer Effects of Imperata cylindrica Leaf Extract on Human Oral Squamous Carcinoma Cell Line SCC-9 in Vitro.

PubMed

Keshava, Rohini; Muniyappa, Nagesh; Gope, Rajalakshmi; Ramaswamaiah, Ananthanarayana Saligrama

2016-01-01

Imperata cylindrica, a tall tufted grass which has multiple pharmacological applications is one of the key ingredients in various traditional medicinal formula used in India. Previous reports have shown that I. cylindrica plant extract inhibited cell proliferation and induced apoptosis in various cancer cell lines. To our knowledge, no studies have been published on the effect of I. cylindrica leaf extract on human oral cancers. The present study was undertaken in order to evaluate the anticancer properties of the leaf extract of I. cylindrica using an oral squamous cell carcinoma cell line SCC-9 as an in vitro model system. A methanol extract from dried leaves of I. cylindrica (ICL) was prepared by standard procedures. Effects of the ICL extract on the morphology of SCC-9 cells was visualized by microscopy. Cytotoxicity was determined by MTT assay. Effects of the ICL extract on colony forming ability of SCC-9 cells was evaluated using clonogenic assay. Cell cycle analysis was performed by flow cytometry and induction of apoptosis was determined by DNA fragmentation assay. The ICL extract treatment caused cytotoxicity and induced cell death in vitro in SCC-9 cells in a dose-dependent manner. This treatment also significantly reduced the clonogenic potential and inhibited cell proliferation by arresting the cell cycle in the G2/M phase. Furthermore, DNA fragmentation assays showed that the observed cell death was caused by apoptosis. This is the first report showing the anticancer activity of the methanol extracts from the leaves of I. cylindrica in human oral cancer cell line. Our data indicates that ICL extract could be considered as one of the lead compounds for the formulation of anticancer therapeutic agents to treat/manage human oral cancers. The natural abundance of I. cylindrica and its wide geographic distribution could render it one of the primary resource materials for preparation of anticancer therapeutic agents.

Induction of apoptosis in human promyelocytic leukemia HL60 cells by an extract from Erythrina suberosa stem bark.

PubMed

Agrawal, Satyam Kumar; Agrawal, Madhunika; Sharma, Parduman Raj; Gupta, Bishan Datt; Arora, Saroj; Saxena, Ajit Kumar

2011-01-01

In this study, the apoptosis-inducing effect of an alcoholic extract from Erythrina suberosa stem bark (ESB) was investigated using human promyelocytic leukemia HL60 cells. Cell viability was estimated by MTT assay. We found that the ESB inhibited cell proliferation in a dose- and time-dependent manner. A series of well-documented morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and nuclear fragmentation, were observed by fluorescence microscopy. The gold standard scanning electron micrographs showed apoptotic bodies and formation of blebs. Cell cycle analysis showed a significant increase in Sub G(0) population of cells above 50 μg/ml. ESB treatment resulted in a dose-dependent increase in annexin V positive cells. Increase in intracellular ROS production up to sixfold was detected in ESB-treated HL60 cells by DCFH-DA assay. Dissipation of mitochondrial membrane potential of intact cells accompanied by increase in cytosolic cytochrome c was observed, which was followed by activation of caspase-9 and -3 but not caspase-8. DNA fragmentation analysis revealed typical ladders as early as 18 h indicative of caspase-3 role in the apoptotic pathway. The overall results suggest that ESB induces mitochondria-mediated intrinsic apoptotic pathway in HL60 cells and might have therapeutic value against human leukemia.

Feedback between intracellular flow, signaling and active stresses in Physarum plasmodial fragments

NASA Astrophysics Data System (ADS)

Zhang, Shun; Guy, Robert; Del Alamo, Juan Carlos

2016-11-01

Physarum polycephalum is a multinucleated slime mold whose endoplasm flows periodically driven by the contraction of its ectoplasm, a dense shell of F-actin cross-linked by myosin molecular motors and attached to the cell membrane. Ectoplasm contractions are regulated by calcium ions whose propagation is in turn governed by the flow. We study experimentally how this feedback leads to auto-oscillation by simultaneously measuring endoplasmic flow speed and rheological properties, the traction stresses between the ectoplasm and its substratum and the distribution of endoplasmic free calcium ions. We find that physarum fragments smaller than 100 microns remain round and stay in place. However, larger fragments break symmetry leading to sustained forward locomotion, in process that is reminiscent of an interfacial instability that seems to settle around two different limit cycles (traveling waves and standing waves). By using different adhesive coatings in the substratum we investigate the role of substratum friction in the emergence of coherent endoplasmic flow patterns and overall physarum fragment locomotion.

Anti-cancer Activity of Osmanthus matsumuranus Extract by Inducing G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma Hep G2 Cells

PubMed Central

Jin, Soojung; Park, Hyun-Jin; Oh, You Na; Kwon, Hyun Ju; Kim, Jeong-Hwan; Choi, Yung Hyun; Kim, Byung Woo

2015-01-01

Background: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). Methods: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. Results: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. Conclusions: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry. PMID:26734586

Novel purification of 1'S-1'-Acetoxychavicol acetate from Alpinia galanga and its cytotoxic plus antiproliferative activity in colorectal adenocarcinoma cell line SW480.

PubMed

Baradwaj, R G; Rao, M V; Senthil Kumar, T

2017-07-01

Alpinia galanga (L.) Willd. is a valuable medicinal crop found in specific tropical regions of southeast Asia. Its crude extracts are well known for their wide medicinal properties and many compounds identified from these extracts are of great interest currently. 1'S-1'-Acetoxychavicol acetate (ACA) obtained from rhizomes of A.galanga is one such well-illustrated compound. This study strives to progress and simplifies the purification protocol for ACA from A.galanga rhizomes. It also studies the cytotoxicity and antiproliferative activity of ACA against Dukes' type B, colorectal adenocarcinoma (SW480). HPLC standardisation was carried out for purification of ACA from rhizomes of Alpinia galanga. MTT assay was executed to estimate the IC 50 value of ACA against SW480 cell line. This value was used to study the apoptosis, nuclear morphological changes and mitochondrial membrane permeability using Acridine orange/ethidium bromide, DAPI, and JC-1 staining. The DNA fragmentation assay was used to substantiate the nuclear fragmentation of DNA observed in the DAPI staining. Further, cell cycle analysis was performed using flow cytometry to study the exact stage of the cell cycle where SW480 cells are arrested due to ACA, western blot analysis of relevant genes were done to further understand at molecular level. A comprehensive 1.89g of 1'S-1'-Acetoxychavicol acetate (ACA) was recovered from 500g of A.galanga rhizomes. ACA significantly suppressed the proliferation of SW480 cells at an IC 50 of 80μM (48h). The mode of SW480 cell death due to ACA was initially identified as apoptosis and cell cycle halted at G 0 /G 1 checkpoint with considerable DNA damage and mitochondrial depolarization. The expression of p21 was increased and concomitantly Cyclin D was downregulated in ACA treated in comparison to control. This study suggests that 1'S-1'-Acetoxychavicol acetate has potent anti-colorectal adenocarcinoma activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

PubMed

Sartagul, Wugangerile; Zhou, Xin; Yamada, Yuki; Ma, Ning; Tanaka, Katsunori; Furuyashiki, Tomoyuki; Ma, Yan

2014-01-01

DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair.

PubMed

Sunada, Shigeaki; Kanai, Hideki; Lee, Younghyun; Yasuda, Takeshi; Hirakawa, Hirokazu; Liu, Cuihua; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

2016-09-01

High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Effect of Methanolic Leaf Extract of Ocimum basilicum L. on Benzene-Induced Hematotoxicity in Mice

PubMed Central

Saha, S.; Mukhopadhyay, M. K.; Ghosh, P. D.; Nath, D.

2012-01-01

The aim of the present study was to investigate the protective role of methanolic leaf extract of Ocimum basilicum L. against benzene-induced hematotoxicity in Swiss albino mice. GC analysis and subacute toxicity level of the extract were tested. Mice were randomly divided into three groups among which II and III were exposed to benzene vapour at a dose 300 ppm × 6 hr/day × 5 days/week for 2 weeks and group I was control. Group III of this experiment was treated with the leaf methanolic extract at a dose of 100 mg/kg body weight, a dose in nontoxic range. Hematological parameters (Hb%, RBC and WBC counts), cell cycle regulatory proteins expression and DNA fragmentation analysis of bone marrow cells was performed. There was an upregulation of p53 and p21 and downregulation of levels of CDK2, CDK4, CDK6, and cyclins D1 and E in leaf extract-treated group. DNA was less fragmented in group III compared to group II (P < 0.05). The present study indicates that the secondary metabolites of O. basilicum L. methanolic leaf extract, comprising essential oil monoterpene geraniol and its oxidized form citral as major constituents, have modulatory effect in cell cycle deregulation and hematological abnormalities induced by benzene in mice. PMID:22988471

A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification.

PubMed

Xiao, Kunyi; Liu, Juan; Chen, Hui; Zhang, Song; Kong, Jilie

2017-05-15

A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 10 5 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells.

PubMed

Kobayashi, Shintaro; Yoshii, Kentaro; Hirano, Minato; Muto, Memi; Kariwa, Hiroaki

2017-02-01

Reverse genetics systems facilitate investigation of many aspects of the life cycle and pathogenesis of viruses. However, genetic instability in Escherichia coli has hampered development of a reverse genetics system for West Nile virus (WNV). In this study, we developed a novel reverse genetics system for WNV based on homologous recombination in mammalian cells. Introduction of the DNA fragment coding for the WNV structural protein together with a DNA-based replicon resulted in the release of infectious WNV. The growth rate and plaque size of the recombinant virus were almost identical to those of the parent WNV. Furthermore, chimeric WNV was produced by introducing the DNA fragment coding for the structural protein and replicon plasmid derived from various strains. Here, we report development of a novel system that will facilitate research into WNV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Chromosomal Aberrations in Normal and AT Cells Exposed to High Dose of Low Dose Rate Irradiation

NASA Technical Reports Server (NTRS)

Kawata, T.; Shigematsu, N.; Kawaguchi, O.; Liu, C.; Furusawa, Y.; Hirayama, R.; George, K.; Cucinotta, F.

2011-01-01

Ataxia telangiectasia (A-T) is a human autosomally recessive syndrome characterized by cerebellar ataxia, telangiectases, immune dysfunction, and genomic instability, and high rate of cancer incidence. A-T cell lines are abnormally sensitive to agents that induce DNA double strand breaks, including ionizing radiation. The diverse clinical features in individuals affected by A-T and the complex cellular phenotypes are all linked to the functional inactivation of a single gene (AT mutated). It is well known that cells deficient in ATM show increased yields of both simple and complex chromosomal aberrations after high-dose-rate irradiation, but, less is known on how cells respond to low-dose-rate irradiation. It has been shown that AT cells contain a large number of unrejoined breaks after both low-dose-rate irradiation and high-dose-rate irradiation, however sensitivity for chromosomal aberrations at low-dose-rate are less often studied. To study how AT cells respond to low-dose-rate irradiation, we exposed confluent normal and AT fibroblast cells to up to 3 Gy of gamma-irradiation at a dose rate of 0.5 Gy/day and analyzed chromosomal aberrations in G0 using fusion PCC (Premature Chromosomal Condensation) technique. Giemsa staining showed that 1 Gy induces around 0.36 unrejoined fragments per cell in normal cells and around 1.35 fragments in AT cells, whereas 3Gy induces around 0.65 fragments in normal cells and around 3.3 fragments in AT cells. This result indicates that AT cells can rejoin breaks less effectively in G0 phase of the cell cycle? compared to normal cells. We also analyzed chromosomal exchanges in normal and AT cells after exposure to 3 Gy of low-dose-rate rays using a combination of G0 PCC and FISH techniques. Misrejoining was detected in the AT cells only? When cells irradiated with 3 Gy were subcultured and G2 chromosomal aberrations were analyzed using calyculin-A induced PCC technique, the yield of unrejoined breaks decreased in both normal and AT cells and misrejoined breaks increased in both cell lines. The present study suggests that AT cells begin to rejoin breaks when a certain number of breaks are accumulated and an increased number of exchanges were observed in G0 AT cells, which is similar situation after high-dose-rate irradiation.

N-substituted 1,2-dihydroquinolines as anticancer agents: electronic control of redox stability, assessment of antiproliferative effects, and mechanistic insights.

PubMed

John Victor, Napoleon; Sakthivel, Ramasamy; Muraleedharan, Kannoth Manheri; Karunagaran, Devarajan

2013-10-01

Redox chemotherapy: Antiproliferative activities of a series of N-substituted 1,2-dihydroquinolines capable of causing redox imbalance in cancer cells are presented. Detailed studies showed that these derivatives arrest the cell cycle in the G2/M phase and induce apoptosis through an intrinsic pathway characterized by loss of mitochondrial membrane potential, DNA fragmentation, cytochrome c release, and activation of caspases 9 and 3. Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses.

PubMed

Sacková, Veronika; Kuliková, Lucia; Mikes, Jaromír; Kleban, Ján; Fedorocko, Peter

2005-01-01

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.

Induction of the c-myc protooncogene following antigen binding to hapten-specific B cells

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

Snow, E.C.; Fetherston, J.; Zimmer, S.

1986-03-01

Considerable controversy has centered on the role that the surface immunoglobulin (sIg) receptor for antigen plays during the induction of B cell activation. Stimulation by anti-Ig reagents has been shown to activate G/sub 0/ B cells to enter the cell cycle. The binding of thymus-dependent antigens to hapten-specific B cell populations apparently does not result in the movement of the antigen-binding cells (ABC) into the G/sub 1/ stage of the cell cycle. However, the authors have recently demonstrated that antigen binding to such hapten-specific B cells does result in the initiation of the membrane phosphatidylinositol cycle. In the present experiments,more » hapten-specific B cells (80-90% ABC, 99% in G/sub 0/) were incubated with either the correct hapten-carrier conjugate, with the carrier protein, or only media for 2 hours at 37/sup 0/C. At that time, total cellular RNA was isolated and subsequently analyzed by either dot blots or Northern gel techniques. The blots were probed with a (/sup 32/P)-c-myc SstI-Xhol fragment. The results indicate that hapten carrier stimulation of the hapten-specific B cells induces enhanced transcription of the c-myc gene. These observations lend further support to the premise that antigen binding to the sIg receptor results in the transduction to the cell of important signals and implicates the active participation of sIg during the process of antigen-mediated B cell activation.« less

Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis : Mentha extract as a neuroprotective against gamma irradiation.

PubMed

Hassan, Hanaa A; Hafez, Hani S; Goda, Mona S

2013-01-01

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue (p < 0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl(2) as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl(2) domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation.

Life Cycle Reversal in Aurelia sp.1 (Cnidaria, Scyphozoa)

PubMed Central

He, Jinru; Zheng, Lianming; Zhang, Wenjing; Lin, Yuanshao

2015-01-01

The genus Aurelia is one of the major contributors to jellyfish blooms in coastal waters, possibly due in part to hydroclimatic and anthropogenic causes, as well as their highly adaptive reproductive traits. Despite the wide plasticity of cnidarian life cycles, especially those recognized in certain Hydroza species, the known modifications of Aurelia life history were mostly restricted to its polyp stage. In this study, we document the formation of polyps directly from the ectoderm of degenerating juvenile medusae, cell masses from medusa tissue fragments, and subumbrella of living medusae. This is the first evidence for back-transformation of sexually mature medusae into polyps in Aurelia sp.1. The resulting reconstruction of the schematic life cycle of Aurelia reveals the underestimated potential of life cycle reversal in scyphozoan medusae, with possible implications for biological and ecological studies. PMID:26690755

Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

NASA Technical Reports Server (NTRS)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines.

PubMed

Uchida, Mona; Saeki, Kohei; Maeda, Shingo; Tamahara, Satoshi; Yonezawa, Tomohiro; Matsuki, Naoaki

2016-10-01

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

GENE EXPRESSION PROFILING OF HUMAN LIVER CARCINOMA (HepG2) CELLS EXPOSED TO THE MARINE TOXIN OKADAIC ACID

PubMed Central

Fieber, Lynne A.; Greer, Justin B.; Guo, Fujiang; Crawford, Douglas C.; Rein, Kathleen S.

2012-01-01

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning (DSP). In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis. PMID:23172983

Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions*

PubMed Central

Lamoliatte, Frederic; Bonneil, Eric; Durette, Chantal; Caron-Lizotte, Olivier; Wildemann, Dirk; Zerweck, Johannes; Wenshuk, Holger; Thibault, Pierre

2013-01-01

Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications. PMID:23750026

Signaling at the Golgi During Mitosis

PubMed Central

Colanzi, Antonino; Sütterlin, Christine

2014-01-01

The Golgi complex of mammalian cells is composed of interconnected stacks of flattened cisternae that form a continuous membrane system in the pericentriolar region of the cell. At the onset of mitosis, this so-called Golgi ribbon is converted into small tubular–vesicular clusters in a tightly regulated fragmentation process, which leads to a temporary loss of the physical Golgi–centrosome proximity. Mitotic Golgi breakdown is required for Golgi partitioning into the two daughter cells, cell cycle progression and may contribute to the dispersal of Golgi-associated signaling molecules. Here, we review our current understanding of the mechanisms that control mitotic Golgi reorganization, its biological significance, and assays that are used to study this process. PMID:24295319

The ethyl acetate extract of Phellinus linteus grown on germinated brown rice induces G0/G1 cell cycle arrest and apoptosis in human colon carcinoma HT29 cells.

PubMed

Park, Hye-Jin; Choi, Se Young; Hong, Se Mi; Hwang, Sung Gu; Park, Dong Ki

2010-07-01

It is well known that Phellinus linteus has a variety of biological functions, such as antitumor and immunomodulating activities. In our previous studies, we developed a P. linteus grown on germinated brown rice (PBR) and found that organic solvent extracts of PBR possessed immunomodulating activity to regulate a balance of cytokine network in mice. The components of PBR are ergosterol peroxide, gamma-aminobutyric acid (GABA) and Beta-glucan. In this study, we demonstrate that an organic solvent extract of P. linteus grown on PBR induced apoptotic cell death through the induction of G(0)/G(1) arrest of cell cycle and the apoptosis via DNA fragmentation in human colon carcinoma HT-29 cells. Cell death induced by the extract of P. linteus grown on PBR was shown to be associated with the upregulation of p21(CIP1/WAF1), the downregulation of cyclin D1, anti-apoptotic protein, Bcl-2, the release of cytochrome c, and the activation of caspase-9, caspase-3 and caspase-8. This study suggests that the ethyl acetate extract of P. linteus grown on PBR induces apoptosis accompanied by cell cycle arrest at G(0)/G(1) phase and regulates apoptosis-regulatory proteins, which may be applicable to anticancer therapy.

miR-30a can inhibit DNA replication by targeting RPA1 thus slowing cancer cell proliferation.

PubMed

Zou, Zhenyou; Ni, Mengjie; Zhang, Jing; Chen, Yongfeng; Ma, Hongyu; Qian, Shihan; Tang, Longhua; Tang, Jiamei; Yao, Hailun; Zhao, Chengbin; Lu, Xiongwen; Sun, Hongyang; Qian, Jue; Mao, Xiaoting; Lu, Xulin; Liu, Qun; Zen, Juping; Wu, Hanbing; Bao, Zhaosheng; Lin, Shudan; Sheng, Hongyu; Li, Yunlong; Liang, Yong; Chen, Zhiqiang; Zong, Dan

2016-07-15

Cell proliferation was inhibited following forced over-expression of miR-30a in the ovary cancer cell line A2780DX5 and the gastric cancer cell line SGC7901R. Interestingly, miR-30a targets the DNA replication protein RPA1, hinders the replication of DNA and induces DNA fragmentation. Furthermore, ataxia telangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2) were phosphorylated after DNA damage, which induced p53 expression, thus triggering the S-phase checkpoint, arresting cell cycle progression and ultimately initiating cancer cell apoptosis. Therefore, forced miR-30a over-expression in cancer cells can be a potential way to inhibit tumour development. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

PubMed Central

Mirza, Sameer; Katafiasz, Bryan J.; Kumar, Rakesh; Wang, Jun; Mohibi, Shakur; Jain, Smrati; Gurumurthy, Channabasavaiah Basavaraju; Pandita, Tej K.; Dave, Bhavana J.; Band, Hamid; Band, Vimla

2012-01-01

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3−/− cells. Notably, Ada3−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3fl/fl and Ada3−/− cells confirmed higher residual DNA damage in Ada3−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability. PMID:23095635

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

NASA Technical Reports Server (NTRS)

Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

2000-01-01

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

Centchroman induces redox-dependent apoptosis and cell-cycle arrest in human endometrial cancer cells.

PubMed

Shyam, Hari; Singh, Neetu; Kaushik, Shweta; Sharma, Ramesh; Balapure, Anil K

2017-04-01

Centchroman (CC) or Ormeloxifene has been shown to induce apoptosis and cell cycle arrest in various types of cancer cells. This has, however, not been addressed for endometrial cancer cells where its (CC) mechanism of action remains unclear. This study focuses on the basis of antineoplasticity of CC by blocking the targets involved in the cell cycle, survival and apoptosis in endometrial cancer cells. Ishikawa Human Endometrial Cancer Cells were cultured under estrogen deprived medium, exposed to CC and analyzed for proliferation and apoptosis. Additionally, we also analyzed oxidative stress induced by CC. Cell viability studies confirmed the IC 50 of CC in Ishikawa cells to be 20 µM after 48 h treatment. CC arrests the cells in G0/G1 phase through cyclin D1 and cyclin E mediated pathways. Phosphatidylserine externalization, nuclear morphology changes, DNA fragmentation, PARP cleavage, and alteration of Bcl-2 family protein expression clearly suggest ongoing apoptosis in the CC treated cells. Activation of caspase 3 & 9, up-regulation of AIF and inhibition of apoptosis by z-VAD-fmk clearly explains the participation of the intrinsic pathway of programmed cell death. Further, the increase of ROS, loss of MMP, inhibition of antioxidant (MnSOD, Cu/Zn-SOD and GST) and inhibition of apoptosis with L-NAC suggests CC induced oxidative stress leading to apoptosis via mitochondria mediated pathway. Therefore, CC could be a potential therapeutic agent for the treatment of Endometrial Cancer adjunct to its utility as a contraceptive and an anti-breast cancer agent.

A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation.

PubMed

Sato, Yuko; Kujirai, Tomoya; Arai, Ritsuko; Asakawa, Haruhiko; Ohtsuki, Chizuru; Horikoshi, Naoki; Yamagata, Kazuo; Ueda, Jun; Nagase, Takahiro; Haraguchi, Tokuko; Hiraoka, Yasushi; Kimura, Akatsuki; Kurumizaka, Hitoshi; Kimura, Hiroshi

2016-10-09

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Growth inhibitory and proapoptotic effects of l-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat).

PubMed

Meghavarnam, Anil K; Salah, Maryam; Sreepriya, Meenakshisundaram; Janakiraman, Savitha

2017-06-01

The objective of this study was to evaluate the anticancer properties of l-asparaginase purified from fungal isolate Fusarium culmorum ASP-87 against human T-cell leukemia cell line (Jurkat). The growth inhibitory and proapoptotic effects of purified l-asparaginase on Jurkat cell lines were investigated by determining its influence on cell viability, colony formation, DNA fragmentation, and cell cycle progression. The results revealed that purified l-asparaginase showed significant decrease in cell survival with IC 50 value of 90 μg/mL (9 IU/mL). The enzyme inhibited colony formation and showed characteristic laddering pattern on agarose gel thereby confirming the induction of apoptosis. Further, cell cycle analysis revealed that the enzyme induced apoptotic cell death by arresting the growth of cells at G 2 -M phase. However, the enzyme did not elicit any toxic effects on human erythrocytes. l-asparaginase purified from F. culmorum ASP-87 showed significant and selective cytotoxic and apoptotic effects on human T-cell leukemic cells in dose-dependent manner. Results of the study give leads for the anticancer effects of fungal l-asparaginase and its potential usefulness in the chemotherapy of leukemia. © 2016 Société Française de Pharmacologie et de Thérapeutique.

Dihydroartemisinin is an inhibitor of ovarian cancer cell growth.

PubMed

Jiao, Yang; Ge, Chun-min; Meng, Qing-hui; Cao, Jian-ping; Tong, Jian; Fan, Sai-jun

2007-07-01

To investigate the anticancer activity of dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin in a panel of human ovarian cancer cell lines. Cell growth was determined by the MTT viability assay. Apoptosis and cell cycle progression were evaluated by a DNA fragmentation gel electro-phoresis, flow cytometry assay, and TUNEL assay; protein and mRNA expression were analyzed by Western blotting and RT-PCR assay. Artemisinin and its derivatives, including artesunate, arteether, artemether, arteannuin, and DHA, exhibit anticancer growth activities in human ovarian cancer cells. Among them, DHA is the most effective in inhibiting cell growth. Ovarian cancer cell lines are more sensitive (5-10-fold) to DHA treatment compared to normal ovarian cell lines. DHA at micromolar dose levels exhibits a dose- and time-dependent cytotoxicity in ovarian cancer cell lines. Furthermore, DHA induced apoptosis and G2 cell cycle arrest, accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad. The promising results show for the first time that DHA inhibits the growth of human ovarian cancer cells. The selective inhibition of ovarian cancer cell growth, apoptosis induction, and G2 arrest provide in vitro evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of ovarian cancer.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

PubMed Central

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

PubMed

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

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.

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

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

Srivastava, Janmejai K.; Department of Urology, University Hospitals of Cleveland, Cleveland, OH 44106; Gupta, Sanjay

2006-07-28

One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation inmore » all three prostate cancer cell lines. The IC{sub 5} values after 24 h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 {mu}g/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 {mu}g/ml. In cell cycle analysis, TRF (10-40 {mu}g/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.« less

Persea declinata (Bl.) Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation

PubMed Central

Wong, Yi Li; Wong, Won Fen; Ali Mohd, Mustafa; Hadi, A. Hamid A.

2014-01-01

Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development. PMID:24808916

Inhibitory activity of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) on transformed cells by human papillomavirus.

PubMed

Hernández-Márquez, Eva; Lagunas-Martínez, Alfredo; Bermudez-Morales, Victor H; Burgete-García, Ana I; León-Rivera, Ismael; Montiel-Arcos, Elizur; García-Villa, Enrique; Gariglio, Patricio; Madrid-Marina V, Vicente; Ondarza-Vidaurreta, Raul N

2014-01-01

In this study, we investigated the effects of the aqueous extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, obtained from three localities (China; and Morelos and Michoacan, Mexico) on cervical cells transformed by human papillomavirus (HeLa and SiHa) and C-33A cancer cells. The cells were plated in DMEM medium supplemented, and were incubated in the presence of different concentrations of G. lucidum for 24 h. Cell proliferation was determined by MTT colorimetric assay and viability by trypan blue assay. Inhibitory dose was determined (IC50) of the three different extracts of G. lucidum in the culture cell lines mentioned above. The apoptosis process was confirmed by nuclear DNA fragmentation and the cell cycle was determined by flow cytometry. The results showed that aqueous extracts G. lucidum obtained from three localities produced inhibition in the proliferation of VPH transformed cells; they also induced apoptosis and cell cycle arrest in HeLa, SiHa, and C-33A cancer cells. Therefore, it was found that aqueous extracts G. lucidum obtained from three different locations produced inhibitory effect on cancer cells and may have a potential therapeutic use for the prevention and treatment of this disease.

The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

PubMed

Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

2017-08-01

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

The critical role of p16/Rb pathway in the inhibition of GH3 cell cycle induced by T-2 toxin.

PubMed

Fatima, Zainab; Guo, Pu; Huang, Deyu; Lu, Qirong; Wu, Qinghua; Dai, Menghong; Cheng, Guyue; Peng, Dapeng; Tao, Yanfei; Ayub, Muhammad; Ul Qamar, Muhammad Tahir; Ali, Muhammad Waqar; Wang, Xu; Yuan, Zonghui

2018-05-01

T-2 toxin is a worldwide trichothecenetoxin and can cause various toxicities.T-2 toxin is involved in G1 phase arrest in several cell lines but molecular mechanism is still not clear. In present study, we used rat pituitary GH3 cells to investigate the mechanism involved in cell cycle arrest against T-2 toxin (40 nM) for 12, 24, 36 and 48 h as compared to control cells. GH3 cells showed a considerable increase in reactive oxygen species (ROS) as well as loss in mitochondrial membrane potential (△Ym) upon exposure to the T-2 toxin. Flow cytometry showed a significant time-dependent increase in percentage of apoptotic cells and gel electrophoresis showed the hallmark of apoptosis oligonucleosomal DNA fragmentation. Additionally, T-2 toxin-induced oxidative stress and DNA damage with a time-dependent significant increased expression of p53 favors the apoptotic process by the activation of caspase-3 in T-2 toxin treated cells. Cell cycle analysis by flow cytometry revealed a time-dependent increase ofG1 cell population along with the significant time-dependent up-regulation of mRNA and protein expression of p16 and p21 and significant down-regulation of cyclin D1, CDK4, and p-RB levels further verify the G1 phase arrest in GH3 cells. Morphology of GH3 cells by TEM clearly showed the damage and dysfunction to mitochondria and the cell nucleus. These findings for the first time demonstrate that T-2 toxin induces G1 phase cell cycle arrest by the involvement of p16/Rb pathway, along with ROS mediated oxidative stress and DNA damage with p53 and caspase cascade interaction, resulting in apoptosis in GH3 cells. Copyright © 2018 Elsevier B.V. All rights reserved.

[On the nervous system of a parasitic cnidarian Polypodium hydriforme].

PubMed

Raĭkova, E V

2013-01-01

Nerve cells in a parasitic cnidarian Polypodium hydriforme at the parasitic and free-living stages of the life cycle have been localized immunocytochemically using antibodies to FMRF-amide, and their ultrastructure has been described. Ganglion cells form a net under epidermis consisting of bi- and tripolar neurons which cross the mesoglea and usually contact muscle cells and cnidocytes. Fusiform sensory and neurosecretory cells, especially characteristic to sensory tentacles, are interspersed among epidermal cells. All three types of nerve cells have dense cored vesicles about 80-120 nm in diameter. The sensory cells demonstrate a sensory flagellum-like immobile structure. Neurosecretory and sensory cells form septate junctions with epidermal cells. Ganglion cells show gap junctions between them. A centriole encircled by a fragment of nuclear envelope which is a marker of ectodermal lineage cells in Polypodium has been described in the cytoplasm of a sensory cell, thus proving the ectodermal nature of the nervous system.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Aqueous extract from pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell show activity against breast cancer cell line MCF-7 and Ehrlich ascites tumor in Balb-C mice.

PubMed

Hilbig, Josiane; Policarpi, Priscila de Britto; Grinevicius, Valdelúcia Maria Alves de Souza; Mota, Nádia Sandrine Ramos Santos; Toaldo, Isabela Maia; Luiz, Marilde Terezinha Bordignon; Pedrosa, Rozangela Curi; Block, Jane Mara

2018-01-30

In Brazil many health disorders are treated with the consumption of different varieties of tea. Shell extracts of pecan nut (Carya illinoinensis), which have significant amounts of phenolic compounds in their composition, are popularly taken as tea to prevent diverse pathologies. Phenolic compounds from pecan nut shell extract have been associated with diverse biological effects but the effect on tumor cells has not been reported yet. The aim of the current work was to evaluate the relationship between DNA fragmentation, cell cycle arrest and apoptosis induced by pecan nut shell extract and its antitumor activity. Cytotoxicity, proliferation, cell death and cell cycle were evaluated in MCF-7 cells by MTT, colony assay, differential coloring and flow cytometry assays, respectively. DNA damage effects were evaluated through intercalation into CT-DNA and plasmid DNA cleavage. Tumor growth inhibition, survival time increase, apoptosis and cell cycle arrest were assessed in Ehrlich ascites tumor in Balb/C mice. The cytotoxic effect of pecan nut shell extracts, the induction of cell death by apoptosis and also the cell cycle arrest in MCF-7 cells have been demonstrated. The survival time in mice with Ehrlich ascites tumor increased by 67%. DNA damage was observed in the CT-DNA, plasmid DNA and comet assays. The mechanism involved in the antitumor effect of pecan nut shell extracts may be related to the activation of key proteins involved in apoptosis cell death (Bcl-XL, Bax and p53) and on the cell cycle regulation (cyclin A, cyclin B and CDK2). These results were attributed to the phenolic profile of the extract, which presented compounds such as gallic, 4-hydroxybenzoic, chlorogenic, vanillic, caffeic and ellagic acid, and catechin, epicatechin, epigallocatechin and epicatechin gallate. The results indicated that pecan nut shell extracts are effective against tumor cells growth and may be considered as an alternative to the treatment of cancer. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Attosecond-recollision-controlled selective fragmentation of polyatomic molecules.

PubMed

Xie, Xinhua; Doblhoff-Dier, Katharina; Roither, Stefan; Schöffler, Markus S; Kartashov, Daniil; Xu, Huailiang; Rathje, Tim; Paulus, Gerhard G; Baltuška, Andrius; Gräfe, Stefanie; Kitzler, Markus

2012-12-14

Control over various fragmentation reactions of a series of polyatomic molecules (acetylene, ethylene, 1,3-butadiene) by the optical waveform of intense few-cycle laser pulses is demonstrated experimentally. We show both experimentally and theoretically that the responsible mechanism is inelastic ionization from inner-valence molecular orbitals by recolliding electron wave packets, whose recollision energy in few-cycle ionizing laser pulses strongly depends on the optical waveform. Our work demonstrates an efficient and selective way of predetermining fragmentation and isomerization reactions in polyatomic molecules on subfemtosecond time scales.

DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome.

PubMed

Avendaño, Conrado; Franchi, Anahí; Duran, Hakan; Oehninger, Sergio

2010-07-01

To evaluate DNA fragmentation in morphologically normal sperm recovered from the same sample used for intracytoplasmic sperm injection (ICSI) and to correlate DNA damage with embryo quality and pregnancy outcome. Prospective study. Academic center. 36 infertile men participating in the ICSI program. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL) assay and morphologic assessment by phase contrast. Simultaneous assessment of sperm morphology and DNA fragmentation by TUNEL assay was performed in the same cell, then the percentage of normal sperm with fragmented DNA (normal SFD) was correlated with embryo quality and pregnancy outcomes. A highly statistically significant negative correlation was found between the percentage of normal SFD and embryo quality. This association was confirmed for the transferred embryos and for the total embryo cohort. The receiver operating characteristics curve analysis demonstrated that the percentage of normal SFD and embryo quality were statistically significant predictors of pregnancy. When the percentage of normal SFD was

Novel fragment-based QSAR modeling and combinatorial design of pyrazole-derived CRK3 inhibitors as potent antileishmanials.

PubMed

Goyal, Sukriti; Dhanjal, Jaspreet K; Tyagi, Chetna; Goyal, Manisha; Grover, Abhinav

2014-07-01

The CRK3 cyclin-dependent kinase of Leishmania plays an important role in regulating the cell-cycle progression at the G2/M phase checkpoint transition, proliferation, and viability inside the host macrophage. In this study, a novel fragment-based QSAR model has been developed using 22 pyrazole-derived compounds exhibiting inhibitory activity against Leishmanial CRK3. Unlike other QSAR methods, this fragment-based method gives flexibility to study the relationship between molecular fragments of interest and their contribution for the variation in the biological response by evaluating cross-term fragment descriptors. Based on the fragment-based QSAR model, a combinatorial library was generated, and top two compounds were reported after predicting their activity. The QSAR model showed satisfactory statistical parameters for the data set (r(2) = 0.8752, q(2) = 0.6690, F-ratio = 30.37, and pred_r(2) = 0.8632) with four descriptors describing the nature of substituent groups and the environment of the substitution site. Evaluation of the model implied that electron-rich substitution at R1 position improves the inhibitory activity, while decline in inhibitory activity was observed in presence of nitrogen at R2 position. The analysis carried out in this study provides a substantial basis for consideration of the designed pyrazole-based leads as potent antileishmanial drugs. © 2014 John Wiley & Sons A/S.

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

PubMed

Chen, Linxu; Xu, Qilong; Tu, Jiagang; Ge, Yihe; Liu, Jun; Liang, Fang Ting

2013-01-01

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

Ardipusilloside I purified from Ardisia pusilla competitively binds VEGFR and induces apoptosis in NCI-H460 cells.

PubMed

Zhang, Yanmin; Qu, Youle; Zhang, Jie; Wang, Xiaojuan

2010-06-01

The present study was to evaluate the effects of Ardipusilloside I isolated from Ardisia pusilla on the growth, vascular endothelial growth factor receptor (VEGFR) expression and apoptosis of NCI-H460 cell line by MTT, ELISA and flow cytometer, respectively. The docking assay between Ardipusilloside I and VEGFR was studied by Sybyl/Sketch module. The change of microstructure was observed by transmission electron microscope (TEM). DNA fragmentation was visualized by agarose gel electrophoresis. The protein expression of Bax and Bcl-2 was detected by immunohistochemistry (IHC). A series of changes were observed in NCI-H460 cell treated by Ardipusilloside I, including microstructure, DNA fragmentation, protein expression of VEGFR, Bax and Bcl-2. The results showed Ardipusilloside I had a good docking with VEGFR and could inhibit growth and induce apoptosis of NCI-H460 cell in a dose-dependent manner. Cell cycle was significantly stopped at the G(1) phase. Under electronic microscope, the morphology of NCI-H460 cell treated with Ardipusilloside I showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body. VEGFR and Bcl-2 expression were decreased and Bax expression was increased. In conclusion, all these results demonstrate that Ardipusilloside I has a good docking with VEGFR and has an inhibitory effect on growth of NCI-H460 cell and can induce its apoptosis.

Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

PubMed

Zhang, X; Xu, Q; Saiki, I

2000-01-01

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

PubMed

Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

2010-06-01

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

Piecing together the fragments: Elucidating edge effects on forest carbon dynamics

NASA Astrophysics Data System (ADS)

Hutyra, L.; Smith, I. A.; Reinmann, A.; Marrs, J.; Thompson, J.

2017-12-01

Forest fragmentation is pervasive throughout the world's forests, impacting growing conditions and carbon dynamics through edge effects that produce gradients in microclimate, biogeochemistry, and stand structure. Despite the majority of the world's forests being <1km from an edge, our understanding of forest carbon dynamics is largely derived from intact forest systems. In the northeastern USA, we find that over 23% of the current forest area is just 30m from an agricultural or developed edge. Edge effects on the carbon cycle vary in their magnitude by biome, but current forest carbon accounting methods and ecosystem models largely do not include edge effects, highlighting an important gap in our understanding of the terrestrial carbon cycle. Characterizing the role of forest fragmentation in regional and global biogeochemical cycles necessitates advancing our understanding of how shifts in microenvironment at the forest edge interact with local prevailing drivers of global change and limitations to microbial activity and forest growth. This study synthesizes the literature related to edge effects and the carbon cycle, considering how fragmentation affects the growing conditions of the world's remaining forests based on risks and opportunities for forests near the edge.

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

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

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

DOE PAGES

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; ...

2015-12-22

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Bögler, Oliver; Duchrow, Michael

2003-01-01

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase. Copyright 2002 John Wiley & Sons, Ltd.

Supercritical carbon dioxide extract of Physalis peruviana induced cell cycle arrest and apoptosis in human lung cancer H661 cells.

PubMed

Wu, Shu-Jing; Chang, Shun-Pang; Lin, Doung-Liang; Wang, Shyh-Shyan; Hou, Fwu-Feuu; Ng, Lean-Teik

2009-06-01

Physalis peruviana L. (PP) is a popular folk medicine used for treating cancer, leukemia, hepatitis, rheumatism and other diseases. In this study, our objectives were to examine the total flavonoid and phenol content of different PP extracts (aqueous: HWEPP; ethanolic: EEPP; supercritical carbon dioxide: SCEPP-0, SCEPP-4 and SCEPP-5) and their antiproliferative effects in human lung cancer H661 cells. Among all the extracts tested, results showed that SCEPP-5 possessed the highest total flavonoid (226.19 +/- 4.15 mg/g) and phenol (100.82 +/- 6.25 mg/g) contents. SCEPP-5 also demonstrated the most potent inhibitory effect on H661 cell proliferation. Using DNA ladder and flow cytometry analysis, SCEPP-5 effectively induced H661 cell apoptosis as demonstrated by the accumulation of Sub-G1 peak and fragmentation of DNA. SCEPP-5 not only induced cell cycle arrest at S phase, it also up-regulated the expression of pro-apoptotic protein (Bax) and down-regulated the inhibitor of apoptosis protein (IAP). Furthermore, the apoptotic induction in H661 cells was found to associate with an elevated p53 protein expression, cytochrome c release, caspase-3 activation and PARP cleavage. Taken together, these results conclude that SCEPP-5 induced cell cycle arrest at S phase, and its apoptotic induction could be mediated through the p53-dependent pathway and modification of Bax and XIAP proteins expression. The results have also provided important pharmacological backgrounds for the potential use of PP supercritical fluid extract as products for cancer prevention.

Dendrobium chrysanthum ethanolic extract induces apoptosis via p53 up-regulation in HeLa cells and inhibits tumor progression in mice.

PubMed

Prasad, Ritika; Rana, Nishant Kumar; Koch, Biplob

2017-06-01

Background Dendrobium is one of the diverse genus of orchid plants. It possesses a number of pharmacological activities and has long been used in traditional system of medicine. The goal of this study was to investigate the apoptosis inducing property of the ethanolic extract from the leaves of Dendrobium chrysanthum, a species of Dendrobium whose anticancer role has not been ascertained yet. Methods To evaluate the anticancer activity of the ethanolic extract of D. chrysanthum in vitro in HeLa (human cervical cancer) cells, cytotoxic activity, generation of reactive oxygen species (ROS), induction of apoptosis and effect on cell cycle were determined. The in vivo study was carried out in Dalton's lymphoma (DL) bearing mice to assess the tumor growth delay. Results Our study demonstrated that the ethanolic extract showed dose-dependent cytotoxicity against HeLa cells. The extract exhibited dose-dependent increase in ROS production as well as apoptotic cell death which was further confirmed through presence of DNA fragmentation. Cell cycle analysis by flow cytometry suggests that the ethanolic extract perturbed cell cycle progression and leads to the delay of the cells in S phase. Further, the real-time PCR studies also showed up-regulation of apoptotic genes p53 and Bax. The in vivo antitumor activity exhibited significant increase in the life span of DL bearing mice as compared to control with significant decrease in abdominal size along with reduced tumor ascites. Conclusions These observations demonstrate the anticancer potential of the D. chrysanthum ethanolic extract mediated through p53-dependent apoptosis.

Effects of 1,3,5-triphenyl-4,5-dihydro-1H-pyrazole derivatives on cell-cycle and apoptosis in human acute leukemia cell lines.

PubMed

Santos Bubniak, Lorena Dos; Gaspar, Pâmela Cristina; de Moraes, Ana Carolina Rabello; Bigolin, Alisson; de Souza, Rubia Karine; Buzzi, Fátima Campos; Corrêa, Rogério; Filho, Valdir Cechinel; Bretanha, Lizandra Czermainski; Micke, Gustavo Amadeu; Nunes, Ricardo José; Santos-Silva, Maria Cláudia

2017-05-01

Pyrazoline is an important 5-membered nitrogen heterocycle that has been extensively researched. Ten derivatives were synthesized and tested for antileukemic effects on 2 human acute leukemia cell lines, K562 and Jurkat. The most cytotoxic of these derivatives, compound 21, was chosen for investigation of cytotoxicity mechanisms. The results obtained with selectivity calculations revealed that compound 21 is more selective for acute leukemia (K562 and Jurkat cell lines) than for other tumor cell lines. Moreover, compound 21 was not cytotoxic to normal cell lines, indicating a potential use in clinical tests. Compound 21 caused a significant cell cycle arrest in the S-phase in Jurkat cells and increased the proportion of cells in the sub G0/G1 phase in both cell lines. Cells treated with compound 21 demonstrated morphological changes characteristic of apoptosis in the EB/AO assay, confirmed by externalization of phosphatidylserine by the annexin V - fluorescein isothiocyanate method and by DNA fragmentation. An investigation of cytotoxicity mechanisms suggests the involvement of an intrinsic apoptosis pathway due to mitochondrial damage and an increase in the ratio of mitochondrial Bax/Bcl2. Pyrazoline 21 obeyed Lipinski's "rule of five" for drug-likeness. Based on these preliminary results, the antileukemic activity of compound 21 makes it a potential anticancer agent.

[Effects of sinensetin on proliferation and apoptosis of human gastric cancer AGS cells].

PubMed

Dong, Yang; Ji, Guang; Cao, Aili; Shi, Jianrong; Shi, Hailian; Xie, Jianqun; Wu, Dazheng

2011-03-01

To study the effects and mechanisms of sinensetin on proliferation and apoptosis of human AGS gastric cancer cells. MTT assay was used to detect the growth inhibition rates of human AGS gastric cancer cells treated with sinsesectin in different concentrations and times. The cell cycle distribution was measured by flow cytometry. The apoptosis was examined by Annexin-FITC/PI staining and DNA fragment analysis. The apoptosis morphology was observed by inverted fluorescence microscope after Hoechst 33342 staining. The protein expressions of p21 and p53 were detected by western blot. MTT assay showed that sinensetin inhibited the growth of AGS gastric cancer cells in a dose- and time-dependent manner. Sinensetin blocked AGS cells in G2/ M and increased the apoptosis rates of AGS cells in a dose-dependent manner. DNA ladder was observed in cells treated with 60 micromol x L(-1) sinensetin for 48 h. The typical apoptotic morphological changes including cell nucleus shrinkage, chromatin condensation and apoptotic bodies were observed when treated with different dose of sinensetin. Western blot showed that sinensetin increased expressions of p53 and p21 in a dose-dependent manner. Sinensetin could inhibit human AGS gastric cancer cells proliferation and induce cell cycle block in G2/M phase and apoptosis. The up regulation of p53 and p21 protein might be one of the mechanisms.

Bis-demethoxy curcumin analog nanoparticles: synthesis, characterization, and anticancer activity in vitro.

PubMed

Francis, Arul Prakash; Murthy, Prakhya Balakishna; Devas, Thiyagarajan

2014-07-01

We have optimized a protocol for the preparation of bisdemethoxy curcumin analog nanoparticles (BDMCA-NP) by the solvent assisted process. The structural similarities between bulk and nano BDMCA were determined by Co-TLC, NMR and F-TIR. This shows that our synthesis protocol enhanced the dispersibility and reduce the size of BDMCA without altering the integrity of functional moieties and structure, which is crucial for anticancer and antioxidant activities. The morphology and size of BDMCA-NP as determined by SEM, HRTEM and DLS was found to be around 80 nm. BDMCA-NP treated breast cancer cell lines (MCF 7) showed cell death as characterized by MTT assay. Flow cytometric analysis of BDMCA-NP treated MCF 7 cell lines showed an increase of cell count in G2/M phase indicates the cell cycle arrest. Western blot analysis revealed the presence of caspase 3, caspase 9, cleaved fragments of PARP and Bax proteins in the BDMCA-NP treated MCF 7 cell lines, but not in untreated cell lines. To recap, we have prepared BDMCA-NP by solvent assisted process, which exerted anticancer activity against breast cancer cells, which may be due to (i) enhanced dispersibility and surface: volume ratio, (ii) apoptosis (iii) mitochondrial pathway induced cell death, (iv) G2/M phase cell cycle arrest and (v) disassembly of mitotic spindle of the cancer cells. Thus, nano BDMCA can be used as a potent anticancer agent.

Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

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

Huang, Li-Wen; Hsieh, Bau-Shan; Cheng, Hsiao-Ling

2012-01-15

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growthmore » and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M phase arrest, followed by apoptosis.« less

Long-term oscillations in the sleep/wake cycle of infants

NASA Astrophysics Data System (ADS)

Diambra, L.; Malta, C. P.; Capurro, A.

2009-11-01

The development of circadian sleep-wakefulness rhythm was investigated by a longitudinal study of six normal infants. We propose an entropy based measure for the sleep/wake cycle fragmentation. Our results confirm that the sleep/wake cycle fragmentation and the sleep/wake ratio decrease, while the circadian power increases during the maturation process of infants. In addition to these expected linear trends in the variables devised to quantify sleep consolidation, circadian power and sleep/wake ratio, we found that they present infradian rhythms in the monthly range.

Can Tissue Cilia Lengths and Urine Cilia Proteins Be Markers of Kidney Diseases?

PubMed

Park, Kwon Moo

2018-05-01

The primary cilium is an organelle which consists of a microtubule in the core and a surrounding cilia membrane, and has long been recognized as a "vestigial organelle". However, new evidence demonstrates that the primary cilium has a notable effect on signal transduction in the cell and is associated with some genetic and non-genetic diseases. In the kidney, the primary cilium protrudes into the Bowman's space and the tubular lumen from the apical side of epithelial cells. The length of primary cilia is dynamically altered during the normal cell cycle, being shortened by retraction into the cell body at the entry of cell division and elongated at differentiation. Furthermore, the length of primary cilia is also dynamically changed in the cells, as a result and/or cause, during the progression of various kidney diseases including acute kidney injury and chronic kidney disease. Notably, recent data has demonstrated that the shortening of the primary cilium in the cell is associated with fragmentation, apart from retraction into the cell body, in the progression of diseases and that the fragmented primary cilia are released into the urine. This data reveals that the alteration of primary cilia length could be related to the progression of diseases. This review will consider if primary cilia length alteration is associated with the progression of kidney diseases and if the length of tissue primary cilia and the presence or increase of cilia proteins in the urine is indicative of kidney diseases.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells.

PubMed

Wei, Ren-Jie; Lin, Su-Shuan; Wu, Wen-Ren; Chen, Lih-Ren; Li, Chien-Feng; Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun; Liang, Shih-Shin; Chien, Shang-Tao; Shiue, Yow-Ling

2016-11-15

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G 2 /M cell cycle regulators, ABT-751 induced G 2 /M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. Copyright © 2016 Elsevier Inc. All rights reserved.

Disruption and molecule degradation of waxy maize starch granules during high pressure homogenization process.

PubMed

Wei, Benxi; Cai, Canxin; Xu, Baoguo; Jin, Zhengyu; Tian, Yaoqi

2018-02-01

The mechanism underlying the fragmentation of waxy maize starch (WMS) granules during high-pressure homogenization (HPH) was studied and the results were interpreted in terms of granular and molecular aspects. The diameter of disrupted starch granules decreased exponentially with increasing HPH pressure, but decreased linearly with increasing of HPH cycles. Scanning electron microscopy revealed a cone-like inside-out disruption pattern through the channels that resulted in separation of blocklets fragments or starch fragments. The M w of amylopectin was reduced by ∼half following treatment at 150MPa with two cycles, or at 100MPa for eight cycles, and the decrease was in accordance with the disruption of starch granules. This indicated that amylopectin was "protected" by blocklets, and the disruption of WMS granules mainly occurred close to the linkage among blocklets. Increasing the HPH pressure appeared to be more effective for breaking starch granules than increasing the number of HPH cycles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Computational model of chromosome aberration yield induced by high- and low-LET radiation exposures.

PubMed

Ponomarev, Artem L; George, Kerry; Cucinotta, Francis A

2012-06-01

We present a computational model for calculating the yield of radiation-induced chromosomal aberrations in human cells based on a stochastic Monte Carlo approach and calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. A previously developed DNA-fragmentation model for high- and low-LET radiation called the NASARadiationTrackImage model was enhanced to simulate a stochastic process of the formation of chromosomal aberrations from DNA fragments. The current version of the model gives predictions of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G(0)/G(1) cell cycle phase during the first cell division after irradiation. As the model can predict smaller-sized deletions and rings (<3 Mbp) that are below the resolution limits of current cytogenetic analysis techniques, we present predictions of hypothesized small deletions that may be produced as a byproduct of properly repaired DNA double-strand breaks (DSB) by nonhomologous end-joining. Additionally, the model was used to scale chromosomal exchanges in two or three chromosomes that were obtained from whole-chromosome FISH painting analysis techniques to whole-genome equivalent values.

Hair cycle control by leptin as a new anagen inducer.

PubMed

Sumikawa, Yasuyuki; Inui, Shigeki; Nakajima, Takeshi; Itami, Satoshi

2014-01-01

Our purpose is to clarify the physiological role of leptin in hair cycle as leptin reportedly causes activation of Stat3, which is indispensable for hair cycling. While hair follicles in dorsal skin of 5-week-old C57/BL6 mice had progressed to late anagen phase, those in dorsal skin of 5-week-old leptin receptor deficient db/db mice remained in the first telogen and later entered the anagen at postnatal day 40, indicating that deficiency in leptin receptor signalling delayed the second hair cycle progression. Next, we shaved dorsal hairs on wild-type mice at postnatal 7 weeks and injected skin with mouse leptin or a mock. After 20 days, although mock injection showed no effect, hair growth occurred around leptin injection area. Human leptin fragment (aa22-56) had similar effects. Although the hair cycle of ob/ob mice was similar to that of wild-type mice, injection of mouse leptin on ob/ob mice at postnatal 7 weeks induced anagen transition. Immunohistochemically, leptin is expressed in hair follicles from catagen to early anagen in wild-type mice, suggesting that leptin is an anagen inducer in vivo. Phosphorylation of Erk, Jak2 and Stat3 in human keratinocytes was stimulated by leptin and leptin fragment. In addition, RT-PCR and ELISA showed that the production of leptin by human dermal papilla cells increased under hypoxic condition, suggesting that hypoxia in catagen/telogen phase promotes leptin production, preparing for entry into the next anagen. In conclusion, leptin, a well-known adipokine, acts as an anagen inducer and represents a new player in hair biology. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Augmented cell survival in eutopic endometrium from women with endometriosis: Expression of c-myc, TGF-beta1 and bax genes

PubMed Central

Johnson, M Cecilia; Torres, Marisa; Alves, Alessandra; Bacallao, Ketty; Fuentes, Ariel; Vega, Margarita; Boric, M Angélica

2005-01-01

Background Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes. Methods Eutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants. Results Cell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0.05). Conclusion An altered expression of c-myc, TGF-beta1 and bax was observed in eutopic endometrium from endometriosis, suggesting its participation in the regulation of cell survival in this disease. The augmented cell viability in eutopic endometrium from these patients as a consequence of a reduction in cell death by apoptosis, and also an increase in cell proliferation indicates that this condition may facilitate the invasive feature of the endometrium. PMID:16150151

Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

PubMed

Lee, Daniel Y; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y; Li, Minhui; Du, William W; Shatseva, Tatiana; Yang, Burton B

2010-10-25

Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

Differential responses to high- and low-dose ultraviolet-B stress in tobacco Bright Yellow-2 cells

PubMed Central

Takahashi, Shinya; Kojo, Kei H.; Kutsuna, Natsumaro; Endo, Masaki; Toki, Seiichi; Isoda, Hiroko; Hasezawa, Seiichiro

2015-01-01

Ultraviolet (UV)-B irradiation leads to DNA damage, cell cycle arrest, growth inhibition, and cell death. To evaluate the UV-B stress–induced changes in plant cells, we developed a model system based on tobacco Bright Yellow-2 (BY-2) cells. Both low-dose UV-B (low UV-B: 740 J m−2) and high-dose UV-B (high UV-B: 2960 J m−2) inhibited cell proliferation and induced cell death; these effects were more pronounced at high UV-B. Flow cytometry showed cell cycle arrest within 1 day after UV-B irradiation; neither low- nor high-UV-B–irradiated cells entered mitosis within 12 h. Cell cycle progression was gradually restored in low-UV-B–irradiated cells but not in high-UV-B–irradiated cells. UV-A irradiation, which activates cyclobutane pyrimidine dimer (CPD) photolyase, reduced inhibition of cell proliferation by low but not high UV-B and suppressed high-UV-B–induced cell death. UV-B induced CPD formation in a dose-dependent manner. The amounts of CPDs decreased gradually within 3 days in low-UV-B–irradiated cells, but remained elevated after 3 days in high-UV-B–irradiated cells. Low UV-B slightly increased the number of DNA single-strand breaks detected by the comet assay at 1 day after irradiation, and then decreased at 2 and 3 days after irradiation. High UV-B increased DNA fragmentation detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay 1 and 3 days after irradiation. Caffeine, an inhibitor of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) checkpoint kinases, reduced the rate of cell death in high-UV-B–irradiated cells. Our data suggest that low-UV-B–induced CPDs and/or DNA strand-breaks inhibit DNA replication and proliferation of BY-2 cells, whereas larger contents of high-UV-B–induced CPDs and/or DNA strand-breaks lead to cell death. PMID:25954287

Distinct compartmentalization of dentin matrix protein 1 fragments in mineralized tissues and cells.

PubMed

Maciejewska, Izabela; Qin, Disheng; Huang, Bingzhen; Sun, Yao; Mues, Gabrielle; Svoboda, Kathy; Bonewald, Lynda; Butler, William T; Feng, Jerry Q; Qin, Chunlin

2009-01-01

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH(2)- and COOH-terminal fragments. The NH(2)-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH(2)- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH(2)- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH(2)-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH(2)-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH(2)-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions. Copyright 2008 S. Karger AG, Basel.

Xanthorrhizol, a natural sesquiterpenoid, induces apoptosis and growth arrest in HCT116 human colon cancer cells.

PubMed

Kang, You-Jin; Park, Kwang-Kyun; Chung, Won-Yoon; Hwang, Jae-Kwan; Lee, Sang Kook

2009-11-01

Xanthorrhizol is a sesquiterpenoid from the rhizome of Curcuma xanthorrhiza. In our previous studies, xanthorrhizol suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, inhibited cancer cell growth, and exerted an anti-metastatic effect in an animal model. However, the exact mechanisms for its inhibitory effects against cancer cell growth have not yet been fully elucidated. In the present study, we investigated the growth inhibitory effect of xanthorrhizol on cancer cells. Xanthorrhizol dose-dependently exerted antiproliferative effects against HCT116 human colon cancer cells. Xanthorrhizol also arrested cell cycle progression in the G0/G1 and G2/M phase and induced the increase of sub-G1 peaks. Cell cycle arrest was highly correlated with the downregulation of cyclin A, cyclin B1, and cyclin D1; cyclin-dependent kinase 1 (CDK1), CDK2, and CDK4; proliferating cell nuclear antigen; and inductions of p21 and p27, cyclin-dependent kinase inhibitors. The apoptosis by xanthorrhizol was markedly evidenced by induction of DNA fragmentation, release of cytochrome c, activation of caspases, and cleavage of poly-(ADP-ribose) polymerase. In addition, xanthorrhizol increased the expression and promoter activity of pro-apoptotic non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). These findings provide one plausible mechanism for the growth inhibitory activity of xanthorrhizol against cancer cells.

Hard clam extracts induce atypical apoptosis in human gastric cancer cells

PubMed Central

Song, Eing-Ju; Chan, Michael W.Y.; Shin, Jyh-Wei; Chen, Che-Chun

2017-01-01

Hard clams (HCs) are a nutritionally high-quality and popular seafood, and are established to be a potent antitumor food. The aim of the present study was to determine whether HC extracts induce apoptosis in the human gastric cancer cell line, AGS. In contrast with previously reported methods of extraction, crude extracts of HC were obtained by freezing and thawing and by a method free of hot water or organic solvents. The composition, quality and properties of the HC extracts were demonstrated to be stable since the extracts that were evaluated by capillary electrophoresis and HPLC analysis at different timepoints were similar. HC extracts also have an inhibitory effect against the survival of AGS cells. Treatment with HC extracts induced a marked sub-G1 DNA peak and reduced the expression of the anti-apoptotic genes BIRC5 and KPNA2. However, hallmarks of classical apoptosis such as DNA fragmentation and apoptotic body formation were not observed, indicating atypical apoptosis. Furthermore, it was revealed that HC extracts interrupted cell cycle progression in AGS cells through altered expression of six cell cycle-associated genes: CDC20, KPNA2, BIRC5, ANAPC2, CDKN1A and RB1. The present findings suggest that HC may contribute to a novel future anticancer agent. PMID:28810604

Antitumor Activity of Ethanolic Extract of Dendrobium formosum in T-Cell Lymphoma: An In Vitro and In Vivo Study

PubMed Central

Prasad, Ritika; Koch, Biplob

2014-01-01

Dendrobium, a genus of orchid, was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. The study was aimed to evaluate the anticancer property of the ethanolic extract of Dendrobium formosum on Dalton's lymphoma. In vitro cytotoxicity was determined by MTT assay, apoptosis was determined by fluorescence microscopy, and cell cycle progression was analysed using flow cytometry; in vivo antitumor activity was performed in Dalton's lymphoma bearing mice. The IC50 value of ethanolic extract was obtained at 350 μg/mL in Dalton's lymphoma cells. Fluorescence microscopy analysis showed significant increase in apoptotic cell death in dose- and time-dependent manner which was further confirmed through the resulting DNA fragmentation. Further, flow cytometry analysis showed that the ethanolic extract arrests the cells in G2/M phase of the cell cycle. The in vivo anticancer activity study illustrates significant increase in the survival time of Dalton's lymphoma bearing mice on treatment with ethanolic extract when compared to control. These results substantiate the antitumor properties of ethanolic extract of Dendrobium formosum and suggest an alternative in treatment of cancer. Further studies are required regarding the isolation and characterization of bioactive components along with the analysis of molecular mechanism involved. PMID:24959588

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells.

PubMed

Urueña, Claudia; Cifuentes, Claudia; Castañeda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

2008-11-18

There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells

PubMed Central

Urueña, Claudia; Cifuentes, Claudia; Castañeda, Diana; Arango, Amparo; Kaur, Punit; Asea, Alexzander; Fiorentino, Susana

2008-01-01

Background There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines. Methods Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS. Results Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed. Conclusion The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent. PMID:19017389

Estrogenic activity of tamoxifen on normal mammary parenchyma in the luteal phase of the menstrual cycle.

PubMed

Facina, G; de Lima, G R; Simões, M J; Novo, N F; Gebrim, L H

1997-01-01

Tamoxifen, an anti-estrogenic drug used in the adjuvant treatment of breast cancer, deserves more investigation for the determination of its efficacy as a prophylactic agent against breast cancer in high risk women. Thus, the action of tamoxifen on the human mammary gland was studied by measuring the number of lysosomes in normal mammary epithelium during the administration of tamoxifen. Tamoxifen was administered only during the luteal phase of the menstrual cycle to avoid interference with corpus luteum formation. A fragment of breast tissue adjacent to a fibroadenoma was obtained during surgery from 35 premenopausal women aged 15 to 37 years who had been eumenorrheic for at least 6 months; 18 of these patients were treated with tamoxifen and 17 were used as controls. Lysosome counts were performed under the light microscope on slides submitted to the acid phosphatase cytochemical technique and the data were analyzed statistically by the Mann-Whitney test. The fragments from the group treated with tamoxifen showed a significant decrease in lysosome numbers. Tamoxifen administered after ovulation significantly decreases the number of lysosomes in the cells of normal mammary epithelium, demonstrating the antiestrogenic effect of the drug on this target tissue.

Lycopene-rich extract from red guava (Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway.

PubMed

Dos Santos, Raimunda C; Ombredane, Alicia S; Souza, Jéssica Maria T; Vasconcelos, Andreanne G; Plácido, Alexandra; Amorim, Adriany das G N; Barbosa, Eder Alves; Lima, Filipe C D A; Ropke, Cristina D; Alves, Michel M M; Arcanjo, Daniel D R; Carvalho, Fernando A A; Delerue-Matos, Cristina; Joanitti, Graziella A; Leite, José Roberto de S A

2018-03-01

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M] +• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC 50 ]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC 50 =1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC 50 ≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway. Copyright © 2017. Published by Elsevier Ltd.

Characterization of a candidate bcl-1 gene.

PubMed Central

Withers, D A; Harvey, R C; Faust, J B; Melnyk, O; Carey, K; Meeker, T C

1991-01-01

The t(11;14)(q13;q32) translocation has been associated with human B-lymphocytic malignancy. Several examples of this translocation have been cloned, documenting that this abnormality joins the immunoglobulin heavy-chain gene to the bcl-1 locus on chromosome 11. However, the identification of the bcl-1 gene, a putative dominant oncogene, has been elusive. In this work, we have isolated genomic clones covering 120 kb of the bcl-1 locus. Probes from the region of an HpaII-tiny-fragment island identified a candidate bcl-1 gene. cDNAs representing the bcl-1 mRNA were cloned from three cell lines, two with the translocation. The deduced amino acid sequence from these clones showed bcl-1 to be a member of the cyclin gene family. In addition, our analysis of expression of bcl-1 in an extensive panel of human cell lines showed it to be widely expressed except in lymphoid or myeloid lineages. This observation may provide a molecular basis for distinct modes of cell cycle control in different mammalian tissues. Activation of the bcl-1 gene may be oncogenic by directly altering progression through the cell cycle. Images PMID:1833629

Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelid): proliferation and migration of neoblasts.

PubMed

Sugio, Mutsumi; Yoshida-Noro, Chikako; Ozawa, Kaname; Tochinai, Shin

2012-05-01

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E. japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast-like cells, which are designated as N-cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5-bromo-2'-deoxyuridine (BrdU)-labeling-experiments, we have shown that neoblasts are slow-cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N-cells proliferate more actively than do neoblasts in intact worms. The results of pulse-chase experiments indicated that neoblast and N-cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej-vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E. japonensis. © 2012 The Authors. Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

PubMed Central

Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

2017-01-01

Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function. PMID:28170432

Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells.

PubMed

de Souza Grinevicius, Valdelúcia Maria Alves; Kviecinski, Maicon Roberto; Santos Mota, Nádia Sandrini Ramos; Ourique, Fabiana; Porfirio Will Castro, Luiza Sheyla Evenni; Andreguetti, Rafaela Rafognato; Gomes Correia, João Francisco; Filho, Danilo Wilhem; Pich, Claus Tröger; Pedrosa, Rozangela Curi

2016-08-02

Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation. The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity. The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice. Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice. The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally, the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The influence of habitat fragmentation on helminth communities in rodent populations from a Brazilian Mountain Atlantic Forest.

PubMed

Cardoso, T S; Simões, R O; Luque, J L F; Maldonado, A; Gentile, R

2016-07-01

The influence of habitat structure on helminth communities of three sigomdontinae rodent species (Akodon cursor, A. montensis and Oligoryzomys nigripes) was investigated in forest fragments within an agricultural landscape in south-eastern Brazil. This is a pionner study correlating the occurrence of helminth species of rodent hosts with microhabitat characteristics. Rodents were collected from 12 fragments and in a continuous conserved area. Up to 13 nematode, three cestode and two trematode species were identified, and habitat fragmentation was found to have more influence on the helminth composition of O. nigripes compared to the other two rodent species. Fragmentation appeared to limit the development of some helminths' life cycles, e.g. with some species such as Trichofreitasia lenti, Protospirura numidica, Cysticercus fasciolaris and Avellaria sp., occurring mostly in areas with less anthropic impact. However, fragmentation did not seem to affect the life cycles of other dominant helminths, such as the trematode Canaania obesa, the nematodes Stilestrongylus lanfrediae, S. eta and S. aculeata, and the cestode Rodentolepis akodontis. The helminth community structure followed a nested pattern of distribution in A. montensis and O. nigripes. Stilestrongylus lanfrediae seemed to be more associated with dense understorey, C. obesa with open canopy and dense understorey, and Guerrerostrongylus zetta with organic matter on the ground. Their presence in each area may be explained by aspects of their life cycles that take place in the external environment outside the host.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

PubMed

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

2016-05-05

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

PubMed Central

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

2016-01-01

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

α-Phellandrene alters expression of genes associated with DNA damage, cell cycle, and apoptosis in murine leukemia WEHI-3 cells.

PubMed

Lin, Jen-Jyh; Yu, Chien-Chih; Lu, Kung-Wen; Chang, Shu-Jen; Yu, Fu-Shun; Liao, Ching-Lung; Lin, Jaung-Geng; Chung, Jing-Gung

2014-08-01

α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

PubMed

Machado, Kátia da Conceição; Sousa, Lívia Queiroz de; Lima, Daisy Jereissati Barbosa; Soares, Bruno Marques; Cavalcanti, Bruno Coêlho; Maranhão, Sarah Sant'Anna; Noronha, Janaina da Costa de; Rodrigues, Domingos de Jesus; Militão, Gardenia Carmen Gadelha; Chaves, Mariana Helena; Vieira-Júnior, Gerardo Magela; Pessoa, Cláudia; Moraes, Manoel Odorico de; Sousa, João Marcelo de Castro E; Melo-Cavalcante, Ana Amélia de Carvalho; Ferreira, Paulo Michel Pinheiro

2018-03-15

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC 50 values ranging from 0.15 (leukemia) to 7.35 (larynx) μM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC 50: 10.88 μM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC 50 : 7.5 μM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

PubMed Central

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A.; Quest, Andrew F.G.; Lavandero, Sergio

2014-01-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM).

PubMed

Russo, Paul; Hood, Brian L; Bateman, Nicholas W; Conrads, Thomas P

2017-01-01

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

Distal Regeneration Involves the Age Dependent Activity of Branchial Sac Stem Cells in the Ascidian Ciona intestinalis.

PubMed

Jeffery, William R

2015-02-01

Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis . Analysis of regenerative potential along the proximal-distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age-related stem cells located in the branchial sac that are a source of precursors for distal body regeneration.

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

PubMed Central

2012-01-01

Background Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations. Results Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres. Conclusions Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms. PMID:22369050

Fragment Length of Circulating Tumor DNA.

PubMed

Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay

2016-07-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

Structure and inhibition analysis of the mouse SAD-B C-terminal fragment.

PubMed

Ma, Hui; Wu, Jing-Xiang; Wang, Jue; Wang, Zhi-Xin; Wu, Jia-Wei

2016-10-01

The SAD (synapses of amphids defective) kinases, including SAD-A and SAD-B, play important roles in the regulation of neuronal development, cell cycle, and energy metabolism. Our recent study of mouse SAD-A identified a unique autoinhibitory sequence (AIS), which binds at the junction of the kinase domain (KD) and the ubiquitin-associated (UBA) domain and exerts autoregulation in cooperation with UBA. Here, we report the crystal structure of the mouse SAD-B C-terminal fragment including the AIS and the kinase-associated domain 1 (KA1) at 2.8 Å resolution. The KA1 domain is structurally conserved, while the isolated AIS sequence is highly flexible and solvent-accessible. Our biochemical studies indicated that the SAD-B AIS exerts the same autoinhibitory role as that in SAD-A. We believe that the flexible isolated AIS sequence is readily available for interaction with KD-UBA and thus inhibits SAD-B activity.

Regulation of apoptosis by low serum in cells of different stages of neoplastic progression: enhanced susceptibility after loss of a senescence gene and decreased susceptibility after loss of a tumor suppressor gene.

PubMed

Preston, G A; Lang, J E; Maronpot, R R; Barrett, J C

1994-08-01

A cell culture model system has been used to study the susceptibility of cells to apoptotic cell death during different stages of neoplastic progression. This system consists of normal diploid Syrian hamster embryo (SHE) cells, two preneoplastic cell lines [tumor suppressor stage I (sup +I) and non-tumor suppressor stage II (sup -II)], and hamster tumor cell lines. Stage I preneoplastic cells are nontumorigenic immortal clones that suppress tumorigenicity when hybridized to tumor cells, whereas stage II cells have lost the ability to suppress tumorigenicity in cell hybrids. We refer to these two types of preneoplastic cells as sup +I and sup -II, respectively. Neoplastic progression is generally associated with cellular alterations in growth factor responsiveness. Therefore, to study the regulation of apoptosis in the system described above, cells were cultured in low serum (0.2%) as a means of withdrawing growth factors. In low serum, normal SHE cells were quiescent (labeling index of 0.2%), with little cell death. The sup +I cells showed a relatively low labeling index (1.6%) but, in contrast to the normal cells, died at a high rate (55% cell loss after 48 h) by apoptosis, as evidenced by morphology, DNA fragmentation, and in situ end-labeling of fragmented DNA. The apoptotic cells did not go through a replicative cycle while in low serum, implying that apoptosis was initiated in the G0/G1 phase of the cell cycle. The sup -II cell line showed a high labeling index (40%) after 48 h, but cell growth was balanced by cell death that occurred at approximately the same rate. The cells died, however, predominantly by necrosis. The tumor cell lines continued to proliferate in low serum, with high labeling indices (ranging from 27% to 43%) and a low level of apoptotic or necrotic cell death. To determine the relative ability of these cells to survive in vivo, normal SHE cells, sup +I cells, and sup -II cells were injected s.c. into nude mice. At 5 or 21 days after injection, the normal SHE cells were readily retrieved from the mice and grew well in culture. In contrast, few sup +I cells were retrieved 5 days after injection and no viable cells were retrieved after 21 days. Sup -II cells were not retrieved at either the 5-day or 21-day harvest, and histological examinations of the sites of injection showed the presence of macrophages, eosinophils, and neutrophils, indicating an inflammatory response associated with necrotic cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis.

PubMed

Dohrmann, Paul R; Manhart, Carol M; Downey, Christopher D; McHenry, Charles S

2011-11-18

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 s to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5'-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 min. Reduction in gap size to 1 nt increased the rate of dissociation 2.5-fold, and complete filling of the gap increased the off-rate an additional 3-fold (t(1/2)~2 min). An exogenous primed template and ATP accelerated dissociation an additional 4-fold in a reaction that required complete filling of the gap. Neither a 5'-triphosphate nor a 5'-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nt) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-cross-linking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating that it serves as the sensor. Copyright © 2011 Elsevier Ltd. All rights reserved.

An improved turbine disk design to increase reliability of aircraft jet engines

NASA Technical Reports Server (NTRS)

Barack, W. N.; Domas, P. A.

1976-01-01

An analytical study was performed on a novel disk design to replace the existing high-pressure turbine, stage 1 disk on the CF6-50 turbofan engine. Preliminary studies were conducted on seven candidate disk design concepts. An integral multidisk design with bore entry of the turbine blade cooling air was selected as the improved disk design. This disk has the unique feature of being redundant such that if one portion of the disk would fail, the remaining portion would prevent the release of large disk fragments from the turbine system. Low cycle fatigue lives, initial defect propagation lives, burst speed, and the kinetic energies of probable disk fragment configurations were calculated, and comparisons were made with the existing disk, both in its current material, IN 718, and with the substitution of an advanced alloy, Rene 95. The design for redundancy approach which necessitated the addition of approximately 44.5 kg (98 lb) to the design disk substantially improved the life of the disk. The life to crack initiation was increased from 30,000 cycles to more than 100,000 cycles. The cycles to failure from initial defect propagation were increased from 380 cycles to 1564 cycles. Burst speed was increased from 126 percent overspeed to 149 percent overspeed. Additionally, the maximum fragment energies associated with a failure were decreased by an order of magnitude.

The use of cell microinjection for the in vivo analysis of viral transcriptional regulatory protein domains.

PubMed

Green, Maurice; Thorburn, Andrew; Kern, Robert; Loewenstein, Paul M

2007-01-01

Microinjection of mammalian cells provides a powerful method for analyzing in vivo functions of viral genes and viral gene products. By microinjection, a controlled amount (ranging from several to many thousands of copies) of a viral or cellular gene, a protein product of a gene, a polypeptide fragment encoding a specific protein domain, or an RNA molecule can be delivered into a target cell and the functional consequences analyzed. Microinjection can be used to deliver antibody targeted to a specific protein domain in order to analyze the requirement of the protein for specific cell functions such as cell cycle progression, transcription of specific genes, or intracellular transport. This chapter describes examples of the successful use of microinjection to probe adenovirus E1A regulatory mechanisms. Detailed methods are provided for manual and semiautomatic microinjection of mammalian cells as well as bioassay protocols for microinjected cells including immunofluorescence, colorimetic, in situ hybridization, and autoradiography.

Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

NASA Technical Reports Server (NTRS)

Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

1995-01-01

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

Changes of ploidy during the Azotobacter vinelandii growth cycle.

PubMed Central

Maldonado, R; Jiménez, J; Casadesús, J

1994-01-01

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory. Images PMID:8021173

Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

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

Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan

2012-05-11

Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colonmore » cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.« less

Fragment Length of Circulating Tumor DNA

PubMed Central

Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

2016-01-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

Radiosensitivity and Induction of Apoptosis by High LET Carbon Ion Beam and Low LET Gamma Radiation: A Comparative Study

PubMed Central

Ghorai, Atanu; Bhattacharyya, Nitai P.; Sarma, Asitikantha; Ghosh, Utpal

2014-01-01

Cancer treatment with high LET heavy ion beam, especially, carbon ion beam (12C), is becoming very popular over conventional radiotherapy like low LET gamma or X-ray. Combination of Poly(ADP-ribose) polymerase (PARP) inhibitor with xenotoxic drugs or conventional radiation (gamma or X-ray) is the newer approach for cancer therapy. The aim of our study was to compare the radiosensitivity and induction of apoptosis by high LET 12C and low LET gamma radiation in HeLa and PARP-1 knocked down cells. We did comet assay to detect DNA breaks, clonogenic survival assay, and cell cycle analysis to measure recovery after DNA damage. We measured apoptotic parameters like nuclear fragmentation and caspase-3 activation. DNA damage, cell killing, and induction of apoptosis were significantly higher for 12C than gamma radiation in HeLa. Cell killing and apoptosis were further elevated upon knocking down of PARP-1. Both 12C and gamma induced G2/M arrest although the 12C had greater effect. Unlike the gamma, 12C irradiation affects DNA replication as detected by S-phase delay in cell cycle analysis. So, we conclude that high LET 12C has greater potential over low LET gamma radiation in killing cells and radiosensitization upon PARP-1 inhibition was several folds greater for 12C than gamma. PMID:25018892

Taxol induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells

NASA Astrophysics Data System (ADS)

Guo, Wen-jing; Chen, Tong-sheng

2010-02-01

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

Differences in the motility of Amoeba proteus isolated fragments are determined by F-actin arrangement and cell nucleus presence.

PubMed

Grebecka, L; Pomorski, P; Lopatowska, A

1995-10-01

Isolated fragments produced by bisection of Amoeba proteus differ by their position in the original cell and by the presence or absence of the cell nucleus. Immediately after the operation, both types of anterior fragments preserve the former motory polarity, and do not interrupt locomotion. In the same time, all posterior fragments stop, round up and fail to react stimuli. In the second phase of experiment, these anterior fragments, which had no nucleus ceased to move, whereas the nucleated posterior ones resumed locomotion. It was demonstrated, that the behaviour of a fragment is primarily determined by the peripheral F-actin distribution, which is different depending on the origin of the fragment either from the anterior or from the posterior cell region. Later, the "inherited" F-actin distribution may be stabilized or reorganized in the presence of the nucleus, or desorganized in its absence.

[Overexpression of SEPP1 inhibits the proliferation and induces cell cycle G2/M arrest of 786-O and 769-P human renal carcinoma cells].

PubMed

Liu, Kan; Zhao, Chaofei; Chen, Jianwen; Wu, Shengpan; Yao, Yuanxin; Wu, Chong; Luo, Guoxiong; Zhang, Xu

2016-06-01

Objective To establish selenoprotein P, plasma 1 (SEPP1) gene recombinant lentiviral vector and investigate the effect of SEPP1 on the proliferation of human clear cell renal cell carcinoma (ccRCC) cells. Methods cDNA sequence of SEPP1 was cloned from the total cDNA of HEK293T cells by PCR. Then, the cDNA fragment was combined with the pLV-EGFP(2A)Puro vector and the constructed plasmid pLV-EGFP(2A)Puro-SEPP1 was transfected into HEK293T cells for packaging the virus. Forty-eight hours after transfected with the virus supernatant, the level of SEPP1 protein in 769-P and 786-O cells were tested by Western blotting. Cells were divided into recombinant lentivirus-infected cells, empty vector lentivirus-infected cells and the blank control cells. Cell proliferation rate was detected by MTS assay, colony forming ability was evaluated by plate clony formation assay and cell cycle change was assayed by flow cytometry after transfected with pLV-EGFP(2A)Puro-SEPP1 or empty pLV-EGFP(2A)Puro vector. Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 was constructed successfully. After being infected by the virus supernatant, the 786-O and 769-P cells expressed EGFP. Compared with the empty vector group and the blank control group, expression level of SEPP1 in the experimental group was much higher. The cell proliferative ability was inhibited in the cells overexpressing SEPP1, and the colony forming ability of SEPP1-overexpressed cells evidently decreased. Cell cycle was arrested in G2/M phase in 786-O cells overexpressing SEPP1. Conclusion The recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 has been constructed successfully. Overexpression of SEPP1 could significantly reduce the proliferation rate of 786-O and 769P cells, and cause G2/M phase arrest of 786-O cells.

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

PubMed

Kumar, Harish; Savaliya, Mihir; Biswas, Subhankar; Nayak, Pawan G; Maliyakkal, Naseer; Manjunath Setty, M; Gourishetti, Karthik; Pai, K Sreedhara Ranganath

2016-08-01

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G0/G1 phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.

Stroma-induced Jagged1 expression drives PC3 prostate cancer cell migration; disparate effects of RIP-generated proteolytic fragments on cell behaviour and Notch signaling

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

Delury, Craig, E-mail: c.delury@lancaster.ac.uk; Hart, Claire, E-mail: claire.hart@manchester.ac.uk; Brown, Mick, E-mail: michael.brown@ics.manchester.ac.uk

The Notch ligand Jagged1 is subject to regulated intramembrane proteolysis (RIP) which yields a soluble ectodomain (sJag) and a soluble Jagged1 intracellular domain (JICD). The full-length Jagged1 protein enhances prostate cancer (PCa) cell proliferation and is highly expressed in metastatic cells. However, little is known regarding the mechanisms by which Jagged1 or its RIP-generated fragments might promote PCa bone metastasis. In the current study we show that bone marrow stroma (BMS) induces Jagged1 expression in bone metastatic prostate cancer PC3 cells and that this enhanced expression is mechanistically linked to the promotion of cell migration. We also show that RIP-generatedmore » Jagged1 fragments exert disparate effects on PC3 cell behaviour and Notch signaling. In conclusion, the expression of both the full-length ligand and its RIP-generated fragments must be considered in tandem when attempting to regulate Jagged1 as a possible PCa therapy. - Highlights: • Bone marrow stroma induces Jagged1 expression in prostate cancer (PCa) PC3 cells. • This enhanced expression of full-length Jagged1 is required for PC3 cell migration. • Proteolytic fragments of Jagged1 exert disparate effects on PC3 cell behaviour. • Effects of fragments on cell behaviour do not correlate with Notch signaling. • Effects of Jagged1 and its fragments on PCa metastasis likely to be complex.« less

Waveform control of orientation-dependent ionization of DCl in few-cycle laser fields.

PubMed

Znakovskaya, I; von den Hoff, P; Schirmel, N; Urbasch, G; Zherebtsov, S; Bergues, B; de Vivie-Riedle, R; Weitzel, K-M; Kling, M F

2011-05-21

Strong few-cycle light fields with stable electric field waveforms allow controlling electrons on time scales down to the attosecond domain. We have studied the dissociative ionization of randomly oriented DCl in 5 fs light fields at 720 nm in the tunneling regime. Momentum distributions of D(+) and Cl(+) fragments were recorded via velocity-map imaging. A waveform-dependent anti-correlated directional emission of D(+) and Cl(+) fragments is observed. Comparison of our results with calculations indicates that tailoring of the light field via the carrier envelope phase permits the control over the orientation of DCl(+) and in turn the directional emission of charged fragments upon the breakup of the molecular ion. © The Owner Societies 2011

Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy

PubMed Central

Chacon, Jessica Ann; Sarnaik, Amod A; Chen, Jie Qing; Creasy, Caitlin; Kale, Charuta; Robinson, John; Weber, Jeffrey; Hwu, Patrick; Pilon-Thomas, Shari; Radvanyi, Laszlo

2014-01-01

Purpose Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy. The expansion of tumor-reactive CD8+ T cells with IL-2 in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8+ T cells recently found to constitute the majority of tumor-specific T cells. Experimental Design We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB co-stimulation. Results We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8+ TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL-2 responsiveness, in the CD8+ T cells in the fragments and emerging from the fragments. Early provision of 4-1BB co-stimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8+ T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8+ TIL outgrowth. Conclusions Our results highlight a previously unrecognized concept in TIL adoptive cell therapy that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. PMID:25472998

atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway

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

Yu Zengli; Xing Ying

2006-08-15

Our previous studies have shown that atRA treatment resulted in cell-cycle block and growth inhibition in mouse embryonic palatal mesenchymal (MEPM). In the current study, gestation day (GD) 13 MEPM cells were used to test the hypothesis that the growth inhibition by atRA is due to apoptosis. The effects of atRA on apoptosis were assessed by performing MTT assay, Cell Death Detection ELISA and flow cytometry, respectively. Data analysis confirmed that atRA treatment induced apoptosis-like cell death, as shown by decreased cell viability and increased fragmented DNA and sub-G1 fraction. atRA-induced apoptosis was associated with upregulation of bcl-2, translocation ofmore » bax protein to the mitochondria from the cytosol, activation of caspase-3 and cytochrome c release into cytosol. atRA-induced apoptosis was abrogated by z-DEVD-fmk, a caspase-3 specific inhibitor, and z-VAD-fmk, a general caspase inhibitor, suggesting that the atRA-induced cell death of MEPM cells occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, atRA treatment caused a strong and sustained activation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38), as well as an early but transient activation of extracellular signal-regulated kinase (ERK). Importantly, atRA-induced DNA fragmentation and capase-3 activation were prevented by pretreatment with the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB202190), but not by pretreatment with MEK inhibitor (U0126). From these results, we suggest that mitogen-activated protein kinase-dependent pathways is involved in the atRA-induced apoptosis of MEPM cells.« less

Characterization of a Null Allelic Mutant of the Rice NAL1 Gene Reveals Its Role in Regulating Cell Division

PubMed Central

Jiang, Dan; Fang, Jingjing; Lou, Lamei; Zhao, Jinfeng; Yuan, Shoujiang; Yin, Liang; Sun, Wei; Peng, Lixiang; Guo, Baotai; Li, Xueyong

2015-01-01

Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogenous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division. PMID:25658704

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway

PubMed Central

Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K.; Kishore, Uday

2018-01-01

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

PubMed

Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K; Kishore, Uday

2018-01-01

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53 mt ), MiaPaCa-2 (p53 mt ), and Capan-2 (p53 wt ) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

Cytotoxicity of flavones and flavonols to a human esophageal squamous cell carcinoma cell line (KYSE-510) by induction of G2/M arrest and apoptosis.

PubMed

Zhang, Qiang; Zhao, Xin-Huai; Wang, Zhu-Jun

2009-08-01

In this study, cytotoxic effects of structurally related flavones and flavonols on a human esophageal squamous cell carcinoma cell line (KYSE-510) were determined, and the molecular mechanisms responsible for their cytotoxic effects were studied. The results of MTT assay showed that flavones (luteolin, apigenin, chrysin) and flavonols (quercetin, kaempferol, myricetin) were able to induce cytotoxicity in KYSE-510 cells in a dose- and time-dependent manner, and the cytotoxic potency of these compounds was in the order of: luteolin>quercetin>chrysin>kaempferol>apigenin>myricetin. Flow cytometry and DNA fragmentation analysis indicated that the cytotoxicity induced by flavones and flavonols was mediated by G(2)/M cell cycle arrest and apoptosis. Furthermore, the expression of genes related to cell cycle arrest and apoptosis was assessed by oligonucleotide microarray, real-time RT-PCR and Western blot. It was shown that the treatment of KYSE-510 cells with these compounds caused G(2)/M arrest through up-regulation of p21(waf1) and down-regulation of cyclin B1 at the mRNA and protein levels, and induced p53-independent mitochondrial-mediated apoptosis through up-regulation of PIG3 and cleavage of caspase-9 and caspase-3. The results of western blot analysis further showed that increases of p63 and p73 protein translation or stability might be contributed to the regulation of p21(waf1), cyclin B1 and PIG3.

Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays.

PubMed

Fernando, M Rohan; Jiang, Chao; Krzyzanowski, Gary D; Ryan, Wayne L

2018-04-12

Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

PubMed

Ju, Young Seok; Tubio, Jose M C; Mifsud, William; Fu, Beiyuan; Davies, Helen R; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J; Tan, Benita K T; Aparicio, Samuel; Span, Paul N; Martens, John W M; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Myklebost, Ola; Flanagan, Adrienne M; Foster, Christopher; Neal, David E; Cooper, Colin; Eeles, Rosalind; Bova, Steven G; Lakhani, Sunil R; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L; Purdie, Colin A; Thompson, Alastair M; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

2015-06-01

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. © 2015 Ju et al.; Published by Cold Spring Harbor Laboratory Press.

The anthracenedione compound bostrycin induces mitochondria-mediated apoptosis in the yeast Saccharomyces cerevisiae.

PubMed

Xu, Chunling; Wang, Jiafeng; Gao, Ye; Lin, Huangyu; Du, Lin; Yang, Shanshan; Long, Simei; She, Zhigang; Cai, Xiaoling; Zhou, Shining; Lu, Yongjun

2010-05-01

Bostrycin is an anthracenedione with phytotoxic and antibacterial activity that belongs to the large family of quinones. We have isolated bostrycin from the secondary metabolites of a mangrove endophytic fungus, no. 1403, collected from the South China Sea. Using the yeast Saccharomyces cerevisiae as a model, we show that bostrycin inhibits cell proliferation by blocking the cell cycle at G1 phase and ultimately leads to cell death in a time- and dose-dependent manner. Bostrycin-induced lethal cytotoxicity is accompanied with increased levels of intracellular reactive oxygen species and hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and externalization of phosphatidylserine. We further show that bostrycin decreases mitochondrial membrane electric potential and causes mitochondrial destruction during the progression of cell death. Bostrycin-induced cell death was promoted in YCA1 null yeast strain but was partially rescued in AIF1 null mutant both in fermentative and respiratory media, strongly indicating that bostrycin induces apoptosis in yeast cells through a mitochondria-mediated but caspase-independent pathway.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

PubMed Central

Ju, Young Seok; Tubio, Jose M.C.; Mifsud, William; Fu, Beiyuan; Davies, Helen R.; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S.; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R.; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J.; Tan, Benita K.T.; Aparicio, Samuel; Span, Paul N.; Martens, John W.M.; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Flanagan, Adrienne M.; Foster, Christopher; Neal, David E.; Cooper, Colin; Eeles, Rosalind; Lakhani, Sunil R.; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L.; Purdie, Colin A.; Thompson, Alastair M.; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J.; Stratton, Michael R.

2015-01-01

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. PMID:25963125

Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

PubMed

Liu, Jin-Yun; Liu, Zhong; Wang, Dong-Mei; Li, Man-Mei; Wang, Shao-Xiang; Wang, Rui; Chen, Jian-Ping; Wang, Yi-Fei; Yang, De-Po

2011-04-25

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 μM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

PubMed

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

2013-08-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

Delayed luminescence to monitor programmed cell death induced by berberine on thyroid cancer cells

NASA Astrophysics Data System (ADS)

Scordino, Agata; Campisi, Agata; Grasso, Rosaria; Bonfanti, Roberta; Gulino, Marisa; Iauk, Liliana; Parenti, Rosalba; Musumeci, Francesco

2014-11-01

Correlation between apoptosis and UVA-induced ultraweak photon emission delayed luminescence (DL) from tumor thyroid cell lines was investigated. In particular, the effects of berberine, an alkaloid that has been reported to have anticancer activities, on two cancer cell lines were studied. The FTC-133 and 8305C cell lines, as representative of follicular and anaplastic thyroid human cancer, respectively, were chosen. The results show that berberine is able to arrest cell cycle and activate apoptotic pathway as shown in both cell lines by deoxyribonucleic acid fragmentation, caspase-3 cleavage, p53 and p27 protein overexpression. In parallel, changes in DL spectral components after berberine treatment support the hypothesis that DL from human cells originates mainly from mitochondria, since berberine acts especially at the mitochondrial level. The decrease of DL blue component for both cell lines could be related to the decrease of intra-mitochondrial nicotinamide adenine dinucleotide and may be a hallmark of induced apoptosis. In contrast, the response in the red spectral range is different for the two cell lines and may be ascribed to a different iron homeostasis.

Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy.

PubMed

Chacon, Jessica Ann; Sarnaik, Amod A; Chen, Jie Qing; Creasy, Caitlin; Kale, Charuta; Robinson, John; Weber, Jeffrey; Hwu, Patrick; Pilon-Thomas, Shari; Radvanyi, Laszlo

2015-02-01

Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy (ACT). The expansion of tumor-reactive CD8(+) T cells with interleukin-2 (IL2) in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8(+) T cells recently found to constitute the majority of tumor-specific T cells. We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB costimulation. We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8(+) TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL2 responsiveness, in the CD8(+) T cells in the fragments and emerging from the fragments. Early provision of 4-1BB costimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8(+) T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8(+) TIL outgrowth. Our results highlight a previously unrecognized concept in TIL ACT that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. ©2014 American Association for Cancer Research.

Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments.

PubMed Central

Bitensky, M W; Wheeler, M A; Mehta, H; Miki, N

1975-01-01

Activation of adenylate [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells. The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000. MCAF is derived from, and may be identical to the "A fragment" of cholera toxin. Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm. Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase. Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments. Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol. In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD. PMID:1058474

Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

PubMed Central

Lee, Daniel Y.; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y.; Li, Minhui; Du, William W.; Shatseva, Tatiana; Yang, Burton B.

2010-01-01

Background Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3′UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Methods and Findings Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3′UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3′UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3′UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3′UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3′UTR formed smaller tumors compared with cells transfected with a control vector. Conclusion Our results demonstrated that a 3′UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3′UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities. PMID:21049042

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

PubMed Central

Milhausen, M; Gill, P R; Parker, G; Agabian, N

1982-01-01

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems. Images PMID:6294658

Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

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

Jun, Do Youn; Institute of Genetic Engineering, Kyungpook National University, Daegu; Kim, Jun Seok

2007-07-15

To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressingmore » Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.« less

Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells.

PubMed

Müller, Joachim; Sidler, Daniel; Nachbur, Ueli; Wastling, Jonathan; Brunner, Thomas; Hemphill, Andrew

2008-10-15

Thiazolides are a novel class of broad-spectrum anti-infective drugs with promising in vitro and in vivo activities against intracellular and extracellular protozoan parasites. The nitrothiazole-analogue nitazoxanide (NTZ; 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide) represents the thiazolide parent compound, and a number of bromo- and carboxy-derivatives with differing activities have been synthesized. Here we report that NTZ and the bromo-thiazolide RM4819, but not the carboxy-thiazolide RM4825, inhibited proliferation of the colon cancer cell line Caco2 and nontransformed human foreskin fibroblasts (HFF) at or below concentrations the compounds normally exhibit anti-parasitic activity. Thiazolides induced typical signs of apoptosis, such as nuclear condensation, DNA fragmentation and phosphatidylserine exposure. Interestingly, the apoptosis-inducing effect of thiazolides appeared to be cell cycle-dependent and induction of cell cycle arrest substantially inhibited the cell death-inducing activity of these compounds. Using affinity chromatography and mass spectrometry glutathione-S-transferase P1 (GSTP1) from the GST class Pi was identified as a major thiazolide-binding protein. GSTP1 expression was more than 10 times higher in the thiazolide-sensitive Caco2 cells than in the less sensitive HFF cells. The enzymatic activity of recombinant GSTP1 was strongly inhibited by thiazolides. Silencing of GSTP1 using siRNA rendered cells insensitive to RM4819, while overexpression of GSTP1 increased sensitivity to RM4819-induced cell death. Thiazolides may thus represent an interesting novel class of future cancer therapeutics.

Method for identifying mutagenic agents which induce large, multilocus deletions in DNA

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

Bradley, W.E.C.; Belouchi, A.; Dewyse, P.

1993-07-13

A method of identifying a mutagenic agent is described which includes a large, multilocus deletions in DNA in mammalian cells comprising: (i) exposing a class III heterozygous CHO cell line to a potential mutagenic agent under investigation, and allowing any mutation of the cell line to proceed, said cell line being characterized in that a restriction fragment length variation exists in on mutation it becomes resistant to 2,6-diaminopurine and in that the DNA sequence adjacent to the two alleles of the APRT gene such that the DNA sequence adjacent to one of the two alleles can be digested with themore » enzyme BclI but the DNA sequence variation adjacent to the other of the two alleles cannot be digested with BclI, (ii) isolating induced mutations of the cell line deficient in APRT function, (iii) isolating DNA from the induced mutants, (iv) digesting the isolated DNA with BclI enzyme to produce digested fragments including a 19 kb fragment and any 2 kb fragment, which fragments hybridize with the labeled probe derived from DNA fragment PDI, (v) separating any digested fragments, (vi) transferring the separated fragments of (v) to a solid support, (vii) hybridizing the supported separated fragments with a labeled probe derived from the clone DNA fragment PD 1, (viii) determining fragments having undergone loss of the 2 kb band identified by the probe, as an identification of parent mutants in which the loss occurred, and (ix) evaluating the mutating ability of the potential mutagenic agent.« less

Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism.

PubMed

Hastings, Jordan; Kenealey, Jason

2017-01-01

Avenanthramides (AVN) are a relatively unstudied family of phytochemicals that could be novel chemotherapeutics. These compounds, found in oats, are non-toxic to healthy cells and have been shown to reduce viability of human colon and liver cancers in vitro. However, these studies do not elucidate a molecular mechanism for individual AVN. In this study we aim to see the effects of AVN on MDA-MB-231 breast cancer cells. An MTT assay was used to determine cell viability. Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. FloJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test. This study demonstrates that AVN-A, B, and C individually reduce viability in the MDA-MB-231 breast cancer cell line. AVN-C has the most potent decrease in tumor cell viability, decreasing viable cells to below 25% at 400 µM when compared to control after 96 h. We demonstrate that treatment with AVN-C causes DNA fragmentation and accumulation of over 90% of cells into a sub G 1 cell cycle population. Further, we conclude that AVN-C treated cells activate apoptosis because 97% of treated cells stain positive for annexin V while 91% have caspase-3/7 activity, a late marker of apoptosis. Breast cancer cells treated with AVN-C have a decrease in cell viability, an increase in the sub G 1 population, and stain positive for both annexin V and caspase activity, indicating that AVN-C induces apoptosis in breast cancer cells. These compounds may be able to act as chemotherapeutics as demonstrated through future in vivo studies.

The Twin-Cycle Experiential Learning Model: Reconceptualising Kolb's Theory

ERIC Educational Resources Information Center

Bergsteiner, Harald; Avery, Gayle C.

2014-01-01

Experiential learning styles remain popular despite criticisms about their validity, usefulness, fragmentation and poor definitions and categorisation. After examining four prominent models and building on Bergsteiner, Avery, and Neumann's suggestion of a dual cycle, this paper proposes a twin-cycle experiential learning model to overcome…

[Effect of the estrous cycle stage on sensitivity to pheromone 2,5-dimethylpyrazine in the house mouse Mus musculus].

PubMed

Daev, E V; Dukel'skaia, A V; Kazarova, V E; Fil'kina, Ia A

2007-01-01

Frequency of cytogenetic disturbances was estimated in mitotically dividing bone marrow cells of CBA strain female mice after the 24-h long action of pheromone 2,5-dimethylpyrazine (2,5-DMP). The stage of the estrous cycle of each animal was taken into account at the moment of the end of the pheromone action. The analysis was performed using the anatelophase method that allows evaluating frequencies of various types of disturbances--bridges, fragments, delayed chromosomes. The spontaneous level of the mitotic disturbances revealed by the anatelophase method in animals of the control group amounts to 5.4 %. Action of pheromone 2,5-dimethylpyrasine induced the mitosis disturbances detected in the dividing bone marrow cells at the anaphase-telophase stage in the females at the di- + postestrus stage. The corresponding frequency of disturbances after the pheromone action was equal to 9.2%. In the female in estrus, the mitotic disturbance level amounted 6.7%, which did not differ statistically significantly from control. It is suggested that differences in the female mouse hormonal state at different estrous cycle stages affect sensitivity to olfactory signals. Mechanisms of the revealed effect and significance of the differences in sensitivity to pheromone for reproductive processes are discussed.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

PubMed

Vanli, Güliz; Peltzer, Nieves; Dubuis, Gilles; Widmann, Christian

2014-12-01

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N. Copyright © 2014 Elsevier Inc. All rights reserved.

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections.

PubMed

Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner; Lykkemark, Simon; Poulsen, Pi Camilla; Overgaard, Laura Falkensteen; Petersen, Helene Bundgaard; Petersen, Ole William; Kristensen, Peter

2016-01-01

Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against small subpopulations of breast cancer cells. Selections were performed against a subpopulation of breast cancer cells expressing CD271+, as these previously have been indicated to be potential breast cancer stem cells. The selected antibody fragments were screened by phage ELISA on both breast cancer and myoepithelial cells. The antibody fragments were validated and evaluated by immunohistochemistry experiments. Our study revealed an antibody fragment, LH8, specific for breast cancer cells. Immunohistochemistry results indicate that this particular antibody fragment binds an antigen that exhibits differential expression in different breast cancer subpopulations. Further studies characterizing this antibody fragment, the subpopulation it binds and the cognate antigen may unearth novel biomarkers of clinical relevance. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Scorpion (Androctonus crassicauda) venom limits growth of transformed cells (SH-SY5Y and MCF-7) by cytotoxicity and cell cycle arrest.

PubMed

Zargan, Jamil; Sajad, Mir; Umar, Sadiq; Naime, Mohammad; Ali, Shakir; Khan, Haider A

2011-08-01

The purpose of study was to examine the cytotoxic and anti-cancer properties along with addressing the plausible pathway followed by scorpion venom to reduce cell viability in SH-SY5Y and MCF-7 cells. Following exposure of cells with scorpion venom, cytotoxicity was estimated using MTT and lactate dehydrogenase assays. Apoptotic effects were measured by assessment of mitochondrial membrane potential, reactive nitrogen species, DNA fragmentation, and caspase-3 activity whereas antiproliferative effect was assayed using BrdU incorporation. Our results indicate that scorpion venom causes suppression of proliferation by arresting S-phase and induction of apoptosis through increased nitric oxide production, caspase-3 activity and depolarization of mitochondrial membrane. Induction of apoptosis and arrest of DNA synthesis are critical determinant factors for development of anti cancer drugs. These properties may lead to isolation of effective molecule(s) with potential anticancer activity from scorpion venom of Androctonus crassicauda. Copyright © 2011 Elsevier Inc. All rights reserved.

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Distal regeneration involves the age dependent activity of branchial sac stem cells in the ascidian Ciona intestinalis

PubMed Central

2014-01-01

Abstract Tunicates have high capacities for regeneration but the underlying mechanisms and their relationship to life cycle progression are not well understood. Here we investigate the regeneration of distal structures in the ascidian tunicate Ciona intestinalis. Analysis of regenerative potential along the proximal−distal body axis indicated that distal organs, such as the siphons, their pigmented sensory organs, and the neural complex, could only be replaced from body fragments containing the branchial sac. Distal regeneration involves the formation of a blastema composed of cells that undergo cell proliferation prior to differentiation and cells that differentiate without cell proliferation. Both cell types originate in the branchial sac and appear in the blastema at different times after distal injury. Whereas the branchial sac stem cells are present in young animals, they are depleted in old animals that have lost their regeneration capacity. Thus Ciona adults contain a population of age‐related stem cells located in the branchial sac that are a source of precursors for distal body regeneration. PMID:25893097

Slow down to stay alive: HER4 protects against cellular stress and confers chemoresistance in neuroblastoma.

PubMed

Hua, Yingqi; Gorshkov, Kirill; Yang, Yanwen; Wang, Wenyi; Zhang, Nianxiang; Hughes, Dennis P M

2012-10-15

Neuroblastoma (NBL) is a common pediatric solid tumor, and outcomes for patients with advanced neuroblastoma remain poor despite extremely aggressive treatment. Chemotherapy resistance at relapse contributes heavily to treatment failure. The poor survival of patients with high-risk NBL prompted this investigation into novel treatment options with the objective of gaining a better understanding of resistance mechanisms. On the basis of previous work and on data from publicly available studies, the authors hypothesized that human epidermal growth factor receptor 4 (Her4) contributes to resistance. Her4 expression was reduced with small-hairpin RNA (shRNA) to over express intracellular HER4, and the authors tested its impact on tumor cell survival under various culture conditions. The resulting changes in gene expression after HER4 knockdown were measured by using a messenger RNA (mRNA) array. HER4 expression was up-regulated in tumor spheres compared with the expression in monolayer culture. With HER4 knockdown, NBL cells became less resistant to anoikis and serum starvation. Moreover, HER4 knockdown increased the chemosensitivity of NBL cells to cisplatin, doxorubicin, etoposide, and activated ifosfamide. In mRNA array analysis, HER4 knockdown predominately altered genes related to cell cycle regulation. In NBL spheres compared with monolayers, cell proliferation was decreased, and cyclin D expression was reduced. HER4 knockdown reversed cyclin D suppression. Overexpressed intracellular HER4 slowed the cell cycle and induced chemoresistance. The current results indicated that HER4 protects NBL cells from multiple exogenous apoptotic stimuli, including anoikis, nutrient deficiency, and cytotoxic chemotherapy. The intracellular fragment of HER4 was sufficient to confer this phenotype. HER4 functions as a cell cycle suppressor, maintaining resistance to cellular stress. The current findings indicate that HER4 overexpression may be associated with refractory disease, and HER4 may be an important therapeutic target. Copyright © 2012 American Cancer Society.

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Ultra High Resolution Linear Ion Trap Orbitrap Mass Spectrometer (Orbitrap Elite) Facilitates Top Down LC MS/MS and Versatile Peptide Fragmentation Modes*

PubMed Central

Michalski, Annette; Damoc, Eugen; Lange, Oliver; Denisov, Eduard; Nolting, Dirk; Müller, Mathias; Viner, Rosa; Schwartz, Jae; Remes, Philip; Belford, Michael; Dunyach, Jean-Jacques; Cox, Juergen; Horning, Stevan; Mann, Matthias; Makarov, Alexander

2012-01-01

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s—increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides. PMID:22159718

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis.

PubMed

Nezis, Ioannis P; Shravage, Bhupendra V; Sagona, Antonia P; Lamark, Trond; Bjørkøy, Geir; Johansen, Terje; Rusten, Tor Erik; Brech, Andreas; Baehrecke, Eric H; Stenmark, Harald

2010-08-23

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.

Dynamics in the tomato root transcriptome on infection with the potato cyst nematode Globodera rostochiensis.

PubMed

Swiecicka, Magdalena; Filipecki, Marcin; Lont, Dieuwertje; Van Vliet, Joke; Qin, Ling; Goverse, Aska; Bakker, Jaap; Helder, Johannes

2009-07-01

Plant parasitic nematodes infect roots and trigger the formation of specialized feeding sites by substantial reprogramming of the developmental process of root cells. In this article, we describe the dynamic changes in the tomato root transcriptome during early interactions with the potato cyst nematode Globodera rostochiensis. Using amplified fragment length polymorphism-based mRNA fingerprinting (cDNA-AFLP), we monitored 17 600 transcript-derived fragments (TDFs) in infected and uninfected tomato roots, 1-14 days after inoculation with nematode larvae. Six hundred and twenty-four TDFs (3.5%) showed significant differential expression on nematode infection. We employed GenEST, a computer program which links gene expression profiles generated by cDNA-AFLP and databases of cDNA sequences, to identify 135 tomato sequences. These sequences were grouped into eight functional categories based on the presence of genes involved in hormone regulation, plant pathogen defence response, cell cycle and cytoskeleton regulation, cell wall modification, cellular signalling, transcriptional regulation, primary metabolism and allocation. The presence of unclassified genes was also taken into consideration. This article describes the responsiveness of numerous tomato genes hitherto uncharacterized during infection with endoparasitic cyst nematodes. The analysis of transcriptome profiles allowed the sequential order of expression to be dissected for many groups of genes and the genes to be connected with the biological processes involved in compatible interactions between the plant and nematode.

Bladder transitional cell carcinoma and BK virus in a young kidney transplant recipient.

PubMed

Pino, L; Rijo, E; Nohales, G; Frances, A; Ubre, A; Arango, O

2013-02-01

Kidney transplant recipients have a heightened risk of developing neoplasms. Immunosuppressive treatments decrease the incidence of transplant rejection but increase the risk of infections, including BK virus (BKV). This infection is acquired in childhood and remains latent in the renal and urinary epithelium. In cases of immunodeficiency, BKV has been implicated as a tumor virus, but the role of BKV in cancer is a controversial topic and is difficult to determine. In the tumor cells, it is possible to detect fragments of the viral genome that could alter the control mechanisms of the cell cycle and DNA repair. We report the case of a kidney transplant recipient who developed BKV nephropathy and carcinoma of the bladder, supporting a possible role for BKV in the oncogenic pathway in this clinical setting, but the role of BKV in cancer remains a controversial topic and difficult to determine. © 2012 John Wiley & Sons A/S.

CED-9 and mitochondrial homeostasis in C. elegans muscle

PubMed Central

Tan, Frederick J.; Husain, Michelle; Manlandro, Cara Marie; Koppenol, Marijke; Fire, Andrew Z.; Hill, R. Blake

2009-01-01

Summary Mitochondrial homeostasis reflects a dynamic balance between membrane fission and fusion events thought essential for mitochondrial function. We report here that altered expression of the C. elegans BCL2 homolog CED-9 affects both mitochondrial fission and fusion. Although striated muscle cells lacking CED-9 have no alteration in mitochondrial size or ultrastructure, these cells appear more sensitive to mitochondrial fragmentation. By contrast, increased CED-9 expression in these cells produces highly interconnected mitochondria. This mitochondrial phenotype is partially suppressed by increased expression of the dynamin-related GTPase DRP-1, with suppression dependent on the BH3 binding pocket of CED-9. This suppression suggests that CED-9 directly regulates DRP-1, a model supported by our finding that CED-9 activates the GTPase activity of human DRP1. Thus, CED-9 is capable of regulating the mitochondrial fission-fusion cycle but is not essential for either fission or fusion. PMID:18827010

I-motif DNA structures are formed in the nuclei of human cells

NASA Astrophysics Data System (ADS)

Zeraati, Mahdi; Langley, David B.; Schofield, Peter; Moye, Aaron L.; Rouet, Romain; Hughes, William E.; Bryan, Tracy M.; Dinger, Marcel E.; Christ, Daniel

2018-06-01

Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.

Electrochemically-driven large amplitude pH cycling for acid-base driven DNA denaturation and renaturation.

PubMed

Wang, Yong-Chun; Lin, Cong-Bin; Su, Jian-Jia; Ru, Ying-Ming; Wu, Qiao; Chen, Zhao-Bin; Mao, Bing-Wei; Tian, Zhao-Wu

2011-06-15

In this paper, we present an electrochemically driven large amplitude pH alteration method based on a serial electrolytic cell involving a hydrogen permeable bifacial working electrode such as Pd thin foil. The method allows solution pH to be changed periodically up to ±4~5 units without additional alteration of concentration and/or composition of the system. Application to the acid-base driven cyclic denaturation and renaturation of 290 bp DNA fragments is successfully demonstrated with in situ real-time UV spectroscopic characterization. Electrophoretic analysis confirms that the denaturation and renaturation processes are reversible without degradation of the DNA. The serial electrolytic cell based electrochemical pH alteration method presented in this work would promote investigations of a wide variety of potential-dependent processes and techniques.

Relationship between plant growth and cytological effect in root apical meristem after exposure of wheat dry seeds to carbon ion beams

NASA Astrophysics Data System (ADS)

Liu, Qingfang; Wang, Zhuanzi; Zhou, Libin; Qu, Ying; Lu, Dong; Yu, Lixia; Du, Yan; Jin, Wenjie; Li, Wenjian

2013-06-01

In order to analyze the relationship between plant growth and cytological effects, wheat dry seeds were exposed to various doses of 12C6+ beams and the biological endpoints reflecting plant growth and root apical meristem (RAM) activities were investigated. The results showed that most of the seeds were able to germinate normally within all dose range, while the plant survival rate descended at higher doses. The seedling growth including root length and seedling height also decreased significantly at higher doses. Mitotic index (MI) in RAM had no changes at 10 and 20 Gy and decreased obviously at higher doses and the proportion of prophase cells had the same trend with MI. These data suggested that RAM cells experienced cell cycle arrest, which should be responsible for the inhibition of root growth after exposure to higher doses irradiation. Moreover, various types of chromosome aberrations (CAs) were observed in the mitotic cells. The frequencies of mitotic cells with lagging chromosomes and these with anaphase bridges peaked around 60 Gy, while the frequencies of these with fragments increased as the irradiation doses increased up to 200 Gy. The total frequencies of mitotic cells with CAs induced by irradiation increased significantly with the increasing doses. The serious damage of mitotic chromosomes maybe caused cell cycle arrest or cell death. These findings suggested that the influences of 12C6+ beams irradiation on plant growth were related to the alternation of mitotic activities and the chromosomal damages in RAM.

Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest.

PubMed

Bortolotto, Luis Felipe Buso; Barbosa, Flávia Regina; Silva, Gabriel; Bitencourt, Tamires Aparecida; Beleboni, Rene Oliveira; Baek, Seung Joon; Marins, Mozart; Fachin, Ana Lúcia

2017-01-01

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA 1 ) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis. Copyright © 2016. Published by Elsevier Masson SAS.

Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation

PubMed Central

Murakawa, Tomokazu; Yamaguchi, Osamu; Hashimoto, Ayako; Hikoso, Shungo; Takeda, Toshihiro; Oka, Takafumi; Yasui, Hiroki; Ueda, Hiromichi; Akazawa, Yasuhiro; Nakayama, Hiroyuki; Taneike, Manabu; Misaka, Tomofumi; Omiya, Shigemiki; Shah, Ajay M.; Yamamoto, Akitsugu; Nishida, Kazuhiko; Ohsumi, Yoshinori; Okamoto, Koji; Sakata, Yasushi; Otsu, Kinya

2015-01-01

Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells. PMID:26146385

Pretreatment of low dose radiation reduces radiation-induced apoptosis in mouse lymphoma (EL4) cells.

PubMed

Kim, J H; Hyun, S J; Yoon, M Y; Ji, Y H; Cho, C K; Yoo, S Y

1997-06-01

Induction of an adaptive response to ionizing radiation in mouse lymphoma (EL4) cells was studied by using cell survival fraction and apoptotic nucleosomal DNA fragmentation as biological end points. Cells in early log phase were pre-exposed to low dose of gamma-rays (0.01 Gy) 4 or 20 hrs prior to high dose gamma-ray (4, 8 and 12 Gy for cell survival fraction analysis; 8 Gy for DNA fragmentation analysis) irradiation. Then cell survival fractions and the extent of DNA fragmentation were measured. Significant adaptive response, increase in cell survival fraction and decrease in the extent of DNA fragmentation were induced when low and high dose gamma-ray irradiation time interval was 4 hr. Addition of protein or RNA synthesis inhibitor, cycloheximide or 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRFB), respectively during adaptation period, the period from low dose gamma-ray irradiation to high dose gamma-ray irradiation, was able to inhibit the induction of adaptive response, which is the reduction of the extent DNA fragmentation in irradiated EL4 cells. These data suggest that the induction of adaptive response to ionizing radiation in EL4 cells required both protein and RNA synthesis.

Radiation breakage of DNA: a model based on random-walk chromatin structure

NASA Technical Reports Server (NTRS)

Ponomarev, A. L.; Sachs, R. K.

2001-01-01

Monte Carlo computer software, called DNAbreak, has recently been developed to analyze observed non-random clustering of DNA double strand breaks in chromatin after exposure to densely ionizing radiation. The software models coarse-grained configurations of chromatin and radiation tracks, small-scale details being suppressed in order to obtain statistical results for larger scales, up to the size of a whole chromosome. We here give an analytic counterpart of the numerical model, useful for benchmarks, for elucidating the numerical results, for analyzing the assumptions of a more general but less mechanistic "randomly-located-clusters" formalism, and, potentially, for speeding up the calculations. The equations characterize multi-track DNA fragment-size distributions in terms of one-track action; an important step in extrapolating high-dose laboratory results to the much lower doses of main interest in environmental or occupational risk estimation. The approach can utilize the experimental information on DNA fragment-size distributions to draw inferences about large-scale chromatin geometry during cell-cycle interphase.

Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?

PubMed Central

Polettini, Jossimara; Behnia, Faranak; Taylor, Brandie D.; Saade, George R.; Taylor, Robert N.; Menon, Ramkumar

2015-01-01

Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]2) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]2). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term. PMID:26397719

Regenerative hair waves in aging mice and extra-follicular modulators follistatin, dkk1, and sfrp4.

PubMed

Chen, Chih-Chiang; Murray, Philip J; Jiang, Ting Xin; Plikus, Maksim V; Chang, Yun-Ting; Lee, Oscar K; Widelitz, Randall B; Chuong, Cheng-Ming

2014-08-01

Hair cycling is modulated by factors both intrinsic and extrinsic to hair follicles. Cycling defects lead to conditions such as aging-associated alopecia. Recently, we demonstrated that mouse skin exhibits regenerative hair waves, reflecting a coordinated regenerative behavior in follicle populations. Here, we use this model to explore the regenerative behavior of aging mouse skin. Old mice (>18 months) tracked over several months show that with progressing age, hair waves slow down, wave propagation becomes restricted, and hair cycle domains fragment into smaller domains. Transplanting aged donor mouse skin to a young host can restore donor cycling within a 3 mm range of the interface, suggesting that changes are due to extracellular factors. Therefore, hair stem cells in aged skin can be reactivated. Molecular studies show that extra-follicular modulators Bmp2, Dkk1, and Sfrp4 increase in early anagen. Further, we identify follistatin as an extra-follicular modulator, which is highly expressed in late telogen and early anagen. Indeed, follistatin induces hair wave propagation and its level decreases in aging mice. We present an excitable medium model to simulate the cycling behavior in aging mice and illustrate how the interorgan macroenvironment can regulate the aging process by integrating both "activator" and "inhibitor" signals.

Expression of the NH2-Terminal Fragment of RasGAP in Pancreatic β-Cells Increases Their Resistance to Stresses and Protects Mice From Diabetes

PubMed Central

Yang, Jiang-Yan; Walicki, Jöel; Jaccard, Evrim; Dubuis, Gilles; Bulat, Natasa; Hornung, Jean-Pierre; Thorens, Bernard; Widmann, Christian

2009-01-01

OBJECTIVE Our laboratory has previously established in vitro that a caspase-generated RasGAP NH2-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic β-cells in a physiological setting. RESEARCH DESIGN AND METHODS A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS Pancreatic β-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor κB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo β-cell apoptosis. CONCLUSIONS Fragment N efficiently increases the overall resistance of β-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools. PMID:19696184

The PCC assay can be used to predict radiosensitivity in biopsy cultures irradiated with different types of radiation.

PubMed

Suzuki, Masao; Tsuruoka, Chizuru; Nakano, Takashi; Ohno, Tatsuya; Furusawa, Yoshiya; Okayasu, Ryuichi

2006-12-01

The aim of this study was to identify potential biomarkers for radiosensitivity using the relationship between cell killing and the yield of excess chromatin fragments detected with the premature chromosome condensation (PCC) technique. This method was applied to primary cultured cells obtained from biopsies from patients. Six primary culture biopsies were obtained from 6 patients with carcinoma of the cervix before starting radiotherapy. The cultures were irradiated with two different LET carbon-ion beams (LET = 13 keV/microm, 77.1+/-2.8 keV/microm) and 200 kV X-rays. The carbon-ion beams were produced by Heavy Ion Medical Accelerator in Chiba (HIMAC). PCC was performed using the polyethylene glycol-mediated cell fusion technique. The yield of excess chromatin fragments were measured by counting the number of unrejoined chromatin fragments detected with the PCC technique after a 24-h post-irradiation incubation period. Obtained results indicated that cultures which were more sensitive to killing were also more susceptible to the induction of excess chromatin fragments. Furthermore there was a good correlation between cell killing and excess chromatin fragments among the 6 cell cultures examined. There is also evidence that the induction of excess chromatin fragments increased with increasing LET as well as cell-killing effect in the same cell culture. The data reported here support the idea that the yield of excess chromatin fragments detected with the PCC technique might be useful for predicting the radiosensitivity of cells contained in tumor tissue, and to predict responses to different radiation types.

Iron overload promotes mitochondrial fragmentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway.

PubMed

Zheng, Qingqing; Zhao, Youshan; Guo, Juan; Zhao, Sida; Fei, Chengming; Xiao, Chao; Wu, Dong; Wu, Lingyun; Li, Xiao; Chang, Chunkang

2018-05-03

Iron overload (IO) has been reported to contribute to mesenchymal stromal cell (MSC) damage, but the precise mechanism has yet to be clearly elucidated. In this study, we found that IO increased cell apoptosis and lowered cell viability in MSCs, accompanied by extensive mitochondrial fragmentation and autophagy enhancement. All these effects were reactive oxygen species (ROS) dependent. In MSCs with IO, the ATP concentrations were significantly reduced due to high ROS levels and low electron respiratory chain complex (ETC) II/III activity. Reduced ATP phosphorylated AMP-activated protein kinase (AMPK). Activation of AMPK kinase complexes triggered mitochondrial fission. Moreover, gene knockout of AMPK via CRISPR/Cas9 reduced cell apoptosis, enhanced cell viability and attenuated mitochondrial fragmentation and autophagy caused by IO in MSCs. Further, AMPK-induced mitochondrial fragmentation of MSCs with IO was mediated via phosphorylation of mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for the GTPase dynamin-related protein 1 (Drp1). Gene knockdown of MFF reversed AMPK-induced mitochondrial fragmentation in MSCs with IO. In addition, MSCs from IO patients with myelodysplastic syndrome (MDS) showed increased cell apoptosis, decreased cell viability, higher ROS levels, lower ATP concentrations and increased mitochondrial fragmentation compared with MSCs from non-IO patients. In addition, iron chelation or antioxidant weakened the activity of the AMPK/MFF/Drp1 pathway in MDS-MSCs with IO from several patients, accompanied by attenuation of mitochondrial fragmentation and autophagy. Taken together, the AMPK/MFF/Drp1 pathway has an important role in the damage to MDS-MSCs caused by IO.

Dynamics and fragmentation of thick-shelled microbubbles.

PubMed

May, Donovan J; Allen, John S; Ferrara, Katherine W

2002-10-01

Localized delivery could decrease the systemic side effects of toxic chemotherapy drugs. The unique delivery agents we examine consist of microbubbles with an outer lipid coating, an oil layer, and a perfluorobutane gas core. These structures are 0.5-12 microm in radius at rest. Oil layers of these acoustically active lipospheres (AALs) range from 0.3-1.5 microm in thickness and thus the agents can carry a large payload compared to nano-scale drug delivery systems. We show that triacetin-based drug-delivery vehicles can be fragmented using ultrasound. Compared with a lipid-shelled contrast agent, the expansion of the drug-delivery vehicle within the first cycle is similar, and a subharmonic component is demonstrated at an equivalent radius, frequency, and driving pressure. For the experimental conditions explored here, the pulse length required for destruction of the drug-delivery vehicle is significantly greater, with at least five cycles required, compared with one cycle for the contrast agent. For the drug-delivery vehicle, the observed destruction mechanism varies with the initial radius, with microbubbles smaller than resonance size undergoing a symmetric collapse and producing a set of small, equal-sized fragments. Between resonance size and twice resonance size, surface waves become visible, and the oscillations become asymmetrical. For agents larger than twice the resonance radius, the destruction mechanism changes to a pinch-off, with one fragment containing a large fraction of the original volume.

Redox-Sensitive Cerium Oxide Nanoparticles Protect Human Keratinocytes from Oxidative Stress Induced by Glutathione Depletion.

PubMed

Singh, Ragini; Karakoti, Ajay S; Self, William; Seal, Sudipta; Singh, Sanjay

2016-11-22

Cerium oxide nanoparticles (CeNPs) have gathered much attention in the biomedical field due to its unique antioxidant property. It can protect cells and tissues from oxidative stress induced damage due to its autoregenerative redox cycle. Our study explores the antioxidant and antigenotoxic behavior of PEGylated CeNPs toward oxidative insult produced by buthionine sulfoximine (BSO) in human keratinocytes (HaCaT cells). BSO inhibits the γ-glutamylcysteinesynthetase (γ-GCS) enzyme and thus acts as a glutathione (GSH) depleting agent to modulate the cellular redox potential. GSH is a natural ROS scavenger present in the mammalian cells, and its depletion causes generation of reactive oxygen species (ROS). In this study, we challenged HaCaT cells (keratinocytes) with BSO to alter the redox potential within the cell and monitored toxicity, ROS generation, and nuclear fragmentation. We also followed changes in expressions of related proteins and genes. We found that PEGylated CeNPs can protect HaCaT cells from BSO-induced oxidative damage. BSO-exposed cells, preincubated with PEGylated CeNPs, showed better cell survival and significant decrease in the intracellular levels of ROS. We also observed decrease in lactate dehydrogenase (LDH) release and nuclear fragmentation in CeNP-treated cells that were challenged with BSO as compared to treatment with BSO alone. Exposure of HaCaT cells with BSO leads to altered expression of antioxidant genes and proteins, i.e., thioredoxin reductase (TrxR) and peroxiredoxin 6 (Prx6) whereas, in our study, pretreatment of PEGylated CeNPs reduces the need for induction of genes that produce enzymes involved in the defense against oxidative stress. Since, growing evidence argued the involvement of ROS in mediating death of mammalian cells in several ailments, our finding reinforces the use of PEGylated CeNPs as a potent pharmacological agent under the lower cellular GSH/GSSG ratios for the treatment of diseases mediated by free radicals.

Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

PubMed Central

2016-01-01

Rapid progress in disease biomarker discovery has increased the need for robust detection technologies. In the past several years, the designs of many immunoaffinity reagents have focused on lowering costs and improving specificity while also promoting stability. Antibody fragments (scFvs) have long been displayed on the surface of yeast and phage libraries for selection; however, the stable production of such fragments presents challenges that hamper their widespread use in diagnostics. Membrane and cell wall proteins similarly suffer from stability problems when solubilized from their native environment. Recently, cell envelope compositions that maintain membrane proteins in native or native-like lipid environment to improve their stability have been developed. This cell envelope composition approach has now been adapted toward stabilizing antibody fragments by retaining their native cell wall environment. A new class of immunoaffinity reagents has been developed that maintains antibody fragment attachment to yeast cell wall. Herein, we review recent strategies that incorporate cell wall fragments with functional scFvs, which are designed for easy production while maintaining specificity and stability when in use with simple detection platforms. These cell wall based antibody fragments are globular in structure, and heterogeneous in size, with fragments ranging from tens to hundreds of nanometers in size. These fragments appear to retain activity once immobilized onto biosensor surfaces for the specific and sensitive detection of pathogen antigens. They can be quickly and economically generated from a yeast display library and stored lyophilized, at room temperature, for up to a year with little effect on stability. This new format of scFvs provides stability, in a simple and low-cost manner toward the use of scFvs in biosensor applications. The production and “panning” of such antibody cell wall composites are also extremely facile, enabling the rapid adoption of stable and inexpensive affinity reagents for emerging infectious threats. PMID:27762541

Habitat fragmentation and its lasting impact on Earth’s ecosystems

PubMed Central

Haddad, Nick M.; Brudvig, Lars A.; Clobert, Jean; Davies, Kendi F.; Gonzalez, Andrew; Holt, Robert D.; Lovejoy, Thomas E.; Sexton, Joseph O.; Austin, Mike P.; Collins, Cathy D.; Cook, William M.; Damschen, Ellen I.; Ewers, Robert M.; Foster, Bryan L.; Jenkins, Clinton N.; King, Andrew J.; Laurance, William F.; Levey, Douglas J.; Margules, Chris R.; Melbourne, Brett A.; Nicholls, A. O.; Orrock, John L.; Song, Dan-Xia; Townshend, John R.

2015-01-01

We conducted an analysis of global forest cover to reveal that 70% of remaining forest is within 1 km of the forest’s edge, subject to the degrading effects of fragmentation. A synthesis of fragmentation experiments spanning multiple biomes and scales, five continents, and 35 years demonstrates that habitat fragmentation reduces biodiversity by 13 to 75% and impairs key ecosystem functions by decreasing biomass and altering nutrient cycles. Effects are greatest in the smallest and most isolated fragments, and they magnify with the passage of time. These findings indicate an urgent need for conservation and restoration measures to improve landscape connectivity, which will reduce extinction rates and help maintain ecosystem services. PMID:26601154

Caspase-3/7-mediated Cleavage of β2-spectrin is Required for Acetaminophen-induced Liver Damage

PubMed Central

Baek, Hye Jung; Lee, Yong Min; Kim, Tae Hyun; Kim, Joo-Young; Park, Eun Jung; Iwabuchi, Kuniyoshi; Mishra, Lopa; Kim, Sang Soo

2016-01-01

The ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration. Conclusions: β2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage. PMID:26884715

Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride.

PubMed

Barbieri, F; Sparatore, F; Cagnoli, M; Bruzzo, C; Novelli, F; Alama, A

2001-03-14

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

Phosphorylation of Nlp by Plk1 negatively regulates its dynein-dynactin-dependent targeting to the centrosome.

PubMed

Casenghi, Martina; Barr, Francis A; Nigg, Erich A

2005-11-01

When cells enter mitosis the microtubule (MT) network undergoes a profound rearrangement, in part due to alterations in the MT nucleating and anchoring properties of the centrosome. Ninein and the ninein-like protein (Nlp) are centrosomal proteins involved in MT organisation in interphase cells. We show that the overexpression of these two proteins induces the fragmentation of the Golgi, and causes lysosomes to disperse toward the cell periphery. The ability of Nlp and ninein to perturb the cytoplasmic distribution of these organelles depends on their ability to interact with the dynein-dynactin motor complex. Our data also indicate that dynactin is required for the targeting of Nlp and ninein to the centrosome. Furthermore, phosphorylation of Nlp by the polo-like kinase 1 (Plk1) negatively regulates its association with dynactin. These findings uncover a mechanism through which Plk1 helps to coordinate changes in MT organisation with cell cycle progression, by controlling the dynein-dynactin-dependent transport of centrosomal proteins.

Timeframe Dependent Fragment Ions Observed in In-Source Decay Experiments with β-Casein Using MALDI MS.

PubMed

Sekiya, Sadanori; Nagoshi, Keishiro; Iwamoto, Shinichi; Tanaka, Koichi; Takayama, Mitsuo

2015-09-01

The fragment ions observed with time-of-flight (TOF) and quadrupole ion trap (QIT) TOF mass spectrometers (MS) combined with matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiments of phosphorylated analytes β-casein and its model peptide were compared from the standpoint of the residence timeframe of analyte and fragment ions in the MALDI ion source and QIT cell. The QIT-TOF MS gave fragment c-, z'-, z-ANL, y-, and b-ions, and further degraded fragments originating from the loss of neutrals such as H(2)O, NH(3), CH(2)O (from serine), C2H4O (from threonine), and H(3)PO(4), whereas the TOF MS merely showed MALDI source-generated fragment c-, z'-, z-ANL, y-, and w-ions. The fragment ions observed in the QIT-TOF MS could be explained by the injection of the source-generated ions into the QIT cell or a cooperative effect of a little internal energy deposition, a long residence timeframe (140 ms) in the QIT cell, and specific amino acid effects on low-energy CID, whereas the source-generated fragments (c-, z'-, z-ANL, y-, and w-ions) could be a result of prompt radical-initiated fragmentation of hydrogen-abundant radical ions [M + H + H](+) and [M + H - H](-) within the 53 ns timeframe, which corresponds to the delayed extraction time. The further degraded fragment b/y-ions produced in the QIT cell were confirmed by positive- and negative-ion low-energy CID experiments performed on the source-generated ions (c-, z'-, and y-ions). The loss of phosphoric acid (98 u) from analyte and fragment ions can be explained by a slow ergodic fragmentation independent of positive and negative charges.

Timeframe Dependent Fragment Ions Observed in In-Source Decay Experiments with β-Casein Using MALDI MS

NASA Astrophysics Data System (ADS)

Sekiya, Sadanori; Nagoshi, Keishiro; Iwamoto, Shinichi; Tanaka, Koichi; Takayama, Mitsuo

2015-09-01

The fragment ions observed with time-of-flight (TOF) and quadrupole ion trap (QIT) TOF mass spectrometers (MS) combined with matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiments of phosphorylated analytes β-casein and its model peptide were compared from the standpoint of the residence timeframe of analyte and fragment ions in the MALDI ion source and QIT cell. The QIT-TOF MS gave fragment c-, z'-, z-ANL, y-, and b-ions, and further degraded fragments originating from the loss of neutrals such as H2O, NH3, CH2O (from serine), C2H4O (from threonine), and H3PO4, whereas the TOF MS merely showed MALDI source-generated fragment c-, z'-, z-ANL, y-, and w-ions. The fragment ions observed in the QIT-TOF MS could be explained by the injection of the source-generated ions into the QIT cell or a cooperative effect of a little internal energy deposition, a long residence timeframe (140 ms) in the QIT cell, and specific amino acid effects on low-energy CID, whereas the source-generated fragments (c-, z'-, z-ANL, y-, and w-ions) could be a result of prompt radical-initiated fragmentation of hydrogen-abundant radical ions [M + H + H]+ and [M + H - H]- within the 53 ns timeframe, which corresponds to the delayed extraction time. The further degraded fragment b/y-ions produced in the QIT cell were confirmed by positive- and negative-ion low-energy CID experiments performed on the source-generated ions (c-, z'-, and y-ions). The loss of phosphoric acid (98 u) from analyte and fragment ions can be explained by a slow ergodic fragmentation independent of positive and negative charges.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

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

Wei, Ren-Jie

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosismore » in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells. • ABT-751 induces early autophagy and delays apoptosis. • ABT-751 induces autophagy via modulations of nuclear TP53 and AKT/MTOR pathways. • ABT-751-induced apoptosis is ROS-, mitochondria- and caspase-dependent. • Inhibition of autophagy enhances ABT-751-induced apoptosis.« less

Polyamine analog TBP inhibits proliferation of human K562 chronic myelogenous leukemia cells by induced apoptosis

PubMed Central

WANG, QING; WANG, YAN-LIN; WANG, KAI; YANG, JIAN-LIN; CAO, CHUN-YU

2015-01-01

The aim of the present study was to investigate the effects of the novel polyamine analog tetrabutyl propanediamine (TBP) on the growth of K562 chronic myelogenous leukemia (CML) cells and the underlying mechanism of these effects. MTT was used for the analysis of cell proliferation and flow cytometry was performed to analyze cell cycle distribution. DNA fragmentation analysis and Annexin V/propidium iodide double staining were used to identify apoptotic cells. The activity of the key enzymes in polyamine catabolism was detected using chemiluminescence. TBP can induce apoptosis and significantly inhibit K562 cell proliferation in a time- and dose-dependent manner. TBP treatment significantly induced the enzyme activity of spermine oxidase and acetylpolyamine oxidase in K562 cells, and also enhanced the inhibitory effect of the antitumor drug doxorubicin on K562 cell proliferation. As a novel polyamine analog, TBP significantly inhibited proliferation and induced apoptosis in K562 cells by upregulating the activity of the key enzymes in the polyamine catabolic pathways. TBP also increased the sensitivity of the K562 cells to the antitumor drug doxorubicin. These data indicate an important potential value of TBP for clinical therapy of human CML. PMID:25435975

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells.

PubMed

Yoon, Jung Mi; Koppula, Sushruta; Huh, Se Jong; Hur, Sun Jin; Kim, Chan Gil

2015-07-24

Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

In situ end labeling of fragmented DNA in induced ovarian atresia.

PubMed

D'Herde, K; De Pestel, G; Roels, F

1994-01-01

Apoptosis is studied in a model of induced follicular atresia in the ovary of Japanese quail (Coturnix coturnix japonica) by in situ end labeling of DNA fragments in granulosa cells using two different techniques (incorporation of labeled nucleotides by DNA polymerase I or terminal deoxynucleotidyl transferase). The most remarkable observation related to apoptosis in this model is the predominant cytoplasmic localization of labeled DNA fragments, while DNA fragmentation appears to be absent from compacted chromatin masses of apoptotic nuclei and apoptotic nuclear fragments. Unstained apoptotic bodies are present adjacent to stained ones, so that their detection rate on hematoxylin + eosin stained sections is better than on the in situ end-labeled sections. This suggests that DNA fragmentation is a late even or not obligatory in apoptotic granulosa cell death. In contrast to similar studies on atretic granulosa in mammalian models, the process of apoptosis is asynchronous in the granulosal epithelium, with a majority of nuclei with normal chromatin configuration remaining negative for DNA fragmentation. Finally it is shown that the techniques used are not specific for apoptosis, as DNA fragmentation in necrotic granulosa cells is detected as well.

Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing

PubMed Central

1983-01-01

We examined the ability of a set of cloned chicken ovalbumin (cOVA)- specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I- region molecules by T cells. PMID:6193218

Fragmentation of cancer cells

NASA Astrophysics Data System (ADS)

Vanapalli, Siva; Kamyabi, Nabiollah

Tumor cells have to travel through blood capillaries to be able to metastasize and colonize in distant organs. Among the numerous cells that are shed by the primary tumor, very few survive in circulation. In vivo studies have shown that tumor cells can undergo breakup at microcapillary junctions affecting their survival. It is currently unclear what hydrodynamic and biomechanical factors contribute to fragmentation and moreover how different are the breakup dynamics of highly and weakly metastatic cells. In this study, we use microfluidics to investigate flow-induced breakup of prostate and breast cancer cells. We observe several different modes of breakup of cancer cells, which have striking similarities with breakup of viscous drops. We quantify the breakup time and find that highly metastatic cancer cells take longer to breakup than lowly metastatic cells suggesting that tumor cells may dynamically modify their deformability to avoid fragmentation. We also identify the role that cytoskeleton and membrane plays in the breakup process. Our study highlights the important role that tumor cell fragmentation plays in cancer metastasis. Cancer Prevention and Research Institute of Texas.

Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer.

PubMed

Ferries, Samantha; Perkins, Simon; Brownridge, Philip J; Campbell, Amy; Eyers, Patrick A; Jones, Andrew R; Eyers, Claire E

2017-09-01

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high-throughput phosphoproteomics. Here we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. In addition to evaluating multiple fragmentation regimes (HCD, EThcD, and neutral-loss-triggered ET(ca/hc)D) and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high-resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlight that a charge-state-dependent fragmentation regime and a multiple algorithm search strategy are likely to be of benefit for confident large-scale phosphosite localization.

Three-Dimensional Analysis of Peeled Internal Limiting Membrane Using Focused Ion Beam/Scanning Electron Microscopy

PubMed Central

Murata, Kazuhisa; Hayashi, Ken; Nakamura, Kei-ichiro

2018-01-01

Purpose To reevaluate the effect of internal limiting membrane peeling during vitrectomy on the Müller cell damage, we examined the ultrastructure of the internal limiting membrane by using focused ion beam/scanning electron microscopy (FIB/SEM). Methods A total of 12 internal limiting membranes obtained during surgery in both the macular hole and the idiopathic epiretinal membrane groups were processed for observation by FIB/SEM. Three-dimensional structures of the internal limiting membrane were analyzed. Results The number of cell fragments in the macular hole group was 5.07 ± 1.03 per unit area of internal limiting membrane (100 μm2). The total volume of cell fragments was 3.54 ± 1.24 μm3/100 μm2. In contrast, the number of cell fragments in the epiretinal membrane group was 12.85 ± 3.45/100 μm2, and the total volume of cell fragments was 10.45 ± 2.77 μm3/100 μm2. Data for both values were significantly higher than those observed in the macular hole group (P = 0.0024 and P = 0.0022, respectively, Mann-Whitney U test). No statistical difference was found for the mean volume of the cell fragment between the two groups. Conclusions All of the internal limiting membrane examined in this study showed cell fragments on the retinal surface of the internal limiting membrane. As compared with macular hole, epiretinal membrane exhibited a higher number and total volume of cell fragments, indicating that internal limiting membrane peeling for epiretinal membrane might have a higher risk of causing inner retinal damage. Translational Relevance FIB/SEM was a useful tool for three-dimensional quantitative analysis of the internal limiting membrane. PMID:29423341

High resolution analysis of tropical forest fragmentation and its impact on the global carbon cycle

NASA Astrophysics Data System (ADS)

Brinck, Katharina; Fischer, Rico; Groeneveld, Jürgen; Lehmann, Sebastian; Dantas de Paula, Mateus; Pütz, Sandro; Sexton, Joseph O.; Song, Danxia; Huth, Andreas

2017-03-01

Deforestation in the tropics is not only responsible for direct carbon emissions but also extends the forest edge wherein trees suffer increased mortality. Here we combine high-resolution (30 m) satellite maps of forest cover with estimates of the edge effect and show that 19% of the remaining area of tropical forests lies within 100 m of a forest edge. The tropics house around 50 million forest fragments and the length of the world's tropical forest edges sums to nearly 50 million km. Edge effects in tropical forests have caused an additional 10.3 Gt (2.1-14.4 Gt) of carbon emissions, which translates into 0.34 Gt per year and represents 31% of the currently estimated annual carbon releases due to tropical deforestation. Fragmentation substantially augments carbon emissions from tropical forests and must be taken into account when analysing the role of vegetation in the global carbon cycle.

Cajanol, a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots, induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway.

PubMed

Luo, Meng; Liu, Xia; Zu, Yuangang; Fu, Yujie; Zhang, Su; Yao, Liping; Efferth, Thomas

2010-10-06

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (DeltaPsim) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC(50) value was 54.05 microM after 72 h treatment, 58.32 microM after 48 h; and 83.42 microM after 24h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Novel anticancer agent, benzyldihydroxyoctenone, isolated from Streptomyces sp. causes G1 cell cycle arrest and induces apoptosis of HeLa cells.

PubMed

Lee, Chul-Hoon; Lim, Haeyoung; Moon, Sangik; Shin, Choonshik; Kim, Seunghyun; Kim, Bum-Joon; Lim, Yoongho

2007-06-01

In the course of screening for anticancer agents, a novel active compound, F3-2-5, was isolated from culture broth of Streptomyces sp., KACC91015. Its structure was identified using nuclear magnetic resonance, mass spectrometry, and molecular modeling experiments, and confirmed by total synthesis. The growth of various human cancer cell lines was inhibited in a dose-dependent manner by 0.06-0.48 mM F3-2-5 over 24 h. Its IC(50) values were estimated at 37 microM on HeLa, 72 microM on A549, and 190 microM on HT-29 cells. However, F3-2-5 had no antiproliferative effect on normal lymphocytes and normal fibroblasts used as controls. Moreover, it affected cell cycle regulation and caused apoptosis of the HeLa cells; chromatin condensation and DNA fragmentation were observed in cells exposed to 80 microM F3-2-5. Western blot analysis revealed that F3-2-5 inhibited phosphorylation of retinoblastoma protein (pRb) and reduced expression of cyclin-dependent kinase-4 and -6, and cyclin D1 and E, while levels of p53 and p21(WAF1/CIP1) increased. Taken together, these findings show that F3-2-5 inhibits proliferation of HeLa cells by inducing G(1) phase arrest as a consequence of inhibition of pRb phosphorylation following up-regulation of p21(WAF1/CIP1) and p53. Furthermore, apoptosis in HeLa cells treated with F3-2-5 was associated with an increase in Bax and p53, leading to release of cytochrome c, activation of caspase-3, and -8, and cleavage of poly (ADP-ribose) polymerase.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Genome expansion via lineage splitting and genome reduction in the cicada endosymbiont Hodgkinia.

PubMed

Campbell, Matthew A; Van Leuven, James T; Meister, Russell C; Carey, Kaitlin M; Simon, Chris; McCutcheon, John P

2015-08-18

Comparative genomics from mitochondria, plastids, and mutualistic endosymbiotic bacteria has shown that the stable establishment of a bacterium in a host cell results in genome reduction. Although many highly reduced genomes from endosymbiotic bacteria are stable in gene content and genome structure, organelle genomes are sometimes characterized by dramatic structural diversity. Previous results from Candidatus Hodgkinia cicadicola, an endosymbiont of cicadas, revealed that some lineages of this bacterium had split into two new cytologically distinct yet genetically interdependent species. It was hypothesized that the long life cycle of cicadas in part enabled this unusual lineage-splitting event. Here we test this hypothesis by investigating the structure of the Ca. Hodgkinia genome in one of the longest-lived cicadas, Magicicada tredecim. We show that the Ca. Hodgkinia genome from M. tredecim has fragmented into multiple new chromosomes or genomes, with at least some remaining partitioned into discrete cells. We also show that this lineage-splitting process has resulted in a complex of Ca. Hodgkinia genomes that are 1.1-Mb pairs in length when considered together, an almost 10-fold increase in size from the hypothetical single-genome ancestor. These results parallel some examples of genome fragmentation and expansion in organelles, although the mechanisms that give rise to these extreme genome instabilities are likely different.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

PubMed

Yu, Bing; Ni, Ming; Li, Wen-Han; Lei, Ping; Xing, Wei; Xiao, Dai-Wen; Huang, Yu; Tang, Zhen-Jie; Zhu, Hui-Fen; Shen, Guan-Xin

2005-07-14

To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

PubMed

Pan, W-R; Chen, P-W; Chen, Y-L S; Hsu, H-C; Lin, C-C; Chen, W-J

2013-01-01

Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 μM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Forest-climate interactions in fragmented tropical landscapes.

PubMed

Laurance, William F

2004-03-29

In the tropics, habitat fragmentation alters forest-climate interactions in diverse ways. On a local scale (less than 1 km), elevated desiccation and wind disturbance near fragment margins lead to sharply increased tree mortality, thus altering canopy-gap dynamics, plant community composition, biomass dynamics and carbon storage. Fragmented forests are also highly vulnerable to edge-related fires, especially in regions with periodic droughts or strong dry seasons. At landscape to regional scales (10-1000 km), habitat fragmentation may have complex effects on forest-climate interactions, with important consequences for atmospheric circulation, water cycling and precipitation. Positive feedbacks among deforestation, regional climate change and fire could pose a serious threat for some tropical forests, but the details of such interactions are poorly understood.

Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions

NASA Technical Reports Server (NTRS)

Durzan, D. J.

1999-01-01

Haploid and diploid cell suspensions of Taxus spp. were examined for their adaptive plasticity in response to simulated microgravity, unit gravity, and hypergravity. Cell suspensions produced the taxane, paclitaxel, (TAXOL (R)), which is useful for the treatment of various cancers. Amyloplasts contributed to taxane ring biosynthesis and to drug release at the cell wall. Drug-producing cells reacted as gravisensing osmotic tensiometers. In stressed cells, amyloplasts docked and fused in clusters to sites on the plasmalemma before taxane discharge into the culture medium. In simulated microgravity and compared to all other treatments, taxane production was reduced nearly 100-fold. The percent paclitaxel of total taxanes remained 3-to 6-fold greater, and biomass doubled. When p53-independent programmed cell death was induced, taxanes were released into the culture medium as free molecules (soluble and insoluble) or bound to membranes, nuclear fragments, xylan residues, and other particulate materials. Unit gravity and especially hypergravity promoted xylogenesis and significant drug overproduction. A model relating families of >touch = (TCH), taxane early response (TER), nuclear cycling, and apoptosis-regulating genes to gravisensing, cell wall modifications, and to taxane recovery accounted for most but not all of the observations.

A study of the effects of citrate-coated silver nanoparticles on RAW 264.7 cells using a toolbox of cytotoxic endpoints

NASA Astrophysics Data System (ADS)

Bastos, V.; Duarte, I. F.; Santos, C.; Oliveira, H.

2017-05-01

Citrate-coated silver nanoparticles (citrate-AgNPs) are among the most commonly used nanomaterials, widely present in industrial and biomedical products. In this study, the cytotoxicity of 30-nm citrate-AgNPs on the macrophage cell line RAW 264.7 was evaluated, using a battery of cytotoxicity endpoints (viability, oxidative stress, and cytostaticity/clastogenicity), at 24 and 48 h of exposure. Citrate-AgNPs decreased cell proliferation and viability only at 75 μg/mL, suggesting a low sensitivity of RAW cells to lower doses of these AgNPs. After 24 h of exposure, ROS content decreased in cells exposed to 60 μg/mL AgNPs (IC20 value), corroborating the high tolerance of these cells to citrate-AgNPs. However, these cells suffered an impairment of the cell cycle, shown by an increase at the sub-G1 phase. This increase of the sub-G1 population was correlated with an increase of DNA fragmentation, suggesting an increase of apoptosis. Thus, our data are important to understand the effects of low concentrations (IC20) of citrate-AgNPs on in vitro vital macrophage functions.

Construction of recombinant FGFR1 containing full-length gene and its potential application.

PubMed

Zhou, Yali; Luo, Wenjuan; Zheng, Lei; Li, Miao; Zhang, Yanmin

2010-07-01

FGFR1, one of the four fibroblast growth factor receptors, has been found to be over-expressed in many cancers. In this study, a full-length expression plasmid for FGFR1 was obtained by fragment amplification. The amplified PCR product was then digested and inserted into the pcDNA3.1(+) vector. A recombinant eukaryotic expression vector containing the complete CDS region of FGFR1 was successfully constructed. After it was transfected to Hek293 cell, the expression of the FGFR1 receptor in recombinant Hek293/FGFR1 was 18 times higher than that of Hek293 cell. The biological activities of high expression FGFR1 cell (Hek293/FGFR1) were verified by FCM, immunofluorescent, RT-PCR, western blot and cell cycle analysis. Then, Hek293/FGFR1 was used to screen taspine with cell membrane chromatography (CMC). Finally, we analyzed the effects of taspine on Hek293/FGFR1 cell and MCF-7 cell. In conclusion, Hek293/FGFR1 was successfully constructed. The results demonstrate that taspine can down-regulate phosphorylation of FGFR1 and ERK, and inhibit Hek293/FGFR1 and MCF-7 cell proliferation. Copyright 2010 Elsevier Inc. All rights reserved.

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

PubMed

Klement, Maximilian; Zheng, Jiyun; Liu, Chengcheng; Tan, Heng-Liang; Wong, Victor Vai Tak; Choo, Andre Boon-Hwa; Lee, Dong-Yup; Ow, Dave Siak-Wei

2017-02-10

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

PubMed Central

Dille, Stephanie; Kleinschnitz, Eva-Maria; Kontchou, Collins Waguia; Nölke, Thilo

2015-01-01

The Chlamydiales are an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species is Chlamydia trachomatis. Many uncertainties remain as to how Chlamydia organizes its intracellular development and replication. The discovery of new Chlamydiales species from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to “chlamydial” development. We used this approach in the infection of human cells with Waddlia chondrophila, a species from the family Waddliaceae whose natural host is uncertain. Compared to C. trachomatis, W. chondrophila had slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for the C. trachomatis inclusion. C. trachomatis infection generates proteolytic activity by the protease Chlamydia protease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case of W. chondrophila. Unlike Chlamydia, W. chondrophila did not protect against staurosporine-induced apoptosis. C. trachomatis infection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent from W. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development of Chlamydiales in vitro but may be linked to host adaptation of Chlamydia and the higher virulence of C. trachomatis. PMID:26056386

p57KIP2 expression and loss of heterozygosity during immortal conversion of cultured human mammary epithelial cells

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

Nijjar, Tarlochan; Wigington, Don; Garbe, James C.

1999-08-01

The authors have uncovered a novel role for the cyclin-dependent kinase inhibitor, p57KIP2, during the immortalization of cultured human mammary epithelial cells (HMEC). HMEC immortalized following chemical carcinogen exposure initially expressed little or no telomerase activity, and their telomeres continued to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined and exhibited slow heterogeneous growth, and contained many non-proliferative cells. These conditionally immortal HMEC cultures accumulated large quantities of p57 protein. With continued passage, the conditionally immortal cell populations very graduall2048nverted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerasemore » activity, and stabilization of telomere length. The fully immortal good growing HMEC did not accumulate p57 in G0 or during the cell cycle. DNA and RNA analysis of mass populations and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically short telomeres was repeatedly accompanied by loss of the expressed p57 allele, and transient expression of the previously imprinted allele. Conditionally immortal 184A1 with mean TRF > 3 kb infected with retroviruses containing the p57 gene exhibited premature slow heterogeneous growth. Conversely, exogenous expression of hTERT, the catalytic subunit of telomerase, in 184A1 with mean TRF > 3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling populations. These data indicate that in HMEC which have overcome replicative senescence, p57 may provide an additional barrier against indefinite proliferation. Overcoming p57 mediated growth inhibition in these cells may be crucial for acquisition of the unlimited growth potential thought to be critical for malignant progression.« less

Presence of bile acids in human follicular fluid and their relation with embryo development in modified natural cycle IVF.

PubMed

Nagy, R A; van Montfoort, A P A; Dikkers, A; van Echten-Arends, J; Homminga, I; Land, J A; Hoek, A; Tietge, U J F

2015-05-01

Are bile acids (BA) and their respective subspecies present in human follicular fluid (FF) and do they relate to embryo quality in modified natural cycle IVF (MNC-IVF)? BA concentrations are 2-fold higher in follicular fluid than in serum and ursodeoxycholic acid (UDCA) derivatives were associated with development of top quality embryos on Day 3 after fertilization. Granulosa cells are capable of synthesizing BA, but a potential correlation with oocyte and embryo quality as well as information on the presence and role of BA subspecies in follicular fluid have yet to be investigated. Between January 2001 and June 2004, follicular fluid and serum samples were collected from 303 patients treated in a single academic centre that was involved in a multicentre cohort study on the effectiveness of MNC-IVF. Material from patients who underwent a first cycle of MNC-IVF was used. Serum was not stored from all patients, and the available material comprised 156 follicular fluid and 116 matching serum samples. Total BA and BA subspecies were measured in follicular fluid and in matching serum by enzymatic fluorimetric assay and liquid chromatography-mass spectrometry, respectively. The association of BA in follicular fluid with oocyte and embryo quality parameters, such as fertilization rate and cell number, presence of multinucleated blastomeres and percentage of fragmentation on Day 3, was analysed. Embryos with eight cells on Day 3 after oocyte retrieval were more likely to originate from follicles with a higher level of UDCA derivatives than those with fewer than eight cells (P < 0.05). Furthermore, follicular fluid levels of chenodeoxycholic derivatives were higher and deoxycholic derivatives were lower in the group of embryos with fragmentation compared with those without (each P < 0.05). Levels of total BA were 2-fold higher in follicular fluid compared with serum (P < 0.001), but had no predictive value for oocyte and embryo quality. Only samples originating from first cycle MNC-IVF were used, which resulted in 14 samples only from women with an ongoing pregnancy, therefore further prospective studies are required to confirm the association of UDCA with IVF pregnancy outcomes. The inter-cycle variability of BA levels in follicular fluid within individuals has yet to be investigated. We checked for macroscopic signs of contamination of follicular fluid by blood but the possibility that small traces of blood were present within the follicular fluid remains. Finally, although BA are considered stable when stored at -20°C, there was a time lag of 10 years between the collection and analysis of follicular fluid and serum samples. The favourable relation between UDCA derivatives in follicular fluid and good embryo development and quality deserves further prospective research, with live birth rates as the end-point. This work was supported by a grant from the Netherlands Organisation for Scientific Research (VIDI Grant 917-56-358 to U.J.F.T.). No competing interests are reported. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

PubMed

Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

2010-01-01

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

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

Microtubule antagonists activate programmed cell death (apoptosis) in cultured rat hepatocytes.

PubMed Central

Tsukidate, K.; Yamamoto, K.; Snyder, J. W.; Farber, J. L.

1993-01-01

We investigated the mechanism of lethal injury following the disruption of microtubules in cultured hepatocytes treated with vinblastine (VBL) or colchicine (COL). These agents kill hepatocytes by a process readily distinguished from two well-known pathways that lead to a loss of viability, namely, oxidative stress and inhibition of mitochondrial electron transport. Cell killing with VBL and COL was accompanied by fragmentation of DNA. Both the loss of viability and the fragmentation of DNA were prevented by the inhibition of protein synthesis within 6 hours following exposure to VBL or COL. Cell death and the fragmentation of DNA were also prevented when Ca2+ was removed from the culture medium. By contrast, the inhibition of protein kinase C prevented cell killing by VBL or COL, but did not alter the extent of DNA fragmentation. The requirements here for protein synthesis, extracellular Ca2+, and protein kinase C activity define a model of apoptosis, or programmed cell death, that seems to involve mechanisms that can be dissociated from the fragmentation of DNA. Images Figure 2 PMID:8362985

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence

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

Ma, Liwei; Zhao, Wenting; Zheng, Quanhui

2016-01-15

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. Themore » data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-β-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-β-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence. - Highlights: • The ribosomal L1 domain and lysine-rich region of CSIG were expressed. • They are critical for CSIG to regulate proliferation and senescence. • CSIG and its domains exhibit different subcellular distribution.« less

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

PubMed

Gordeev, Serguei A; Bykova, Tatiana V; Zubova, Svetlana G; Bystrova, Olga A; Martynova, Marina G; Pospelov, Valery A; Pospelova, Tatiana V

2015-12-29

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

YC-1 induces G0/G1 phase arrest and mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells.

PubMed

Lee, Miau-Rong; Lin, Chingju; Lu, Chi-Cheng; Kuo, Sheng-Chu; Tsao, Je-Wei; Juan, Yu-Ning; Chiu, Hong-Yi; Lee, Fang-Yu; Yang, Jai-Sing; Tsai, Fuu-Jen

2017-06-01

Oral cancer is a serious and fatal disease. Cisplatin is the first line of chemotherapeutic agent for oral cancer therapy. However, the development of drug resistance and severe side effects cause tremendous problems clinically. In this study, we investigated the pharmacologic mechanisms of YC-1 on cisplatin-resistant human oral cancer cell line, CAR. Our results indicated that YC-1 induced a concentration-dependent and time-dependent decrease in viability of CAR cells analyzed by MTT assay. Real-time image analysis of CAR cells by IncuCyte™ Kinetic Live Cell Imaging System demonstrated that YC-1 inhibited cell proliferation and reduced cell confluence in a time-dependent manner. Results from flow cytometric analysis revealed that YC-1 promoted G 0 /G 1 phase arrest and provoked apoptosis in CAR cells. The effects of cell cycle arrest by YC-1 were further supported by up-regulation of p21 and down-regulation of cyclin A, D, E and CDK2 protein levels. TUNEL staining showed that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G 0 /G 1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future. © Author(s) 2017. This article is published with open access by China Medical University.

Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Bernardini, Jame N.; Stam, Christina N.

2010-01-01

The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells.

Feeding group responses of a Neotropical termite assemblage to rain forest fragmentation.

PubMed

Davies, Richard G

2002-10-01

Biomass collapse and its associated microclimatic stresses within recently isolated rain forest fragments may negatively affect species diversity of most resident taxa. However, for some decomposer organisms, increased resource availability via accompanying tree die-off may effect positive responses, at least for a time, with implications for rates of nutrient cycling and greenhouse gas release. This study investigates the early effects of forest fragmentation on a Neotropical termite assemblage. Numbers of encounters (surrogate for relative abundance) and species richness of wood and leaf-litter feeders, soil feeders, and the whole assemblage, were studied across true forest islands and mainland sites at a hydroelectric reservoir in French Guiana. Results showed no overall effect of fragmentation on either total termite encounters or species richness. However, numbers of encounters and species richness of wood and leaf-litter feeders showed positive responses to forest fragmentation. By contrast, soil feeders showed a negative response for numbers of encounters and no significant effect for species richness. Environmental data suggest that increased tree die-off, and other edge effects associated with biomass collapse, were underway at the time of sampling. Resulting increase in resource availability may therefore explain the positive influence on wood and leaf-litter feeders. A possible decrease in predation pressure from ants with decrease in island size was not tested for, but was a likely effect of the flooded matrix habitat. Fragmentation effects on soil feeder encounters may be due to the energetic and microclimatic constraints of feeding lower down the humification gradient of termite food substrates, but were not sufficient to affect species richness. The patterns revealed suggest that rates of wood decomposition following tree die-off, and of soil nutrient cycling, under different rain forest fragmentation scenarios, merit further study.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

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

Belakavadi, Madesh; Prabhakar, B.T.; Salimath, Bharathi P.

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptoticmore » effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.« less

Vitamin C in synergism with cisplatin induces cell death in cervical cancer cells through altered redox cycling and p53 upregulation.

PubMed

Leekha, Ankita; Gurjar, Bahadur S; Tyagi, Aakriti; Rizvi, Moshahid A; Verma, Anita K

2016-12-01

Cervical cancer is the second most prevalent cancer in women worldwide. Survival of patients has been improved by cisplatin-based chemotherapy, but its effectiveness is limited due to its adverse effects on many tissues, especially nephrotoxicity. To optimize the efficacy of CDDP, we propose a combination therapy using natural products with minimal side effects. Vitamin C being a natural antioxidant is capable of selectively targeting cancer cells at pharmacological concentrations. Vitamin C synergistically enhances the activity of chemotherapeutic agents without increasing toxicity to normal cells. Therefore, we exploited co-therapy with cisplatin and vitamin C to kill cervical cancer cells. We elucidated the role of CDDP and VC on cervical cancer cell line (SiHa) by using cell growth assays, DNA fragmentation analysis, comet assay, in vitro morphological assessment of apoptosis (AO/EB and DAPI staining), ROS analysis by DCFDA, flow cytometry, biochemical assays (GST, GSH, NO, catalase, TPA) and Western blotting. Our results clearly demonstrated that CDDP and VC treatment exhibited ameliorative effect on induction of cell death by p53 overexpression and generation of hydrogen peroxide in SiHa cells, thereby reducing the dosage of CDDP required to induce cell death in cancer cells. These studies provide novel approaches to combat cisplatin resistance in cervical cancer.

Enhancing the intense field control of molecular fragmentation.

PubMed

Anis, Fatima; Esry, B D

2012-09-28

We describe a pump-probe scheme with which the spatial asymmetry of dissociating molecular fragments-as controlled by the carrier-envelope phase of an intense few-cycle laser pulse-can be enhanced by an order of magnitude or more. We illustrate the scheme using extensive, full-dimensional calculations for dissociation of H(2)(+) and include the averaging necessary for comparison with experiment.

Contrasting patterns of nest survival and postfledging survival in ovenbirds and Acadian flycatchers in Missouri forest fragments

Treesearch

Julianna M. A. Jenkins; Frank R. Thompson; John Faaborg

2016-01-01

We can improve our ability to assess population viability and forecast population growth under different scenarios by understanding factors that limit population parameters in each stage of the annual cycle. Postfledging mortality rates may be as variable as nest survival across regions and fragmentation gradients, although factors that negatively impact nest survival...

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.

Molecular Dynamics Simulations of a Cyclic DP-240 Amylose Fragment in a Periodic Cell: Glass Transition Temperature and Water Diffusion

USDA-ARS?s Scientific Manuscript database

Molecular dynamics simulations using AMB06C, an in-house carbohydrate force field, (NPT ensembles, 1atm) were carried out on a periodic cell that contained a cyclic-DP-240 amylose fragment and TIP3P water molecules. Molecular conformation and movement of the amylose fragment and water molecules at ...

Dimethylaminoethanol affects the viability of human cultured fibroblasts.

PubMed

Gragnani, Alfredo; Giannoccaro, Fabiana Bocci; Sobral, Christiane S; Moraes, A A F; França, Jeronimo P; Ferreira, A T; Ferreira, Lydia Masako

2007-01-01

In clinical practice, dimethylaminoethanol (DMAE) has been used in the fight against wrinkles and flaccidity in the cervicofacial region. The firming action of DMAE is explained by the fact that its molecule, considered to be a precursor of acetylcholine, alters muscle contraction. However, no experimental studies have confirmed this theory. Because the actual mechanism of DMAE action was not defined and there were no references in the literature regarding its direct action on fibroblasts, this study was performed to evaluate the direct action of DMAE on cultured human fibroblasts. Human fibroblasts obtained from discarded fragments of total skin from patients undergoing plastic or reconstructive surgical procedures performed within the Plastic Surgery Division at the Federal University of São Paulo were used for this study. The explant technique was used. The culture medium was supplemented with different concentrations of DMAE on the fourth cell passage, and the cell proliferation rate, cytosolic calcium levels, and cell cycle were evaluated. Statistical analysis was performed using analysis of variance (ANOVA) followed by a Newman-Keuls test for multiple comparisons. A decrease in fibroblast proliferation was associated with an increase in DMAE concentration. A longer treatment time with trypsin was required for the groups treated with DMAE in a dose-dependent manner. In the presence of DMAE, cytosolic calcium increased in a dose-dependent manner. Apoptosis also increased in groups treated with DMAE. Dimethylaminoethanol reduced the proliferation of fibroblasts, increased cytosolic calcium, and changed the cell cycle, causing an increase in apoptosis in cultured human fibroblasts.

Cadmium-induced cyto- and genotoxicity are organ-dependent in lettuce.

PubMed

Monteiro, Cristina; Santos, Conceição; Pinho, Sónia; Oliveira, Helena; Pedrosa, Tiago; Dias, Maria Celeste

2012-07-16

Cadmium is a priority pollutant. Its mechanisms and effects within different plant organs remain unclear. Here, cyto-genotoxicity biomarkers were evaluated in roots and leaves after Cd exposure (0, 1, 10, and 50 μM) of the model crop Lactuca sativa L. (cv. "Reine de Mai"). Overall, superoxide dismutase (SOD) and catalase (CAT) activities were stimulated in leaves, where Cd accumulation was lower in comparison to that in roots. In roots, SOD and peroxidase (POX, APX) activities were stimulated. Moreover, in both organs glutathione reductase (GR) was not affected by Cd. Overall, the H(2)O(2) content increased in both organs, while the total antioxidant capacity decreased in leaves and increased in roots with Cd concentrations. In both organs, lipid and protein oxidation rose with consequent increase of membrane permeability. Simultaneously, the comet assay showed that tail moment, tail length, and % tail DNA were maximum for 1 μM. For 10 μM, shorter tails were found suggesting induced Cd-DNA adducts that lead to DNA-DNA/DNA-protein cross-links, and/or formation of longer DNA fragments, and/or impairment of DNA repair mechanisms, while at 50 μM, nucleoids sensitivity to the technique was evident. This result was consistent with the maximum micronuclei frequency found for the 10 μM Cd dose in roots, suggesting that the surviving cells in this organ had an increase of mitotic catastrophe and that DNA repair systems for blocking cell cycle were dysfunctional. In lower Cd concentrations, root cells might have developed strategies to repair damaged DNA by blocking the cell cycle at specific checkpoints, thus avoiding mitotic catastrophe. Roots at 1 μM showed a cell cycle blockage trend at the G(2) checkpoint, while those at higher concentrations presented S phase delay. We finally discuss a general model of Cd-organ interaction covering these cyto- and genotoxic effects and the potential use of this cultivar in phytoremediation strategies.

Luminescent threat: toxicity of light stick attractors used in pelagic fishery

PubMed Central

de Oliveira, Tiago Franco; da Silva, Amanda Lucila Medeiros; de Moura, Rafaela Alves; Bagattini, Raquel; de Oliveira, Antonio Anax Falcão; de Medeiros, Marisa Helena Gennari; Di Mascio, Paolo; de Arruda Campos, Ivan Pérsio; Barretto, Fabiano Prado; Bechara, Etelvino José Henriques; de Melo Loureiro, Ana Paula

2014-01-01

Light sticks (LS) are sources of chemiluminescence commonly used in pelagic fishery, where hundreds are discarded and reach the shores. Residents from fishing villages report an improper use of LS contents on the skin. Given the scarce information regarding LS toxicity, the effects of LS solutions in cell cultures were evaluated herein. Loss of viability, cell cycle changes and DNA fragmentation were observed in HepG2 cell line and skin fibroblasts. A non-cytotoxic LS concentration increased the occurrence of the mutagenic lesion 1,N6-εdAdo in HepG2 DNA by three-fold. Additionally, in vitro incubations of spent LS contents with DNA generated dGuo-LS adducts, whose structure elucidation revealed the presence of a reactive chlorinated product. In conclusion, the LS contents were found to be highly cyto- and genotoxic. Our data indicate an urgent need for LS waste management guidelines and for adequate information regarding toxic outcomes that may arise from human exposure. PMID:24942522

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

PubMed

Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

2010-12-01

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo

PubMed Central

1986-01-01

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self- reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes. PMID:3941154

Mycobacterium tuberculosis Cell Wall Fragments Released upon Bacterial Contact with the Human Lung Mucosa Alter the Neutrophil Response to Infection

PubMed Central

Scordo, Julia M.; Arcos, Jesús; Kelley, Holden V.; Diangelo, Lauren; Sasindran, Smitha J.; Youngmin, Ellie; Wewers, Mark D.; Wang, Shu-Hua; Balada-Llasat, Joan-Miquel; Torrelles, Jordi B.

2017-01-01

In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis (M.tb) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site. PMID:28373877

Mycobacterium tuberculosis Cell Wall Fragments Released upon Bacterial Contact with the Human Lung Mucosa Alter the Neutrophil Response to Infection.

PubMed

Scordo, Julia M; Arcos, Jesús; Kelley, Holden V; Diangelo, Lauren; Sasindran, Smitha J; Youngmin, Ellie; Wewers, Mark D; Wang, Shu-Hua; Balada-Llasat, Joan-Miquel; Torrelles, Jordi B

2017-01-01

In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis ( M.tb ) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site.

RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

PubMed

Yu, W; Sanders, B G; Kline, K

1997-01-01

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

Regulation of apoptosis of interleukin 2-dependent mouse T-cell line by protein tyrosine phosphorylation and polyamines.

PubMed

Min, A; Hasuma, T; Yano, Y; Matsui-Yuasa, I; Otani, S

1995-12-01

We examined the effect of inhibitors of tyrosine kinase and tyrosine phosphatase on DNA fragmentation, protein tyrosine phosphorylation, and polyamine metabolism in the murine T-cell line CTLL-2. When cells were exposed to herbimycin A, a specific inhibitor of tyrosine kinase (Uehara et al., 1989, Biochem. Biophys. Res. Commun., 163:803-809), in the presence of interleukin 2 (IL-2), DNA was degraded into oligonucleosomal fragments in a dose-dependent fashion. Genistein, another inhibitor of tyrosine kinase (Akiyama et al., 1987, J. Biol. Chem., 262:5592-5596), had similar effects. Exposure of CTLL-2 cells to vanadate, a tyrosine phosphatase inhibitor, blocked with the DNA fragmentation induced by herbimycin A. Tyrosine phosphorylation of 55 Kd protein was inhibited by herbimycin A, and the inhibition was reduced by vanadate. Ornithine decarboxylase (ODC) activity decreased rapidly after herbimycin A was added to CTLL-2 cell cultures, while vanadate increased ODC activity. The exogenous addition of putrescine or spermine, but not that of spermidine, attenuated herbimycin A-induced DNA fragmentation. These findings suggest that phosphorylation of tyrosine residues of 55 Kd protein prevents DNA fragmentation and that polyamines are involved in regulation of apoptosis.

Sertoli cell index and spermatic reserves in adult captive African lions (Panthera leo, Linnaeus, 1758).

PubMed

de Barros, João Bosco Gonçalves; de Paula, Tarcízio Antônio Rego; da Matta, Sérgio Luis Pinto; Fonseca, Cláudio César; Leite, Flaviana Lima Guião; Rossi, João Luiz; de Oliveira, Priscila Carvalho; da Costa, Eduardo Paulino

2007-12-01

The intrinsic yield of spermatogenesis and the supporting indexes of the Sertoli cells are the best indicators for the spermatic production capacity in a species. The aim of the present study was to quantify the intrinsic yield of the spermatogenetic process, as well as the Sertoli cell index and spermatic reserves. Testicular fragments of five adult African lions was fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 microm thickness. In the seminiferous epithelium of the African lions, 10.3 primary spermatocytes at pre-leptotene phase are produced by the type-A spermatogonia. During meiotic divisions, only 2.7 spermatids were produced from the primary spermatocytes. The general spermatogenesis production in the African lions was approximately 22.1 cells, and each Sertoli cell was able to sustain and maintain approximately 14.9 cells of the germinative line, from which 7.9 are round spermatids. A total of 103x10(6) spermatozoa are produced by each testis gram at each cycle of the seminiferous epithelium. The spermatic reserve of lion is below the amplitude observed in mammals.

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites.

PubMed

Lungu, Cristiana; Pinter, Sabine; Broche, Julian; Rathert, Philipp; Jeltsch, Albert

2017-09-21

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live-cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing defined epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.Tools for imaging epigenetic modifications can shed light on the regulation of epigenetic processes. Here, the authors present a fluorescence complementation approach for detection of DNA and histone methylation at endogenous genomic sites allowing following of dynamic changes of these marks by live-cell microscopy.

Multifunctional biosynthesized silver nanoparticles exhibiting excellent antimicrobial potential against multi-drug resistant microbes along with remarkable anticancerous properties.

PubMed

Jha, Diksha; Thiruveedula, Prasanna Kumar; Pathak, Rajiv; Kumar, Bipul; Gautam, Hemant K; Agnihotri, Shrish; Sharma, Ashwani Kumar; Kumar, Pradeep

2017-11-01

This study demonstrates the therapeutic potential of silver nanoparticles (AgNPs), which were biosynthesized using the extracts of Citrus maxima plant. Characterization through UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), Fourier Transform Infrared spectroscopy (FTIR), X-ray Diffraction (XRD) and Transmission Electron Microscopy (TEM) confirmed the formation of AgNps in nano-size range. These nanoparticles exhibited enhanced antioxidative activity and showed commendable antimicrobial activity against wide range of microbes including multi-drug resistant bacteria that were later confirmed by TEM. These particles exhibited minimal toxicity when cytotoxicity study was performed on normal human lung fibroblast cell line as well as human red blood cells. It was quite noteworthy that these particles showed remarkable cytotoxicity on human fibrosarcoma and mouse melanoma cell line (B16-F10). Additionally, the apoptotic topographies of B16-F10 cells treated with AgNps were confirmed by using acridine orange and ethidium bromide dual dye staining, caspase-3 assay, DNA fragmentation assay followed by cell cycle analysis using fluorescence-activated cell sorting. Taken together, these results advocate promising potential of the biosynthesized AgNps for their use in therapeutic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Single-ion triblock copolymer electrolytes based on poly(ethylene oxide) and methacrylic sulfonamide blocks for lithium metal batteries

NASA Astrophysics Data System (ADS)

Porcarelli, Luca; Aboudzadeh, M. Ali; Rubatat, Laurent; Nair, Jijeesh R.; Shaplov, Alexander S.; Gerbaldi, Claudio; Mecerreyes, David

2017-10-01

Single-ion conducting polymer electrolytes represent the ideal solution to reduce concentration polarization in lithium metal batteries (LMBs). This paper reports on the synthesis and characterization of single-ion ABA triblock copolymer electrolytes comprising PEO and poly(lithium 1-[3-(methacryloyloxy)propylsulfonyl]-1-(trifluoromethylsulfonyl)imide) blocks, poly(LiMTFSI). Block copolymers are prepared by reversible addition-fragmentation chain transfer polymerization, showing low glass transition temperature (-55 to 7 °C) and degree of crystallinity (51-0%). Comparatively high values of ionic conductivity are obtained (up to ≈ 10-4 S cm-1 at 70 °C), combined with a lithium-ion transference number close to unity (tLi+ ≈ 0.91) and a 4 V electrochemical stability window. In addition to these promising features, solid polymer electrolytes are successfully tested in lithium metal cells at 70 °C providing long lifetime up to 300 cycles, and stable charge/discharge cycling at C/2 (≈100 mAh g-1).

Fragmentation scaling of percolation clusters in two and three dimensions: Large-cell Monte Carlo RG approach

NASA Astrophysics Data System (ADS)

Cheon, M.; Chang, I.

1999-04-01

The scaling behavior for a binary fragmentation of critical percolation clusters is investigated by a large-cell Monte Carlo real-space renormalization group method in two and three dimensions. We obtain accurate values of critical exponents λ and phi describing the scaling of fragmentation rate and the distribution of fragments' masses produced by a binary fragmentation. Our results for λ and phi show that the fragmentation rate is proportional to the size of mother cluster, and the scaling relation σ = 1 + λ - phi conjectured by Edwards et al. to be valid for all dimensions is satisfied in two and three dimensions, where σ is the crossover exponent of the average cluster number in percolation theory, which excludes the other scaling relations.

The incomplete anti-Rh antibody agglutination mechanism of trypsinized ORh+ red cells.

PubMed Central

Margni, R A; Leoni, J; Bazzurro, M

1977-01-01

The capacity for binding to trypsinized and non-trypsinized ORh+ red cells, of the IgG incomplete anti-Rh antibody and its F(ab')2 and Fc fragments has been investigated. An analysis has also been made of the capacity of non-specific human IgG, aggregated non-specific human IgG, human IgM (19S) and IgM (7S), and of fragments Fcgamma, Fcmu and Fc5mu to inhibit the agglutination of trypsinized ORh+ red cells by the IgG incomplete anti-Rh antibody. The results obtained indicate that these antibodies behave in a similar manner to that of nonprecipitating antibodies, and that the agglutination of trypsinized red cells seems to be a mixed reaction due to the interaction of an Fab fragment with its Rh antigenic determinant present in the surface of a red cell and the Fc of the same molecule with a receptor for Fc present in adjacent red cells. The trypsin treatment apparently results in the liberation of occult Fc receptors. It has also been demonstrated that in the agglutination of ORh+ red cells by IgG incomplete anti-Rh antibody in the presence of albumin, interaction must occur in some manner between the albumin and the Fc fragment since the F(ab')2 fragment does not give rise to agglutination under such conditions. Images Figure 1 PMID:415968

Cloning of nascent monkey DNA synthesized early in the cell cycle.

PubMed

Kaufmann, G; Zannis-Hadjopoulos, M; Martin, R G

1985-04-01

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells

PubMed Central

Domínguez, Fernando; Cejudo, Francisco J.

2006-01-01

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed. PMID:16613587

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

PubMed Central

Tyedmers, Jens

2012-01-01

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI+] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates. PMID:22449721

Acacia catechu ethanolic bark extract induces apoptosis in human oral squamous carcinoma cells.

PubMed

Lakshmi, Thangavelu; Ezhilarasan, Devaraj; Vijayaragavan, Rajagopal; Bhullar, Sukhwinder Kaur; Rajendran, Ramasamy

2017-01-01

Oral cancer is in approximately 30% of all cancers in India. This study was conducted to evaluate the cytotoxic activity of ethanolic extract of Acacia catechu bark (ACB) against human squamous cell carcinoma cell line-25 (SCC-25). Cytotoxic effect of ACB extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide assay. A. catechu extract was treated SCC-25 cells with 25 and 50 μg/mL for 24 h. Apoptosis markers such as caspases-8 and 9, bcl-2, bax, and cytochrome c (Cyt-c) were done by RT-PCR. Morphological changes of ACB treated cells were evaluated using acridine orange/ethidium bromide (AO/EB) dual staining. Nuclear morphology and DNA fragmentation were evaluated using propidium iodide (PI) staining. Further, cell cycle analysis was performed using flow cytometry. A. catechu treatment caused cytotoxicity in SCC-25 cells with an IC 50 of 52.09 μg/mL. Apoptotic marker gene expressions were significantly increased on ACB treatment. Staining with AO/EB and PI shows membrane blebbing and nuclear membrane distortion, respectively, and it confirms the apoptosis induction in SCC-25 cells. These results suggest that ACB extract can be used as a modulating agent in oral squamous cell carcinoma.

In search of new lead compounds for trypanosomiasis drug design: A protein structure-based linked-fragment approach

NASA Astrophysics Data System (ADS)

Verlinde, Christophe L. M. J.; Rudenko, Gabrielle; Hol, Wim G. J.

1992-04-01

A modular method for pursuing structure-based inhibitor design in the framework of a design cycle is presented. The approach entails four stages: (1) a design pathway is defined in the three-dimensional structure of a target protein; (2) this pathway is divided into subregions; (3) complementary building blocks, also called fragments, are designed in each subregion; complementarity is defined in terms of shape, hydrophobicity, hydrogen bond properties and electrostatics; and (4) fragments from different subregions are linked into potential lead compounds. Stages (3) and (4) are qualitatively guided by force-field calculations. In addition, the designed fragments serve as entries for retrieving existing compounds from chemical databases. This linked-fragment approach has been applied in the design of potentially selective inhibitors of triosephosphate isomerase from Trypanosoma brucei, the causative agent of sleeping sickness.

A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections.

PubMed

Bavia, Lorena; Mosimann, Ana Luiza Pamplona; Aoki, Mateus Nóbrega; Duarte Dos Santos, Claudia Nunes

2016-05-28

The family Flaviviridae comprises a wide variety of viruses that are distributed worldwide, some of which are associated with high rates of morbidity and mortality. There are neither vaccines nor antivirals for most flavivirus infections, reinforcing the importance of research on different aspects of the viral life cycle. During infection, cytoplasmic accumulation of RNA fragments mainly originating from the 3' UTRs, which have been designated subgenomic flavivirus RNAs (sfRNAs), has been detected. It has been shown that eukaryotic exoribonucleases are involved in viral sfRNA production. Additionally, viral and human small RNAs (sRNAs) have also been found in flavivirus-infected cells, especially microRNAs (miRNAs). miRNAs were first described in eukaryotic cells and in a mature and functional state present as single-stranded 18-24 nt RNA fragments. Their main function is the repression of translation through base pairing with cellular mRNAs, besides other functions, such as mRNA degradation. Canonical miRNA biogenesis involves Drosha and Dicer, however miRNA can also be generated by alternative pathways. In the case of flaviviruses, alternative pathways have been suggested. Both sfRNAs and miRNAs are involved in viral infection and host cell response modulation, representing interesting targets of antiviral strategies. In this review, we focus on the generation and function of viral sfRNAs, sRNAs and miRNAs in West Nile, dengue, Japanese encephalitis, Murray Valley encephalitis and yellow fever infections, as well as their roles in viral replication, translation and cell immune response evasion. We also give an overview regarding other flaviviruses and the generation of cellular miRNAs during infection.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

PubMed

Makino, K; Takeichi, O; Imai, K; Inoue, H; Hatori, K; Himi, K; Saito, I; Ochiai, K; Ogiso, B

2018-06-01

To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). The concentrations of short-chain fatty acids (SCFAs) in P. endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P. endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P. endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P. endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. The concentrations of n-butyric acid in P. endodontalis culture supernatants were significantly higher than those of other SCFAs (P=0.0173). Using B-95-8-221 Luc cells treated with P. endodontalis culture supernatants, the luciferase assay demonstrated that P. endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P. endodontalis culture supernatants. P. endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P. endodontalis cells in each specimen. n-butyric acid produced by P. endodontalis reactivated latent EBV. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae.

PubMed

Lewinska, Anna; Miedziak, Beata; Kulak, Klaudia; Molon, Mateusz; Wnuk, Maciej

2014-06-01

The nucleolus is speculated to be a regulator of cellular senescence in numerous biological systems (Guarente, Genes Dev 11(19):2449-2455, 1997; Johnson et al., Curr Opin Cell Biol 10(3):332-338, 1998). In the budding yeast Saccharomyces cerevisiae, alterations in nucleolar architecture, the redistribution of nucleolar protein and the accumulation of extrachromosomal ribosomal DNA circles (ERCs) during replicative aging have been reported. However, little is known regarding rDNA stability and changes in nucleolar activity during chronological aging (CA), which is another yeast aging model used. In the present study, the impact of aberrant cell cycle checkpoint control (knock-out of BUB1, BUB2, MAD1 and TEL1 genes in haploid and diploid hemizygous states) on CA-mediated changes in the nucleolus was studied. Nucleolus fragmentation, changes in the nucleolus size and the nucleolus/nucleus ratio, ERC accumulation, expression pattern changes and the relocation of protein involved in transcriptional silencing during CA were revealed. All strains examined were affected by oxidative stress, aneuploidy (numerical rather than structural aberrations) and DNA damage. However, the bub1 cells were the most prone to aneuploidy events, which may contribute to observed decrease in chronological lifespan. We postulate that chronological aging may be affected by redox imbalance-mediated chromosome XII instability leading to both rDNA instability and whole chromosome aneuploidy. CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation. Moreover, the rDNA content of chronologically aging cells may be a factor determining the subsequent replicative lifespan. Taken together, we demonstrated that the nucleolus state is also affected during CA in yeast.

The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

PubMed

Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

2016-02-01

In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

The Behavior of Lipid Debris Left on Cell Surfaces from Microbubble Based Ultrasound Molecular Imaging

PubMed Central

Ibsen, Stuart; Shi, Guixin; Schutt, Carolyn; Shi, Linda; Suico, Kyle-David; Benchimol, Michael; Serra, Viviana; Simberg, Dmitri; Berns, Michael; Esener, Sadik

2014-01-01

Lipid monolayer coated microbubbles are currently being developed to identify vascular regions that express certain surface proteins as part of the new technique of ultrasound molecular imaging. The microbubbles are functionalized with targeting ligands which bind to the desired cells holding the microbubbles in place as the remaining unbound microbubbles are eliminated from circulation. Subsequent scanning with ultrasound can detect the highly reflectant microbubbles that are left behind. The ultrasound scanning and detection process results in the destruction of the microbubble, creating lipid fragments from the monolayer. Here we demonstrate that microbubbles targeted to 4T1 murine breast cancer cells and human umbilical cord endothelial cells leave behind adhered fragments of the lipid monolayer after exposure to ultrasound with peak negative pressures of 0.18 and 0.8 MPa. Most of the observed fragments were large enough to be resistant to receptor mediated endocytosis. The fragments were not observed to incorporate into the lipid membrane of the cell over a period of 96 min. They were not observed to break into smaller pieces or significantly change shape but they were observed to undergo translation and rotation across the cell surface as the cells migrated over the substrate. These large fragments will apparently remain on the surface of the targeted cells for significant periods of time and need to be considered for their potential effects on blood flow through the microcapillaries and potential for immune system recognition. PMID:25059435

(Z)-3,5,4'-Trimethoxystilbene Limits Hepatitis C and Cancer Pathophysiology by Blocking Microtubule Dynamics and Cell Cycle Progression

PubMed Central

Nguyen, Charles B.; Kotturi, Hari; Waris, Gulam; Mohammed, Altaf; Chandrakesan, Parthasarathy; May, Randal; Sureban, Sripathi; Weygant, Nathaniel; Qu, Dongfeng; Rao, Chinthalapally V.; Dhanasekaran, Danny N.; Bronze, Michael S.; Houchen, Courtney W.; Ali, Naushad

2016-01-01

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Chronic hepatitis C virus (HCV) infection causes induction of several tumor/cancer stem cell (CSC) markers and is known to be a major risk factor for development of HCC. Therefore, drugs that simultaneously target viral replication and CSC properties are needed for a risk-free treatment of advanced stage liver diseases including HCC. Here, we demonstrated that (Z)-3,5,4’-trimethoxystilbene (Z-TMS) exhibits potent anti-tumor and anti-HCV activities without exhibiting cytotoxicity to human hepatocytes in vitro or in mice livers. Diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) extensively induced expression of DCLK1 (a CSC marker) in the livers of C57BL/6 mice following hepatic injury. Z-TMS exhibited hepatoprotective effects against DEN/CCl4-induced injury by reducing DCLK1 expression and improving histological outcomes. The drug caused bundling of DCLK1 with microtubules and blocked cell cycle progression at G2/M phase in hepatoma cells via downregulation of CDK1, induction of p21cip1/waf1 expression, and inhibition of Akt (Ser473) phosphorylation. Z-TMS also inhibited proliferation of erlotinib-resistant lung adenocarcinoma cells (H1975) bearing the T790M EGFR mutation most likely by promoting autophagy and nuclear fragmentation. In conclusion, Z-TMS appears to be a unique therapeutic agent targeting HCV and concurrently eliminating cells with neoplastic potential during chronic liver diseases including HCC. It may also be a valuable drug for targeting drug-resistant carcinomas and cancers of the lungs, pancreas, colon, and intestine in which DCLK1 is involved in tumorigenesis. PMID:27287718

Expression of two isoforms of CD44 in human endometrium.

PubMed

Behzad, F; Seif, M W; Campbell, S; Aplin, J D

1994-10-01

The distribution of the cell-surface adhesion glycoprotein CD44 in human endometrium was examined by immunofluorescence using six monoclonal antibodies to epitopes common to all forms of the molecule, and by reverse transcription-polymerase chain reaction (RT-PCR). Immunoreactivity was observed throughout the menstrual cycle in stroma, vessels, glandular, and luminal epithelium. Variations in staining intensity were observed, especially in the epithelial compartment. CD44 was also expressed strongly by decidualized stromal cells of first-trimester pregnancy. No systematic variation of immunoreactivity was observed with stages of the normal cycle, but a fraction (25%) of the specimens lacked reactivity in the epithelium. To determine the molecular size of the epithelial isoform, an immunoprecipitation technique was developed using surface-radioiodinated, detergent-extracted glands. This indicated the presence at the cell surface of a single dominant CD44E species with an approximate molecular mass of 130 kDa. RT-PCR was used to investigate the isoforms present in whole endometrial tissue, isolated gland fragments, and Ishikawa endometrial carcinoma cells. Complementary DNA produced from total endometrial mRNA was PCR-amplified across the splice junction between exons 5 and 15. Transcripts corresponding to the hyaluronate receptor CD44H as well as a larger isoform were identified. CD44H was absent, or very scarce, in cDNA from purified gland epithelium. In contrast, Ishikawa cells expressed this form abundantly. The glands and Ishikawa cells also expressed CD44E containing sequences encoded by exons 12, 13, and 14. These data demonstrate the presence of CD44 in human endometrium and decidua, and show that different isoforms of CD44 are associated with tissue compartments in which different functional roles can be anticipated.

ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

PubMed

Abbruzzese, Genevieve; Becker, Sarah F; Kashef, Jubin; Alfandari, Dominique

2016-07-15

The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. Copyright © 2015. Published by Elsevier Inc.

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

Apoptosis Process in Mouse Leydig Cells during Postnatal Development

NASA Astrophysics Data System (ADS)

Salles Faria, Maria José; Simões, Zilá Paulino; Luz; Orive Lunardi, Laurelucia; Hartfelder, Klaus

2003-02-01

The development of Leydig cells in mammals has been widely described as a biphasic pattern with two temporally mature Leydig cell populations, fetal stage followed by the adult generation beginning at puberty. In the present study, mouse Leydig cells were examined for apoptosis during postnatal testis development using electron microscopy and in situ DNA fragmentation by terminal deoxynucleotidyl transferase staining (TdT). Both the morphological study and the DNA fragmentation analysis showed that cellular death by apoptosis did not occur in Leydig cells during the neonatal, prepubertal, puberty, and adult periods. From these results, we suggest that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes. In contrast, in the prepubertal and puberty stages, fragmentation of apoptotic DNA was detected in germ cells present in some seminiferous tubules.

Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA

PubMed Central

Chakrabortty, Sudipto K.; Prakash, Ashwin; Nechooshtan, Gal; Hearn, Stephen; Gingeras, Thomas R.

2015-01-01

Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion. PMID:26392588

ULTRASTRUCTURAL CHARACTERIZATION OF THE LYTIC CYCLE OF AN INTRANUCLEAR VIRUS INFECTING THE DIATOM CHAETOCEROS CF. WIGHAMII(BACILLARIOPHYCEAE) FROM CHESAPEAKE BAY, USA(1).

PubMed

Eissler, Yoanna; Wang, Kui; Chen, Feng; Eric Wommack, K; Wayne Coats, D

2009-08-01

Numerous microalgal species are infected by viruses that have the potential to control phytoplankton dynamics by reducing host populations, preventing bloom formation, or causing the collapse of blooms. Here we describe a virus infecting the diatom Chaetoceros cf. wighamii Brightw. from the Chesapeake Bay. To characterize the morphology and lytic cycle of this virus, we conducted a time-course experiment, sampling every 4 h over 72 h following viral inoculation. In vivo fluorescence began to decline 16 h after inoculation and was reduced to <19% of control cultures by the end of experiment. TEM confirmed infection within the first 8 h of inoculation, as indicated by the presence of virus-like particles (VLP) in the nuclei. VLP were present in two different arrangements: rod-like structures that appeared in cross-section as paracrystalline arrays of hexagonal-shaped profiles measuring 12 ± 2 nm in diameter and uniformly electron-dense hexagonal-shaped particles measuring ∼ 22-28 nm in diameter. Nuclei containing paracrystalline arrays were most prevalent early in the infection cycle, while cells containing VLP increased and then declined toward the end of the cycle. The proportion of nuclei containing both paracrystalline arrays and VLP remained relatively constant. This pattern suggests that rod-like paracrystalline arrays fragmented to produce icosahedral VLP. C. cf. wighamii nuclear inclusion virus (CwNIV) is characterized by a high burst size (averaged 26,400 viruses per infected cell) and fast generation time that could have ecological implications on C. cf. wighamii population control. © 2009 Phycological Society of America.

Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila.

PubMed

Nezis, Ioannis P; Shravage, Bhupendra V; Sagona, Antonia P; Johansen, Terje; Baehrecke, Eric H; Stenmark, Harald

2010-11-01

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.

Neurotensin may function as a regulatory peptide in small cell lung cancer.

PubMed

Davis, T P; Crowell, S; McInturff, B; Louis, R; Gillespie, T

1991-01-01

Neurotensin (NT) has been postulated to act as a modulatory agent in the central nervous system. Besides its presence in mammalian brain, NT is produced by small cell carcinoma of the lung (SCLC) and cell lines derived from these tumors. Receptors have also been characterized in some SCLC cell lines leading to the suggestion that NT could regulate the growth of SCLC in an autocrine fashion similar to bombesin/GRP. Previously, we had reported that a 10 nM dose of NT and NT(8-13), but not NT(1-8), elevated cytosolic Ca2+, indicating that SCLC NT receptors may use Ca2+ as a second messenger. Using intact SCLC cells we report that time-course incubations with NT lead to the formation of the amino-terminal fragment NT(1-8) and small amounts of the C-terminal fragment NT(9-13). These fragments are formed by metalloendopeptidase 3.4.24.15 cleaving enzyme at the Arg8-Arg9 bond of NT. Significant levels of soluble 3.4.24.15 (10-17 nmoles/mg Pr-/min) are present in SCLC cell lines. Using the in vitro clonogenic assay we tested the effect of 0.5, 5.0 and 10.0 nM doses of NT, NT(1-8) and NT(8-13) on SCLC clonal growth. NT and the C-terminal fragment NT(8-13) stimulated colony formation whereas the N-terminal fragment did not. In summary, NT may function as a regulatory peptide in SCLC through the formation of peptide fragments.

Mutational Landscape of cfDNA Identifies Distinct Molecular Features Associated With Therapeutic Response to First-Line Platinum-Based Doublet Chemotherapy in Patients with Advanced NSCLC

PubMed Central

Jiang, Tao; Li, Xuefei; Wang, Jianfei; Su, Chunxia; Han, Wenbo; Zhao, Chao; Wu, Fengying; Gao, Guanghui; Li, Wei; Chen, Xiaoxia; Li, Jiayu; Zhou, Fei; Zhao, Jing; Cai, Weijing; Zhang, Henghui; Du, Bo; Zhang, Jun; Ren, Shengxiang; Zhou, Caicun; Yu, Hui; Hirsch, Fred R.

2017-01-01

Rationale To investigate whether the mutational landscape of circulating cell-free DNA (cfDNA) could predict and dynamically monitor the response to first-line platinum-based chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC). Methods Eligible patients were included and blood samples were collected from a phase III trial. Both cfDNA fragments and fragmented genomic DNA were extracted for enrichment in a 1.15M size panel covering exon regions of 1,086 genes. Molecular mutational burden (MMB) was calculated to investigate the relationship between molecular features of cfDNA and response to chemotherapy. Results In total, 52 eligible cases were enrolled and their blood samples were prospectively collected at baseline, every cycle of chemotherapy and time of disease progression. At baseline, alterations of 17 genes were found. Patients with partial response (PR) had significantly lower baseline MMB of these genes than those patients with either stable disease (SD) (P = 0.0006) or progression disease (PD) (P = 0.0074). Further analysis revealed that the mutational landscape of cfDNA from pretreatment blood samples were distinctly different among patients with PR vs. SD/PD. For patients with baseline TP53 mutation, those with PR experienced a significant reduction in MMB whereas patients with SD or PD experienced an increase after two, three or four cycles of chemotherapy. Furthermore, patients with low MMB had superior response rate and significantly longer progression-free survival than those with high MMB. Conclusion This study indicated that the mutational landscape of cfDNA has potential clinical value to predict the therapeutic response to first-line platinum-based doublet chemotherapy in NSCLC patients. At the single gene level, dynamic change of molecular mutational burden of TP53 is valuable to monitor efficacy (and, therefore, might aid in early recognition of resistance and relapse) in patients harboring this mutation at baseline. PMID:29187901

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

PubMed

Fiscus, R R; Leung, C P; Yuen, J P; Chan, H C

2001-01-01

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines. Copyright 2001 Academic Press.

CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia

PubMed Central

Díaz-Chico, Juan Carlos; McNaughton-Smith, Grant; Jiménez-Alonso, Sandra; Hueso-Falcón, Idaira; Montero, Juan Carlos; Blanco, Raquel; León, Javier; Rodríguez-González, Germán; Estévez-Braun, Ana; Pandiella, Atanasio; Díaz-Chico, Bonifacio Nicolás; Fernández-Pérez, Leandro

2017-01-01

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies. PMID:27557509

CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia.

PubMed

Guerra, Borja; Martín-Rodríguez, Patricia; Díaz-Chico, Juan Carlos; McNaughton-Smith, Grant; Jiménez-Alonso, Sandra; Hueso-Falcón, Idaira; Montero, Juan Carlos; Blanco, Raquel; León, Javier; Rodríguez-González, Germán; Estévez-Braun, Ana; Pandiella, Atanasio; Díaz-Chico, Bonifacio Nicolás; Fernández-Pérez, Leandro

2017-05-02

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment.

PubMed

Banerjee, Aditi; Ahmed, Hafiz; Yang, Peixin; Czinn, Steven J; Blanchard, Thomas G

2016-07-05

The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

Inhibition of Mdm2 Sensitizes Human Retinal Pigment Epithelial Cells to Apoptosis

PubMed Central

Ray, Ramesh M.; Chaum, Edward; Johnson, Dianna A.; Johnson, Leonard R.

2011-01-01

Purpose. Because recent studies indicate that blocking the interaction between p53 and Mdm2 results in the nongenotoxic activation of p53, the authors sought to investigate whether the inhibition of p53-Mdm2 binding activates p53 and sensitizes human retinal epithelial cells to apoptosis. Methods. Apoptosis was evaluated by the activation of caspases and DNA fragmentation assays. The Mdm2 antagonist Nutlin-3 was used to dissociate p53 from Mdm2 and, thus, to increase p53 activity. Knockdown of p53 expression was accomplished by using p53 siRNA. Results. ARPE-19 and primary RPE cells expressed high levels of the antiapoptotic proteins Bcl-2 and Bcl-xL. Exposure of these cells to camptothecin (CPT) or TNF-α/ cycloheximide (CHX) failed to induce apoptosis. In contrast, treatment with the Mdm2 antagonist Nutlin-3 in the absence of CPT or TNF-α/CHX increased apoptosis. Activation of p53 in response to Nutlin-3 also increased levels of Noxa, p53-upregulated modulator of apoptosis (PUMA), and Siva-1, decreased expression of Bcl-2 and Bcl-xL, and simultaneously increased caspases-9 and -3 activities and DNA fragmentation. Knockdown of p53 decreased the basal expression of p21Cip1 and Bcl-2, inhibited the Nutlin-3–induced upregulation of Siva-1 and PUMA expression, and consequently inhibited caspase-3 activation. Conclusions. These results indicate that the normally available pool of intracellular p53 is predominantly engaged in the regulation of cell cycle checkpoints by p21Cip1 and does not trigger apoptosis in response to DNA-damaging agents. However, the blockage of p53 binding to Mdm2 frees a pool of p53 that is sufficient, even in the absence of DNA-damaging agents, to increase the expression of proapoptotic targets and to override the resistance of RPE cells to apoptosis. PMID:21345989

Antitumor effect and toxicity of free rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles in mice bearing breast cancer.

PubMed

Carneiro, Marcella Lemos Brettas; Peixoto, Raphael C A; Joanitti, Graziela A; Oliveira, Ricardo G S; Telles, Luis A M; Miranda-Vilela, Ana L; Bocca, Anamélia L; Vianna, Leonora M S; da Silva, Izabel C R; de Souza, Aparecido R; Lacava, Zulmira G M; Báo, Sônia N

2013-02-16

Magnetic fluids containing superparamagnetic iron oxide nanoparticles represent an attractive platform as nanocarriers in chemotherapy. Recently, we developed a formulation of maghemite nanoparticles coated with rhodium (II) citrate, which resulted in in vitro cytotoxicity enhanced up to 4.6 times when compared to free rhodium (II) citrate formulation on breast carcinoma cells. In this work, we evaluate the antitumor activity and toxicity induced by these formulations in Balb/c mice bearing orthotopic 4T1 breast carcinoma. Mice were evaluated with regard to the treatments' toxicity through analyses of hemogram, serum levels of alanine aminotransferase, iron, and creatinine; DNA fragmentation and cell cycle of bone marrow cells; and liver, kidney and lung histology. In addition, the antitumor activity of rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate was verified by tumor volume reduction, histology and immunohistochemistry. Regarding the treatments' toxicity, no experimental groups had alterations in levels of serum ALT or creatinine, and this suggestion was corroborated by the histopathologic examination of liver and kidney of mice. Moreover, DNA fragmentation frequency of bone marrow cells was lower than 15% in all experimental groups. On the other hand, the complexes rhodium (II) citrate-functionalized maghemite and free rhodium (II) citrate led to a marked growth inhibition of tumor and decrease in CD31 and Ki-67 staining. In summary, we demonstrated that both rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate formulations exhibited antitumor effects against 4T1 metastatic breast cancer cell line following intratumoral administration. This antitumor effect was followed by inhibition of both cell proliferation and microvascularization and by tumor tissue injury characterized as necrosis and fibrosis. Remarkably, this is the first published report demonstrating the therapeutic efficacy of maghemite nanoparticles coated with rhodium (II) citrate. This treatment prolonged the survival period of treated mice without inducing apparent systemic toxicity, which strengthens its use for future breast cancer therapeutic applications.

Degradation Mechanisms of Electrochemically Cycled Graphite Anodes in Lithium-ion Cells

NASA Astrophysics Data System (ADS)

Bhattacharya, Sandeep

This research is aimed at developing advanced characterization methods for studying the surface and subsurface damage in Li-ion battery anodes made of polycrystalline graphite and identifying the degradation mechanisms that cause loss of electrochemical capacity. Understanding microstructural aspects of the graphite electrode degradation mechanisms during charging and discharging of Li-ion batteries is of key importance in order to design durable anodes with high capacity. An in-situ system was constructed using an electrochemical cell with an observation window, a large depth-of-field digital microscope and a micro-Raman spectrometer. It was revealed that electrode damage by removal of the surface graphite fragments of 5-10 mum size is the most intense during the first cycle that led to a drastic capacity drop. Once a solid electrolyte interphase (SEI) layer covered the electrode surface, the rate of graphite particle loss decreased. Yet, a gradual loss of capacity continued by the formation of interlayer cracks adjacent to SEI/graphite interfaces. Deposition of co-intercalation compounds, LiC6, Li2CO3 and Li2O, near the crack tips caused partial closure of propagating graphite cracks during cycling and reduced the crack growth rate. Bridging of crack faces by delaminated graphite layers also retarded crack propagation. The microstructure of the SEI layer, formed by electrochemical reduction of the ethylene carbonate based electrolyte, consisted of ˜5-20 nm sized crystalline domains (containing Li2CO3, Li2O 2 and nano-sized graphite fragments) dispersed in an amorphous matrix. During the SEI formation, two regimes of Li-ion diffusion were identified at the electrode/electrolyte interface depending on the applied voltage scan rate (dV/dt). A low Li-ion diffusion coefficient ( DLi+) at dV/dt < 0.05 mVs-1 produced a tubular SEI that uniformly covered the graphite surface and prevented damage at 25°C. At 60°C, a high D Li+ formed a Li2CO3-enriched SEI and ensued a 28% increase in the battery capacity at 25°C. On correlating the microscopic information to the electrochemical performance, novel Li2CO3-coated electrodes were fabricated that were durable. The SEI formed on pre-treated electrodes reduced the strain in the graphite lattice from 0.4% (for uncoated electrodes) to 0.1%, facilitated Li-ion diffusion and hence improved the capacity retention of Li-ion batteries during long-term cycling.

ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site

PubMed Central

Kashef, Jubin; Alfandari, Dominique

2015-01-01

The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell–cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. PMID:26206614

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

PubMed Central

Zavašnik-Bergant, Tina; Bergant Marušič, Martina

2016-01-01

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii. PMID:26960148

Effect of cyclic treatment on the formation of a fragmented structure in a sparingly alloyed martensitic steel

NASA Astrophysics Data System (ADS)

Tabatchikova, T. I.; Yakovleva, I. L.; Delgado Reina, S. Yu.; Plokhikh, A. I.

2016-11-01

Methods of metallography and transmission electron microscopy were used to study the structure of a high-alloy low-carbon steel of martensitic VKS-10 class subjected to cyclic treatment according to different regimes. It has been found that the warm deformation in the α state at 700°C causes the fragmentation of the structure; however, the decomposition of the α solid solution and the precipitation of coarse carbides leads to a significant decrease in the strength. It has been shown that 12 cycles of treatment, including austenitizing at 1000°C, rolling at 700°C, and subsequent γ → α transformation during rapid cooling do not lead to a noticeable fragmentation of the structure. It has been found that the deformation of the overcooled austenite by rolling carried out using 12 cycles in the range of temperatures of 700-500°C and subsequent γ → α transformation lead to the formation of a fragmented structure with a large fraction of fine grains with a size less than 0.5 μm. This treatment and the subsequent tempering at 530°C for 1 h allow us to increase the strength and hardness of the VKS-10 steel at an insignificant decrease in the plasticity.

The anti-human immunodeficiency virus agent 3'-fluorothymidine induces DNA damage and apoptosis in human lymphoblastoid cells.

PubMed Central

Sundseth, R; Joyner, S S; Moore, J T; Dornsife, R E; Dev, I K

1996-01-01

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo. PMID:8834875

Production of neutron-rich nuclei approaching r-process by gamma-induced fission of 238U at ELI-NP

NASA Astrophysics Data System (ADS)

Mei, Bo; Balabanski, Dimiter; Constantin, Paul; Anh Le, Tuan; Viet Cuong, Phan

2018-05-01

The investigation of neutron-rich exotic nuclei is crucial not only for nuclear physics but also for nuclear astrophysics. Experimentally, only few neutron-rich nuclei near the stability have been studied, however, most neutron-rich nuclei have not been measured due to their small production cross sections as well as short half-lives. At ELI-NP, gamma beams with high intensities will open new opportunities to investigate very neutron-rich fragments produced by photofission of 238U targets in a gas cell. Based on some simulations, a novel gas cell has been designed to produce, stop and extract 238U photofission fragments. The extraction time and efficiency of photofission fragments have been optimized by using SIMION simulations. According to these simulations, a high extraction efficiency and a short extraction time can be achieved for 238U photofission fragments in the gas cell, which will allow one to measure very neutron-rich fragments with short half-lives by using the IGISOL facility proposed at ELI-NP.

DLP1-Dependent Mitochondrial Fragmentation Mediates 1-methyl-4-phenylpyridinium Toxicity in Neurons: Implications for Parkinson's Disease

PubMed Central

Wang, Xinglong; Su, Bo; Liu, Wanhong; He, Xiaohua; Gao, Yuan; Castellani, Rudy J.; Perry, George; Smith, Mark A.; Zhu, Xiongwei

2011-01-01

SUMMARY Selective degeneration of nigrostriatal dopaminergic neurons in Parkinson disease (PD) can be modeled by the administration of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Since abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this study, we investigated the effect of MPP+ on mitochondrial dynamics and assessed temporal and causal relationship with other toxic effects induced by MPP+ in neuronal cells. In SH-SY5Y cells, MPP+ causes a rapid increase in mitochondrial fragmentation followed by a second wave of increase in mitochondrial fragmentation, along with increased DLP1 expression and mitochondrial translocation. Genetic inactivation of DLP1 completely blocks MPP+-induced mitochondrial fragmentation. Notably, this approach partially rescues MPP+-induced decline in ATP levels and ATP/ADP ratio and increased [Ca2+]i and almost completely prevents increased reactive oxygen species production, loss of mitochondrial membrane potential, enhanced autophagy and cell death, suggesting that mitochondria fragmentation is an upstream event that mediates MPP+-induced toxicity. On the other hand, thiol antioxidant NAC or glutamate receptor antagonist D-AP5 also partially alleviate MPP+-induced mitochondrial fragmentation, suggesting a vicious spiral of events contributes to MPP+-induced toxicity. We further validated our findings in primary rat midbrain dopaminergic neurons that 0.5 μM MPP+ induced mitochondrial fragmentation only in TH-positive dopaminergic neurons in a similar pattern to that in SH-SY5Y cells but had no effects on these mitochondrial parameters in TH-negative neurons. Overall, these findings suggest that DLP1-dependent mitochondrial fragmentation plays a crucial role in mediating MPP+-induced mitochondria abnormalities and cellular dysfunction and may represent a novel therapeutic target for PD. PMID:21615675

Telomere Restriction Fragment (TRF) Analysis.

PubMed

Mender, Ilgen; Shay, Jerry W

2015-11-20

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al. , 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al. , 1990; Ouellette et al. , 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.

PubMed

Halicka, H Dorota; Garcia, Jorge; Li, Jiangwei; Zhao, Hong; Darzynkiewicz, Zbigniew

2017-02-01

Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.

Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.

PubMed

Ma, Cui; Beyer, Andreas M; Durand, Matthew; Clough, Anne V; Zhu, Daling; Norwood Toro, Laura; Terashvili, Maia; Ebben, Johnathan D; Hill, R Blake; Audi, Said H; Medhora, Meetha; Jacobs, Elizabeth R

2018-03-01

We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction. © 2018 American Heart Association, Inc.

Biological effectiveness of nuclear fragments produced by high-energy protons interacting in tissues near the bone- soft tissue interface

NASA Astrophysics Data System (ADS)

Shavers, Mark Randall

1999-12-01

High-energy protons in the galactic cosmic rays (GCR)-or generated by nuclear interactions of GCR heavy-ions with material-are capable of penetrating great thicknesses of shielding to irradiate humans in spacecraft or in lunar or Martian habitats. As protons interact with the nuclei of the elemental constituents of soft tissue and bone, low energy nuclei-target fragments-are emitted into the cells responsible for bone development and maintenance and for hematopoiesis. Leukemogenesis is the principal endpoint of concern because it is the most likely deleterious effect, and it has a short latency period and comparatively low survival rate, although other myelo- proliferative disorders and osteosarcoma also may be induced. A one-dimensional proton-target fragment transport model was used to calculate the energy spectra of fragments produced in bone and soft tissue, and present in marrow cavities at distances from a bone interface. In terms of dose equivalent, the target fragments are as significant as the incident protons. An average radiation quality factor was found to be between 1.8 and 2.6. Biological response to the highly non- uniform energy deposition of the target fragments is such that an alternative approach to conventional predictive risk assessment is needed. Alternative procedures are presented. In vitro cell response and relative biological effectiveness were calculated from the radial dose distribution of each fragment produced by 1-GeV protons using parameters of a modified Ion-Gamma- Kill (IGK) model of radiation action. The modelled endpoints were survival of C3H10t 1/2 and V79 cells, neoplastic transformation of C3H10t1/2 cells, and mutation of the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in V79 cells. The dose equivalent and cell responses increased by 10% or less near the interface. Since RBE increases with decreasing dose in the IGK model, comparisons with quality factors were made at dose levels 0.01 <= D [Gy] <= 2. Applying average quality factors derived herein to GCR exposures results in a <= 5% increase of in average quality. Calculated RBEs indicate that accepted quality factors for high-energy protons may be too low due to the relatively high effectiveness of the low-charged target fragments. Derived RBEs for target fragments increase the calculated biological effectiveness of GCR by 20% to 180%.

Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells.

PubMed

Yang, Jun; Adamian, Michael; Li, Tiansen

2006-02-01

Rootletin, a major structural component of the ciliary rootlet, is located at the basal bodies and centrosomes in ciliated and nonciliated cells, respectively. Here we investigated its potential role in the linkage of basal bodies/centrioles and the mechanism involved in such linkages. We show that rootletin interacts with C-Nap1, a protein restricted at the ends of centrioles and functioning in centrosome cohesion in interphase cells. Their interaction in vivo is supported by their colocalization at the basal bodies/centrioles and coordinated association with the centrioles during the cell cycle. Ultrastructural examinations demonstrate that rootletin fibers connect the basal bodies in ciliated cells and are present both at the ends of and in between the pair of centrioles in nonciliated cells. The latter finding stands in contrast with C-Nap1, which is present only at the ends of the centrioles. Transient expression of C-Nap1 fragments dissociated rootletin fibers from the centrioles, resulting in centrosome separation in interphase. Overexpression of rootletin in cells caused multinucleation, micronucleation, and irregularity of nuclear shape and size, indicative of defects in chromosome separation. These data suggest that rootletin may function as a physical linker between the pair of basal bodies/centrioles by binding to C-Nap1.

Deletion of Pofut1 in Mouse Skeletal Myofibers Induces Muscle Aging-Related Phenotypes in cis and in trans

PubMed Central

Zygmunt, Deborah A.; Singhal, Neha; Kim, Mi-Lyang; Cramer, Megan L.; Crowe, Kelly E.; Xu, Rui; Jia, Ying; Adair, Jessica; Martinez-Pena y Valenzuela, Isabel; Akaaboune, Mohammed; White, Peter; Janssen, Paulus M.

2017-01-01

ABSTRACT Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on α dystroglycan glycosylation. PMID:28265002

Is it the Earth that turns or the Sun that goes behind the mountains? Students' misconceptions about the day/night cycle after reading a science text

NASA Astrophysics Data System (ADS)

Vosniadou, Stella; Skopeliti, Irini

2017-10-01

The present research tested the hypothesis that the reading of science text can create new misconceptions in students with incongruent prior knowledge, and that these new misconceptions will be similar to the fragmented and synthetic conceptions obtained in prior developmental research. Ninety-nine third- and fifth-grade children read and recalled one of two texts that provided scientific or phenomenal explanations of the day/night cycle. All the participants gave explanations of the phenomenon in question prior to reading one of the texts and after they read it. The results showed that the participants who provided explanations of the day/night cycle at pretest incongruent with the scientific explanation recalled less information and generated more invalid inferences. An analysis of the participants' posttest explanations indicated that these readers formed new misconceptions similar to the fragmented and synthetic conceptions obtained in developmental research. The implications of the above for text comprehension and science education research are discussed.

Long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele-specific MYC expression in HeLa cells.

PubMed

Shen, Congle; Liu, Yongzhen; Shi, Shu; Zhang, Ruiyang; Zhang, Ting; Xu, Qiang; Zhu, Pengfei; Chen, Xiangmei; Lu, Fengmin

2017-08-01

Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis-activate the expression of proto-oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV-integrated haplotype, and a long-range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long-range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence-associated acidic β-gal activity in HeLa cells. These data indicate a long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele‐specific MYC expression in HeLa cells

PubMed Central

Shen, Congle; Liu, Yongzhen; Shi, Shu; Zhang, Ruiyang; Zhang, Ting; Xu, Qiang; Zhu, Pengfei; Lu, Fengmin

2017-01-01

Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis‐activate the expression of proto‐oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV‐integrated haplotype, and a long‐range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long‐range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence‐associated acidic β‐gal activity in HeLa cells. These data indicate a long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration. PMID:28470669

1-(Benzenesulfonyl)-1,5-dihydro-4,1-benzoxazepine as a new scaffold for the design of antitumor compounds.

PubMed

Cruz-López, Olga; Ramírez, Alberto; Navarro, Saúl A; García, María A; Marchal, Juan A; Campos, Joaquín M; Conejo-García, Ana

2017-07-01

Bozepinib is a potent and selective anticancer compound which chemical structure is made up of a benzofused seven-membered ring and a purine moiety. We previously demonstrated that the purine fragment does not exert antiproliferative effect per se. A series of 1-(benzenesulfonyl)-4,1-benzoxazepine derivatives were synthesized in order to study the influence of the benzofused seven-membered ring in the biological activity of bozepinib by means of antiproliferative, cell cycle and apoptosis studies. Our results show that the methyleneoxy enamine sulfonyl function is essential in the antitumor activity of the structures and thus, it is a scaffold suitable for further modification with a view to obtain more potent antitumor compounds.

Ethyl acetate fraction of Garcina epunctata induces apoptosis in human promyelocytic cells (HL-60) through the ROS generation and G0/G1 cell cycle arrest: a bioassay-guided approach.

PubMed

Constant Anatole, Pieme; Guru, Santoh Kumar; Bathelemy, Ngamegni; Jeanne, Ngogang; Bhushan, Shashi; Murayama, Tetsuya; Saxena, Ajit Kumar

2013-11-01

Number of deaths due to cancer diseases is increasing in the world. There is an urgent need to develop alternative therapeutic measures against the disease. Our study reports the cytotoxicity activity of Garcina epunctata (gutifferae) in human promyelocytic leukemia cells (HL-60) and prostate cancer cells (PC-3) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and morphological changes associated with apoptosis were examined by flow cytometry and Hoescht staining respectively. The results of in vitro antiproliferative screening of fractions and extract from G. epunctata indicated that three fractions inhibited the viability of PC-3 cells with IC₅₀ varied from 50 to 88 μ/ml while two fractions inhibited the proliferation of HL-60 cells with IC₅₀ range between 47.5 and 12 μg/ml. Among the entire fraction tested, Hex-EtOAc (75:25) showed cytotoxic effects on the two cell lines and EtOAc fraction was most active only HL-60 cells (12 μg/ml). Treatment of HL-60 cells with G. epunctata (20, 50, 100 μg/ml) for 24 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a number of apoptotic bodies containing nuclear fragments were observed in cells treated with 100 μg/ml. The EtOAc fraction of G. epunctata treatment significantly arrested HL-60 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated as well as the loss of membrane mitochondrial potential in a concentration dependant manner. The results demonstrated that the EtOAc fraction of G. epunctata inhibited the proliferation of HL-60 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

Self-polarization and directional motility of cytoplasm.

PubMed

Verkhovsky, A B; Svitkina, T M; Borisy, G G

1999-01-14

Directional cell motility implies the presence of a steering mechanism and a functional asymmetry between the front and rear of the cell. How this functional asymmetry arises and is maintained during cell locomotion is, however, unclear. Lamellar fragments of fish epidermal keratocytes, which lack nuclei, microtubules and most organelles, present a simplified, perhaps minimal, system for analyzing this problem because they consist of little other than the motile machinery enclosed by a membrane and yet can move with remarkable speed and persistence. We have produced two types of cellular fragments: discoid stationary fragments and polarized fragments undergoing locomotion. The organization and dynamics of the actin-myosin II system were isotropic in stationary fragments and anisotropic in the moving fragments. To investigate whether the creation of asymmetry could result in locomotion, a transient mechanical stimulus was applied to stationary fragments. The stimulus induced localized contraction and the formation of an actin-myosin II bundle at one edge of the fragment. Remarkably, stimulated fragments started to undergo locomotion and the locomotion and associated anisotropic organization of the actin-myosin II system were sustained after withdrawal of the stimulus. We propose a model in which lamellar cytoplasm is considered a dynamically bistable system capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive-feedback loop intrinsic to the actin-myosin II machinery: anisotropic organization of the machinery drives translocation, which then reinforces the asymmetry of the machinery, favoring further translocation.

Titer-plate formatted continuous flow thermal reactors: Design and performance of a nanoliter reactor

PubMed Central

Chen, Pin-Chuan; Park, Daniel S.; You, Byoung-Hee; Kim, Namwon; Park, Taehyun; Soper, Steven A.; Nikitopoulos, Dimitris E.; Murphy, Michael C.

2010-01-01

Arrays of continuous flow thermal reactors were designed, configured, and fabricated in a 96-device (12 × 8) titer-plate format with overall dimensions of 120 mm × 96 mm, with each reactor confined to a 8 mm × 8 mm footprint. To demonstrate the potential, individual 20-cycle (740 nL) and 25-cycle (990 nL) reactors were used to perform the continuous flow polymerase chain reaction (CFPCR) for amplification of DNA fragments of different lengths. Since thermal isolation of the required temperature zones was essential for optimal biochemical reactions, three finite element models, executed with ANSYS (v. 11.0, Canonsburg, PA), were used to characterize the thermal performance and guide system design: (1) a single device to determine the dimensions of the thermal management structures; (2) a single CFPCR device within an 8 mm × 8 mm area to evaluate the integrity of the thermostatic zones; and (3) a single, straight microchannel representing a single loop of the spiral CFPCR device, accounting for all of the heat transfer modes, to determine whether the PCR cocktail was exposed to the proper temperature cycling. In prior work on larger footprint devices, simple grooves between temperature zones provided sufficient thermal resistance between zones. For the small footprint reactor array, 0.4 mm wide and 1.2 mm high fins were necessary within the groove to cool the PCR cocktail efficiently, with a temperature gradient of 15.8°C/mm, as it flowed from the denaturation zone to the renaturation zone. With temperature tolerance bands of ±2°C defined about the nominal temperatures, more than 72.5% of the microchannel length was located within the desired temperature bands. The residence time of the PCR cocktail in each temperature zone decreased and the transition times between zones increased at higher PCR cocktail flow velocities, leading to less time for the amplification reactions. Experiments demonstrated the performance of the CFPCR devices as a function of flow velocity, fragment length, and copy number. A 99 bp DNA fragment was successfully amplified at flow velocities from 1 mm/s to 3 mm/s, requiring from 8.16 minutes for 20 cycles (24.48 s/cycle) to 2.72 minutes for 20 cycles (8.16 s/cycle), respectively. Yield compared to the same amplification sequence performed using a bench top thermal cycler decreased nonlinearly from 73% (at 1 mm/s) to 13% (at 3 mm/s) with shorter residence time at the optimal temperatures for the reactions due to increased flow rate primarily responsible. Six different DNA fragments with lengths between 99 bp and 997 bp were successfully amplified at 1 mm/s. Repeatable, successful amplification of a 99 bp fragment was achieved with a minimum of 8000 copies of the DNA template. This is the first demonstration and characterization of continuous flow thermal reactors within the 8 mm × 8 mm footprint of a 96-well micro-titer plate and is the smallest continuous flow PCR to date. PMID:20871807

Revealing the micromechanics driving cellular division: optical manipulation of force-bearing substructure in mitotic cells

NASA Astrophysics Data System (ADS)

Ono, Matthew; Preece, Daryl; Duquette, Michelle; Forer, Arthur; Berns, Michael

2017-08-01

During the anaphase stage of mitosis, a motility force transports genetic material in the form of chromosomes to the poles of the cell. Chromosome deformations during anaphase transport have largely been attributed to viscous drag force, however LaFountain et. al. found that a physical tether connects separating chromosome ends in crane-fly spermatocytes such that a backwards tethering force elongates the separating chromosomes. In the presented study laser microsurgery was used to deduce the mechanistic basis of chromosome elongation in rat-kangaroo cells. In half of tested chromosome pairs, laser microsurgery between separating chromosome ends reduced elongation by 7+/-3% suggesting a source of chromosome strain independent of viscous drag. When microsurgery was used to sever chromosomes during transport, kinetochore attached fragments continued poleward travel while half of end fragments traveled towards the opposite pole and the remaining fragments either did not move or segregated to the proper pole. Microsurgery directed between chromosome ends always ceased cross-polar fragment travel suggesting the laser severed a physical tether transferring force to the fragment. Optical trapping of fragments moving towards the opposite pole estimates an upper boundary on the tethering force of 1.5 pN.

Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method.

PubMed

Ding, Wenwen; Weng, Huanjiao; Jin, Peng; Du, Guocheng; Chen, Jian; Kang, Zhen

2017-05-04

Efficient assembly of multiple DNA fragments is a pivotal technology for synthetic biology. A scarless and sequence-independent DNA assembly method (DATEL) using thermal exonucleases has been developed recently. Here, we present a simplified DATEL (sDATEL) for efficient assembly of unphosphorylated DNA fragments with low cost. The sDATEL method is only dependent on Taq DNA polymerase and Taq DNA ligase. After optimizing the committed parameters of the reaction system such as pH and the concentration of Mg 2+ and NAD+, the assembly efficiency was increased by 32-fold. To further improve the assembly capacity, the number of thermal cycles was optimized, resulting in successful assembly 4 unphosphorylated DNA fragments with an accuracy of 75%. sDATEL could be a desirable method for routine manual and automated assembly.

Bard Denali inferior vena cava filter fracture and embolization resulting in cardiac tamponade: a device failure analysis.

PubMed

Kuo, William T; Robertson, Scott W

2015-01-01

A 46-year-old woman underwent inferior vena cava filter placement before bariatric surgery and returned within 6 months for routine removal. She complained of a 1-week history of severe chest pain, and during retrieval, two fractured filter components were identified including one arm in the right ventricle. The filter body and one fragment were successfully retrieved, but the fragment in the right ventricle was refractory to percutaneous retrieval. During open-heart surgery, the fragment was found traversing through the ventricular wall resulting in cardiac tamponade. Electron microscopic fragment analysis revealed high-cycle metal fatigue indicating the filter design failed to withstand this patient's natural inferior vena cava biomechanical motions. Copyright © 2015 SIR. Published by Elsevier Inc. All rights reserved.

Fission fragment charge and mass distributions in 239Pu(n, f ) in the adiabatic nuclear energy density functional theory

DOE PAGES

Regnier, D.; Dubray, N.; Schunck, N.; ...

2016-05-13

Here, accurate knowledge of fission fragment yields is an essential ingredient of numerous applications ranging from the formation of elements in the r process to fuel cycle optimization for nuclear energy. The need for a predictive theory applicable where no data are available, together with the variety of potential applications, is an incentive to develop a fully microscopic approach to fission dynamics.

Factors Affecting Soil Fauna Feeding Activity in a Fragmented Lowland Temperate Deciduous Woodland

PubMed Central

Simpson, Jake E.; Slade, Eleanor; Riutta, Terhi; Taylor, Michele E.

2012-01-01

British temperate broadleaf woodlands have been widely fragmented since the advent of modern agriculture and development. As a result, a higher proportion of woodland area is now subject to edge effects which can alter the efficiency of ecosystem functions. These areas are particularly sensitive to drought. Decomposition of detritus and nutrient cycling are driven by soil microbe and fauna coactivity. The bait lamina assay was used to assess soil fauna trophic activity in the upper soil horizons at five sites in Wytham Woods, Oxfordshire: two edge, two intermediate and one core site. Faunal trophic activity was highest in the core of the woodland, and lowest at the edge, which was correlated with a decreasing soil moisture gradient. The efficiency of the assay was tested using four different bait flavours: standardised, ash (Fraxinus excelsior L.), oak (Quercus robur L.), and sycamore (Acer pseudoplatanus L.). The standardised bait proved the most efficient flavour in terms of feeding activity. This study suggests that decomposition and nutrient cycling may be compromised in many of the UK's small, fragmented woodlands in the event of drought or climate change. PMID:22235311

Factors affecting soil fauna feeding activity in a fragmented lowland temperate deciduous woodland.

PubMed

Simpson, Jake E; Slade, Eleanor; Riutta, Terhi; Taylor, Michele E

2012-01-01

British temperate broadleaf woodlands have been widely fragmented since the advent of modern agriculture and development. As a result, a higher proportion of woodland area is now subject to edge effects which can alter the efficiency of ecosystem functions. These areas are particularly sensitive to drought. Decomposition of detritus and nutrient cycling are driven by soil microbe and fauna coactivity. The bait lamina assay was used to assess soil fauna trophic activity in the upper soil horizons at five sites in Wytham Woods, Oxfordshire: two edge, two intermediate and one core site. Faunal trophic activity was highest in the core of the woodland, and lowest at the edge, which was correlated with a decreasing soil moisture gradient. The efficiency of the assay was tested using four different bait flavours: standardised, ash (Fraxinus excelsior L.), oak (Quercus robur L.), and sycamore (Acer pseudoplatanus L.). The standardised bait proved the most efficient flavour in terms of feeding activity. This study suggests that decomposition and nutrient cycling may be compromised in many of the UK's small, fragmented woodlands in the event of drought or climate change.

Binding of Soluble Natural Ligands to a Soluble Human T-Cell Receptor Fragment Produced in Escherichia coli

NASA Astrophysics Data System (ADS)

Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.

1994-09-01

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

PubMed

Jorgez, Carolina J; Bischoff, Farideh Z

2009-01-01

Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells

PubMed Central

2011-01-01

Background Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells. Methods Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 oC for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation. Results More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 oC hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 oC was more potent than the essential oil prepared at 78 oC in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression. Conclusions Similar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer. PMID:22171782

Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

PubMed Central

Li, Xiang; Li, Xiang; Wang, Jiaxiong; Ye, Zaiyuan; Li, Ji-Cheng

2012-01-01

Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer. PMID:22745580

Targeting DNA repair with PNKP inhibition sensitizes radioresistant prostate cancer cells to high LET radiation

PubMed Central

Srivastava, Pallavi; Sarma, Asitikantha

2018-01-01

High linear energy transfer (LET) radiation or heavy ion such as carbon ion radiation is used as a method for advanced radiotherapy in the treatment of cancer. It has many advantages over the conventional photon based radiotherapy using Co-60 gamma or high energy X-rays from a Linear Accelerator. However, charged particle therapy is very costly. One way to reduce the cost as well as irradiation effects on normal cells is to reduce the dose of radiation by enhancing the radiation sensitivity through the use of a radiomodulator. PNKP (polynucleotide kinase/phosphatase) is an enzyme which plays important role in the non-homologous end joining (NHEJ) DNA repair pathway. It is expected that inhibition of PNKP activity may enhance the efficacy of the charged particle irradiation in the radioresistant prostate cancer cell line PC-3. To test this hypothesis, we investigated cellular radiosensitivity by clonogenic cell survival assay in PC-3 cells.12Carbon ion beam of62 MeVenergy (equivalent 5.16 MeV/nucleon) and with an entrance LET of 287 kev/μm was used for the present study. Apoptotic parameters such as nuclear fragmentation and caspase-3 activity were measured by DAPI staining, nuclear ladder assay and colorimetric caspase-3method. Cell cycle arrest was determined by FACS analysis. Cell death was enhanced when carbon ion irradiation is combined with PNKPi (PNKP inhibitor) to treat cells as compared to that seen for PNKPi untreated cells. A low concentration (10μM) of PNKPi effectively radiosensitized the PC-3 cells in terms of reduction of dose in achieving the same survival fraction. PC-3 cells underwent significant apoptosis and cell cycle arrest too was enhanced at G2/M phase when carbon ion irradiation was combined with PNKPi treatment. Our findings suggest that combined treatment of carbon ion irradiation and PNKP inhibition could enhance cellular radiosensitivity in a radioresistant prostate cancer cell line PC-3. The synergistic effect of PNKPi and carbon ion irradiation could be used as a promising method for carbon-ion therapy in radioresistant cells. PMID:29320576

Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

PubMed

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

PubMed Central

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania.

PubMed

Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

2012-12-01

Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved.

Vitamin K3 analogs induce selective tumor cytotoxicity in neuroblastoma.

PubMed

Kitano, Toru; Yoda, Hiroyuki; Tabata, Keiichi; Miura, Motofumi; Toriyama, Masaharu; Motohashi, Shigeyasu; Suzuki, Takashi

2012-01-01

We investigated the cytotoxicity of eight vitamin K3 (VK3) analogs against neuroblastoma cell lines (IMR-32, LA-N-1, NB-39, and SK-N-SH) and normal cell lines (human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF)) using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. 2-[(2-Methoxy)ethylthio]-3-methyl-1,4-naphthoquinone (VK3-OCH(3)) showed especially potent cytotoxic activities against neuroblastoma cells compared with normal cells. In a Hoechst 33342 staining experiment, apoptotic morphologies characterized by cell shrinkage, nuclear condensation, and nuclear fragmentation were observed in IMR-32 and LA-N-1 cells after 48 h of treatment with 10(-5) M of VK3-OCH(3). To clarify the molecular mechanisms of apoptosis induced by VK3-OCH(3), we examined the expression of apoptosis related proteins using a Proteome Profiler Array and western blotting. Heme oxygenase (HO)-1 was remarkably increased by VK3-OCH(3) compared with the control (173% in IMR-32 and 170% in LA-N-1 at 24 h). Moreover, caveolin-1 was induced by VK3-OCH(3) at 48 h. In addition, VK3-OCH(3) arrested the cell cycle at the G2/M phase in IMR-32 cells. These results suggest that VK3-OCH(3) exhibited a selective antitumor activity via HO-1-related mechanisms.

DNA damage and polyploidization.

PubMed

Chow, Jeremy; Poon, Randy Y C

2010-01-01

A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy.

PubMed

Dhouib, Rabeb; Ducret, Adrien; Hubert, Pierre; Carrière, Frédéric; Dukan, Sam; Canaan, Stéphane

2011-04-01

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways. Copyright © 2011 Elsevier B.V. All rights reserved.

Replication profile of Saccharomyces cerevisiae chromosome VI.

PubMed

Friedman, K L; Brewer, B J; Fangman, W L

1997-11-01

An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae. Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early. As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.

The life cycle of Phaeocystis (Prymnesiophycaea): evidence and hypotheses

NASA Astrophysics Data System (ADS)

Rousseau, V.; Vaulot, D.; Casotti, R.; Cariou, V.; Lenz, J.; Gunkel, J.; Baumann, M.

1994-04-01

The present paper reviews the literature related to the life cycle of the prymnesiophyte Phaeocystis and its controlling factors and proposes novel hypotheses based on unpublished observations in culture and in the field. We chiefly refer to P. globosa Scherffel as most of the observations concern this species. P. globosa exhibits a complex alternation between several types of free-living cells (non-motile, flagellates, microzoopores and possibly macrozoospores) and colonies for which neither forms nor pathways have been completely identified and described. The different types of Phaeocystis cells were reappraised on the basis of existing microscopic descriptions complemented by unpublished flow cytometric investigations. This analysis revealed the existence of at least three different types of free-living cells identified on the basis of a combination of size, motility and ploidy characteristics: non-motile cells, flagellates and microzoospores. Their respective function within Phaeocystis life cycle, and in particular their involvement in colony formation is not completely understood. Observational evidence shows that Phaeocystis colonies are initiated at the early stage of their bloom each by one free-living cell. The mechanisms controlling this cellular transformation are still uncertain due to the lack of information on the overwintering Phaeocystis fomms and on the cell type responsible for colony induction. The existence of haploid microzoospores released from senescent colonies gives however some support to sexuality involvement at some stages of colony formation. Once colonies are formed, at least two mechanisms were identified as responsible of the spreading of colony form: colony multiplication by colonial division or budding and induction of new colony from colonial cells released in the external medium after colony disruption. The latter mechanism was clearly identified, involving at least two successive cell differentiations in the following sequence: motility development, subsequent flagella loss and settlement to a surface, mucus secretion and colony formation, colonial cell division and colony growth. Aggregate formation, cell motility development and subsequent emigration from the colonies, release of non-motile cells after colony lysis on the other hand, were identified as characteristics for termination of Phaeocystis colony development. These pathways were shown to occur similarly in natural environments. In the early stages of the bloom however, many recently-formed colonies were found on the setae of Chaetoceros spp, suggesting this diatom could play a key-rôle in Phaeocystis bloom inception. Analysis of the possible environmental factors regulating the transition between the different phases of the life cycle, suggested that nutrient status and requirement of a substrate for attachment of free-living cells would be essential for initiation of the colonial form. Physical constraints obviously would be important in determining colony shape and fragmentation although autogenic factors cannot be excluded. Some evidence exists that nutrients regulate colony division, while temperature and nutrient stress would stimulate cell emigration from the colonies.

A novel trypanoplasm-like flagellate Jarrellia atramenti n. g., n. sp. (Kinetoplastida: Bodonidae) and ciliates from the blowhole of a stranded pygmy sperm whale Kogia breviceps (Physeteridae): morphology, life cycle and potential pathogenicity.

PubMed

Poynton, S L; Whitaker, B R; Heinrich, A B

2001-04-10

The successful 6 mo rehabilitation of a stranded juvenile pygmy sperm whale Kogia breviceps afforded the opportunity to study the poorly known protozoan fauna of the upper respiratory tract of cetaceans. Mucus samples were collected by holding either a petri dish or glass slides over the blowhole for 3 to 5 exhalations; preparations were examined as wet mounts, and then stained with Wrights-Giemsa or Gram stain. Blood smears were stained with Wrights-Giemsa. Unidentified spindle-shaped and unidentified broad ciliates, reported from the blowhole of the pygmy sperm whale for the first time, were seen only initially, while yeast-like organisms and bacteria were seen intermittently. Epithelial cells and white blood cells were often present in the blowhole mucus, but red blood cells were never seen. A novel trypanoplasm-like bodonid kinetoplastid biflagellate (Order Kinetoplastida) was commonly encountered in the blowhole mucus, but never in the blood. Both mature flagellates and those undergoing longitudinal binary fission were present. The elongate flagellate had a long whiplash anterior flagellum; the recurrent flagellum was attached along at least two-thirds of the body length, forming a prominent undulating membrane, and the trailing portion was short. The kinetoplast was irregularly fragmented. The flagellates were either free-swimming, or attached to host material via the free portion of the posterior flagellum. The prominent undulating membrane was characteristic of Trypanoplasma, while the fragmented kinetoplast was characteristic of some species of Cryptobia. For the novel bodonid kinetoplastid, with its unique combination of morphological features (prominent undulating membrane and fragmented kinetoplast), we propose the creation of a new genus Jarrellia. We believe this to be the first published description of a flagellate from a marine mammal, and among the first reports of a trypanoplasm-like flagellate from a warm-blooded host. We expect that a diversity of flagellates and ciliates are commonly present in the blowhole of cetaceans. Future studies on the identity of the protozoans and the health of their cetacean hosts, which are readily studied in captivity, are necessary to establish their status as commensals or parasites.

High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

PubMed

Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

2015-06-17

High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

Aberrant meiotic behavior in Agave tequilana Weber var. azul

PubMed Central

Ruvalcaba-Ruiz, Domingo; Rodríguez-Garay, Benjamin

2002-01-01

Background Agave tequilana Weber var. azul, is the only one variety permitted by federal law in México to be used for tequila production which is the most popular contemporary alcoholic beverage made from agave and recognized worldwide. Despite the economic, genetic, and ornamental value of the plant, it has not been subjected to detailed cytogenetic research, which could lead to a better understanding of its reproduction for future genetic improvement. The objective of this work was to study the meiotic behavior in pollen mother cells and its implications on the pollen viability in Agave tequilana Weber var. azul. Results The analysis of Pollen Mother Cells in anaphase I (A-I) showed 82.56% of cells with a normal anaphase and, 17.44% with an irregular anaphase. In which 5.28% corresponded to cells with side arm bridges (SAB); 3.68% cells with one bridge and one fragment; 2.58% of irregular anaphase showed cells with one or two lagging chromosomes and 2.95% showed one acentric fragment; cells with two bridges and cells with two bridges and one acentric fragment were observed in frequencies of 1.60% and 1.35% respectively. In anaphase II some cells showed bridges and fragments too. Aberrant A-I cells had many shrunken or empty pollen grains (42.00%) and 58.00 % viable pollen. Conclusion The observed meiotic irregularities suggest that structural chromosome aberrations have occurred, such as heterozygous inversions, sister chromatid exchanges, deletions and duplications which in turn are reflected in a low pollen viability. PMID:12396234

Aberrant meiotic behavior in Agave tequilana Weber var. azul.

PubMed

Ruvalcaba-Ruiz, Domingo; Rodríguez-Garay, Benjamin

2002-10-23

Agave tequilana Weber var. azul, is the only one variety permitted by federal law in México to be used for tequila production which is the most popular contemporary alcoholic beverage made from agave and recognized worldwide. Despite the economic, genetic, and ornamental value of the plant, it has not been subjected to detailed cytogenetic research, which could lead to a better understanding of its reproduction for future genetic improvement. The objective of this work was to study the meiotic behavior in pollen mother cells and its implications on the pollen viability in Agave tequilana Weber var. azul. The analysis of Pollen Mother Cells in anaphase I (A-I) showed 82.56% of cells with a normal anaphase and, 17.44% with an irregular anaphase. In which 5.28% corresponded to cells with side arm bridges (SAB); 3.68% cells with one bridge and one fragment; 2.58% of irregular anaphase showed cells with one or two lagging chromosomes and 2.95% showed one acentric fragment; cells with two bridges and cells with two bridges and one acentric fragment were observed in frequencies of 1.60% and 1.35% respectively. In anaphase II some cells showed bridges and fragments too. Aberrant A-I cells had many shrunken or empty pollen grains (42.00%) and 58.00 % viable pollen. The observed meiotic irregularities suggest that structural chromosome aberrations have occurred, such as heterozygous inversions, sister chromatid exchanges, deletions and duplications which in turn are reflected in a low pollen viability.

Cytotoxic effects of pyrrolidine dithiocarbamate in small-cell lung cancer cells, alone and in combination with cisplatin.

PubMed

Tahata, Shinichi; Yuan, Bo; Kikuchi, Hidetomo; Takagi, Norio; Hirano, Toshihiko; Toyoda, Hiroo

2014-10-01

The cytocidal effect of pyrrolidine dithiocarbamate (PDTC) was investigated by focusing on cell viability, cell cycle arrest and apoptosis induction in small-cell lung cancer (SCLC) cell lines (NCI-H196 and NCI-H889). PDTC exhibited a much stronger dose-dependent cytotoxic activity against NCI-H196 compared to NCI-H889, while no such activity was observed in normal human embryonal lung fibroblast MRC-5 cells. Cell cycle arrest in S phase paralleled with suppression of c-myc expression without accompanying DNA fragmentation was observed in NCI-H196 cells. A transient increase in the intracellular ROS accompanied with an alteration of expression of oxidative stress-related genes was also confirmed in NCI-H196 cells. Furthermore, the addition of N-acetyl-l-cysteine (NAC), a free radical scavenger, not only abolished PDTC-trigger alterations of expression of these oxidative-related genes, but also almost completely abrogated PDTC-induced reduction in cell viability and morphological changes associated with cell damage. These results thus suggest that PDTC-induced cytotoxicity is attributed to its pro-oxidant activity. PDTC-induced cytotoxicity was further enhanced by CuCl2, however, abolished by bathocuproine disulfonate (BCPS), a non-permeable copper-specific chelator, supporting the plausibility that accumulation of intracellular Cu plays an important role in the cytotoxicity. Importantly, we demonstrated for the first time that PDTC downregulated the expression of ATP7A, known to be responsible for Cu efflux, but did not affect the expression of CTR1, known as a copper uptake transporter. Intriguingly, combination of much lower dose of cisplatin (5 µM) and non-toxic dose of PDTC (0.1 µM) synergistically induced a significant cytotoxicity in NCI-H196 cells. Given that ATP7A plays a critical role in the resistance of platinum-drug (such as cisplatin) representing a first-line treatment for SCLC, PDTC could be a promising candidate of adjunct therapeutic reagent for the patients requiring treatment with platinum-based regimens.

Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

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

Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com; Cheah, Yew-Hoong; Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur

Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action ofmore » xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.« less

ACTIVITY OF DISSOCIATED AND REASSOCIATED 19S ANTI-γ-GLOBULINS

PubMed Central

Schrohenloher, Ralph E.; Kunkel, Henry G.; Tomasi, Thomas B.

1964-01-01

19S anti-γ-globulins were isolated in a high state of purity from the sera of two patients with rheumatoid arthritis. Following reduction with ethyl mercaptan and alkylation by iodoacetamide, fragments were produced which retained the capacity to combine with 7S γ-globulin. The fragments from one of the 19S anti-γ-globulins agglutinated red cells coated with incomplete anti-Rh antibodies. This activity was shown by density gradient ultracentrifugation to be associated with low molecular weight fractions. The agglutination of the coated red cells by the fragments was strongly inhibited by normal and myeloma 7S γ-globulins and showed a greater specificity than the parent 19S material. Analytical ultracentrifuge experiments demonstrated that the fragments from either of the 19S anti-γ-globulins formed complexes with 7S γ-globulin. Reassociation of the dissociated fragments through reformation of disulfide bonds resulted in the formation of fast sedimenting molecules having properties similar to those of the untreated 19S material in respect to precipitation with aggregated γ-globulin and agglutination of coated red cells. PMID:14238936

Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis.

PubMed

Ayotte, Yann; Bilodeau, François; Descoteaux, Albert; LaPlante, Steven R

2018-05-02

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment-based phenotypic lead discovery (FPLD) combines aspects of traditional fragment-based lead discovery (FBLD), which involves the screening of small-molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug-like compounds in cell-based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low-molecular-weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone-marrow-derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction.

PubMed

Chen, Chia-Chun; Liao, Cheng-Hao; Wang, Yao-Horng; Hsu, Yuan-Ming; Huang, Shih-Horng; Chang, Chih-Hung; Fang, Hsu-Wei

2012-03-01

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage-like structure with positive Alcian blue staining in the cartilage fragment-fibrin-MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction. Copyright © 2011 Orthopaedic Research Society.

PARKIN overexpression in human mesenchymal stromal cells from Wharton's jelly suppresses 6-hydroxydopamine-induced apoptosis: Potential therapeutic strategy in Parkinson's disease.

PubMed

Bonilla-Porras, A R; Arevalo-Arbelaez, A; Alzate-Restrepo, J F; Velez-Pardo, C; Jimenez-Del-Rio, M

2018-01-01

Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, ~35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Phytotoxicity and cytogenotoxicity of hydroalcoholic extracts from Solanum muricatum Ait. and Solanum betaceum Cav. (Solanaceae) in the plant model Lactuca sativa.

PubMed

Dos Santos, Fabio Eduardo; Carvalho, Marcos Schleiden Sousa; Silveira, Graciele Lurdes; Correa, Felipe Folgaroli; Cardoso, Maria das Graças; Andrade-Vieira, Larissa Fonseca; Vilela, Luciane Resende

2018-03-05

Plants are rich in biologically active compounds. They can be explored for the production of bioherbicides. In this context, the present work aimed to evaluate the allelopathic effect of hydroalcoholic extracts from two Solanaceae species: Solanum muricatum Ait. and Solanum betaceum Cav. For this end, we conducted phytochemical screening and biological assays, determining the effects of the extracts on germination, early development, cell cycle, and DNA fragmentation in plantlets and meristematic cells of the plant model Lactuca sativa L. (lettuce). The percentage of seeds germinated under effect of S. muricatum extract did not differ from the control, but plantlet growth was reduced at the highest concentrations. For S. betaceum extract, dose dependence was observed for both germination and plantlet development, with the highest concentrations inhibiting germination. The growth curves revealed the concentrations of 2.06 and 1.93 g/L for S. muricatum and S. betaceum extracts, respectively, as those reducing 50% of root growth (RG). At these concentrations, both extracts presented mitodepressive effect, besides inducing significant increase in the frequency of condensed nuclei, associated to DNA fragmentation and cytoplasmic shrinkage. The frequency of chromosome alterations was not significant. We further discuss the mechanisms of action related to the chemical composition of the extracts, which presented organic acids, reducing sugars, proteins, amino acids, and tannins, besides catechins and flavonoids, only found in the extract of S. betaceum.

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

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

Turner, Roberta L.; Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu; Ornelles, David A., E-mail: ornelles@wakehealth.edu

2014-05-15

Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose)more » polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.« less

Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells.

PubMed

Khajeh, Shirin; Tohidkia, Mohammad Reza; Aghanejad, Ayuob; Mehdipour, Tayebeh; Fathi, Farzaneh; Omidi, Yadollah

2018-06-09

Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 V L antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed K D values to be in the nanomolar range (87.9-334 nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and V L G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and V L antibodies with potential therapeutic applications in G17-Gly-responsive tumors.

Diphenyl difluoroketone: a potent chemotherapy candidate for human hepatocellular carcinoma.

PubMed

Liang, Yingjian; Yin, Dalong; Hou, Limin; Zheng, Tongsen; Wang, Jiabei; Meng, Xianzhi; Lu, Zhaoyang; Song, Xuan; Pan, Shangha; Jiang, Hongchi; Liu, Lianxin

2011-01-01

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 µM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 µM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G(2)/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB-regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma.

Diphenyl Difluoroketone: A Potent Chemotherapy Candidate for Human Hepatocellular Carcinoma

PubMed Central

Hou, Limin; Zheng, Tongsen; Wang, Jiabei; Meng, Xianzhi; Lu, Zhaoyang; Song, Xuan; Pan, Shangha; Jiang, Hongchi; Liu, Lianxin

2011-01-01

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 µM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 µM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G2/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB–regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma. PMID:21901145

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

PubMed

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-10-03

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo . Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo . Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro . Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy

PubMed Central

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-01-01

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence. PMID:29108242

Porcine proximal tubular cells (LLC-PK1) are able to tolerate high levels of lithium chloride in vitro: assessment of the influence of 1-20 mM LiCl on cell death and alterations in cell biology and biochemistry.

PubMed

Lucas, Kirsten C; Hart, David A; Becker, Rolf W

2010-01-25

Lithium, a prophylactic drug for the treatment of bipolar disorder, is prescribed with caution due to its side effects, including renal damage. In this study porcine LLC-PK1 renal tubular cells were used to establish the direct toxicity of lithium on proximal cells and gain insights into the molecular mechanisms involved. In the presence of LiCl, cell proliferation exhibited insignificant decreases in a concentration-dependent manner, but once confluent, constant cell numbers were observed. Cell cycle studies indicated a small dose-dependent accumulation of cells in the G2/M stage after 24 h, as well as an increase in cells in the G0/G1 phase after treatment with 1-10 mM LiCl, but not at 20 mM LiCl. No evidence of apoptosis was observed based on cell morphology or DNA fragmentation studies, or evidence of protein expression changes for Bax, Bcl-2, and p53 proteins using immunocytochemistry. In addition caspases 3, 8 and 9 activity remained unaltered between control and lithium-treated cultures. To conclude, exposure to high concentrations of lithium did not result in overt toxic effects to LLC-PK1 renal cells, although LiCl did alter some aspects of cell behaviour, which could potentially influence function over time.

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

PubMed

Verrax, Julien; Stockis, Julie; Tison, Aurélie; Taper, Henryk S; Calderon, Pedro Buc

2006-09-14

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

Fragment-based drug discovery using rational design.

PubMed

Jhoti, H

2007-01-01

Fragment-based drug discovery (FBDD) is established as an alternative approach to high-throughput screening for generating novel small molecule drug candidates. In FBDD, relatively small libraries of low molecular weight compounds (or fragments) are screened using sensitive biophysical techniques to detect their binding to the target protein. A lower absolute affinity of binding is expected from fragments, compared to much higher molecular weight hits detected by high-throughput screening, due to their reduced size and complexity. Through the use of iterative cycles of medicinal chemistry, ideally guided by three-dimensional structural data, it is often then relatively straightforward to optimize these weak binding fragment hits into potent and selective lead compounds. As with most other lead discovery methods there are two key components of FBDD; the detection technology and the compound library. In this review I outline the two main approaches used for detecting the binding of low affinity fragments and also some of the key principles that are used to generate a fragment library. In addition, I describe an example of how FBDD has led to the generation of a drug candidate that is now being tested in clinical trials for the treatment of cancer.

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

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

Woo, Seon Rang; Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705; Park, Jeong-Eun

2012-01-06

Highlights: Black-Right-Pointing-Pointer Under conditions of telomere erosion, cells become extremely sensitive to H{sub 2}O{sub 2}. Black-Right-Pointing-Pointer Chromosomal regions adjacent to telomeres are cleaved by H{sub 2}O{sub 2} under such conditions. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} thus causes multichromosomal fusions and generation of small chromosomal fragments. Black-Right-Pointing-Pointer N-acetylcysteine prevents H{sub 2}O{sub 2}-induced chromosomal aberrations. -- Abstract: During genotoxic stress, reactive oxygen species hydrogen peroxide (H{sub 2}O{sub 2}) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H{submore » 2}O{sub 2}, via generation of multichromosomal fusion and chromosomal fragments. H{sub 2}O{sub 2} caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H{sub 2}O{sub 2} cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H{sub 2}O{sub 2}. Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.« less

Nuclear uptake of an amino-terminal fragment of apolipoprotein E4 promotes cell death and localizes within microglia of the Alzheimer's disease brain.

PubMed

Love, Julia E; Day, Ryan J; Gause, Justin W; Brown, Raquel J; Pu, Xinzhu; Theis, Dustin I; Caraway, Chad A; Poon, Wayne W; Rahman, Abir A; Morrison, Brad E; Rohn, Troy T

2017-01-01

Although harboring the apolipoprotein E4 ( APOE4 ) allele is a well known risk factor in Alzheimer's disease (AD), the mechanism by which it contributes to disease risk remains elusive. To investigate the role of proteolysis of apoE4 as a potential mechanism, we designed and characterized a site-directed cleavage antibody directed at position D151 of the mature form of apoE4 and E3. Characterization of this antibody indicated a high specificity for detecting synthesized recombinant proteins corresponding to the amino acid sequences 1-151 of apoE3 and E4 that would generate the 17 kDa (p17) fragment. In addition, this antibody also detected a ~17 kDa amino-terminal fragment of apoE4 following incubation with collagenase and matrix metalloproteinase-9 (MMP-9), but did not react with full-length apoE4. Application of this amino-terminal apoE cleavage-fragment (nApoECFp17) antibody, revealed nuclear labeling within glial cells and labeling of a subset of neurofibrillary tangles in the human AD brain. A quantitative analysis indicated that roughly 80% of labeled nuclei were microglia. To confirm these findings, cultured BV2 microglia cells were incubated with the amino-terminal fragment of apoE4 corresponding to the cleavage site at D151. The results indicated efficient uptake of this fragment and trafficking to the nucleus that also resulted in significant cell death. In contrast, a similarly designed apoE3 fragment showed no toxicity and primarily localized within the cytoplasm. These data suggest a novel cleavage event by which apoE4 is cleaved by the extracellular proteases, collagenase and MMP-9, generating an amino-terminal fragment that is then taken up by microglia, traffics to the nucleus and promotes cell death. Collectively, these findings provide important mechanistic insights into the mechanism by which harboring the APOE4 allele may elevate dementia risk observed in AD.

Dynamic aspects of the contractile system in Physarum plasmodium. III. Cyclic contraction-relaxation of the plasmodial fragment in accordance with the generation-degeneration of cytoplasmic actomyosin fibrils

PubMed Central

1987-01-01

Plasmodial fragments of Physarum polycephalum, excised from anterior regions of a thin-spread plasmodium, contracted-relaxed cyclicly with a period of 3-5 min. The area of the fragments decreased approximately 10% during contraction. In most cases, there was little endoplasmic streaming which indicates that contractions were synchronized throughout the fragment. By both polarized light and fluorescence microscopy, the organization and distribution of the cytoplasmic actomyosin fibrils in the fragments changed in synchrony with the contraction cycle. The fibrils formed during the contraction phase, and finally became a highly organized framework consisting of a three- dimensional network of numerous fibrils with many converging points (the nodes). During relaxation, the fibrils degenerated and disappeared almost completely, though some very weak fibrils remained near the nodes and the periphery. The results obtained by fluorometry of the fragments, stained with rhodamine-phalloidin, suggested that the G-F transformation of actin is not the main underlying process of the fibrillar formation. PMID:3611192

Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

PubMed Central

Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

2011-01-01

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells. PMID:21217069

Effects of histidin-2-ylidene vs. imidazol-2-ylidene ligands on the anticancer and antivascular activity of complexes of ruthenium, iridium, platinum, and gold.

PubMed

Schmitt, Florian; Donnelly, Kate; Muenzner, Julienne K; Rehm, Tobias; Novohradsky, Vojtech; Brabec, Viktor; Kasparkova, Jana; Albrecht, Martin; Schobert, Rainer; Mueller, Thomas

2016-10-01

Couples of N-heterocyclic carbene complexes of ruthenium, iridium, platinum, and gold, each differing only in the carbene ligand being either 1,3-dimethylimidazol-2-ylidene (IM) or 1,3-dimethyl-N-boc-O-methylhistidin-2-ylidene (HIS), were assessed for their antiproliferative effect on seven cancer cell lines, their interaction with DNA, their cell cycle interference, and their vascular disrupting properties. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays only the platinum complexes were cytotoxic at single-digit micromolar IC 50 concentrations with the (HIS)Pt complex being on average twice as active as the (IM)Pt complex. The former was highly efficacious against cisplatin-resistant HT-29 colon carcinoma cells where the latter had no effect. Both Pt complexes were accumulated by cancer cells and bound to double-helical DNA equally well. Only the (HIS)Pt complex modified the electrophoretic mobility of circular DNA in vitro due to the HIS ligand causing greater morphological changes to the DNA. Both platinum complexes induced accumulation of 518A2 melanoma cells in G2/M and S phase of the cell cycle. A disruption of blood vessels in the chorioallantoic membrane of fertilized chicken eggs was observed for both platinum complexes and the (IM)gold complex. The (HIS)platinum complex was as active as cisplatin in tumor xenografted mice while being tolerated better. We found that the HIS ligand may augment the cytotoxicity of certain antitumoral metal fragments in two ways: by acting as a transmembrane carrier increasing the cellular accumulation of the complex, and by initiating a pronounced distortion and unwinding of DNA. We identified a new (HIS)platinum complex which was highly cytotoxic against cancer cells including cisplatin-resistant ones. Copyright © 2016 Elsevier Inc. All rights reserved.

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells

PubMed Central

Abdullah, Rasedee; Kassim, Nur Kartinee Bt; Rosli, Rozita; Yeap, Swee Keong; Waziri, Peter; Etti, Imaobong Christopher; Bello, Muhammad Bashir

2017-01-01

Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer. PMID:28103302

Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells.

PubMed

Malami, Ibrahim; Abdul, Ahmad Bustamam; Abdullah, Rasedee; Kassim, Nur Kartinee Bt; Rosli, Rozita; Yeap, Swee Keong; Waziri, Peter; Etti, Imaobong Christopher; Bello, Muhammad Bashir

2017-01-01

Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer.

Ethyl acetate fraction from methanol extraction of Vitis thunbergii var. taiwaniana induced G0 /G1 phase arrest via inhibition of cyclins D and E and induction of apoptosis through caspase-dependent and -independent pathways in human prostate carcinoma DU145 cells.

PubMed

Lin, Chia-Hsin; Chan, Hsiao-Sung; Tsay, Hsin-Sheng; Funayama, Shinji; Kuo, Chao-Lin; Chung, Jing-Gung

2018-01-01

Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, belonging to the Vitaceae family and Vitis genus, and widely used as folk herbal medicine. It is traditionally used for the treatment of diarrhea, hypertension, neuroprotection, jaundice, and arthritis. We used the wild-collected VTT and sterilized them to establish the plant tissue culture, and then took the leaves for DNA sequencing to determine its original base. We use methanol to extract VTT in four different solvents: 1-butanol, n-hexane, ethyl acetate, and water. These four preliminary extracts were used to treat human prostate cancer DU145 cells in vitro. We use the flow cytometry to check the cell survival situation. Finally, we found the ethyl acetate layer roughing product (referred VTEA) in human prostate cancer apoptotic effects of cell line DU-145. In the present studies, we use the crude extract of VTT to examine whether or not it can induce apoptosis of DU145 cells in vitro. Viability assays for extracts of VTT treatment showed that it had dose-dependent effect on human prostate cancer DU145 cells. We also found that the extract of VTT induces time-dependent mitochondrial and intrinsic-dependent apoptosis pathways. The in vitro cytotoxic effects were investigated by cell cycle analysis and the determination of apoptotic DNA fragmentation in DU145 cells. The cell cycle analysis showed that extracts of VTT induced a significant increase in the number of cells in G 0 /G 1 phase. The extract of VTT induced chromatin changes and apoptosis of DU145 cells also were confirmed by DAPI and PI staining that were measured by fluorescence microscopy and flow cytometry, respectively. Finally, the expression of relevant proteins was analyzed by Western blot analysis. These results promoted us to further evaluate apoptosis associated proteins and elucidate the possible signal pathway in DU-145 cells after treated with the extract of VTT. © 2017 Wiley Periodicals, Inc.

The Role of Biotin in Bacterial Physiology and Virulence: a Novel Antibiotic Target for Mycobacterium tuberculosis.

PubMed

Salaemae, Wanisa; Booker, Grant W; Polyak, Steven W

2016-04-01

Biotin is an essential cofactor for enzymes present in key metabolic pathways such as fatty acid biosynthesis, replenishment of the tricarboxylic acid cycle, and amino acid metabolism. Biotin is synthesized de novo in microorganisms, plants, and fungi, but this metabolic activity is absent in mammals, making biotin biosynthesis an attractive target for antibiotic discovery. In particular, biotin biosynthesis plays important metabolic roles as the sole source of biotin in all stages of the Mycobacterium tuberculosis life cycle due to the lack of a transporter for scavenging exogenous biotin. Biotin is intimately associated with lipid synthesis where the products form key components of the mycobacterial cell membrane that are critical for bacterial survival and pathogenesis. In this review we discuss the central role of biotin in bacterial physiology and highlight studies that demonstrate the importance of its biosynthesis for virulence. The structural biology of the known biotin synthetic enzymes is described alongside studies using structure-guided design, phenotypic screening, and fragment-based approaches to drug discovery as routes to new antituberculosis agents.

A new mechanism for the formation of regolith on asteroids

NASA Astrophysics Data System (ADS)

Delbo, Marco; Libourel, Guy; Wilkerson, Justin; Murdoch, Naomi; Michel, Patrick; Ramesh, Kt; Ganino, Clement; Verati, Chrystele; Marchi, Simone

2014-11-01

The soil of asteroids, like that of the Moon, and other rocky, airless bodies in the Solar System, is made of a layer of pebbles, sand, and dust called regolith.Previous works suggested that the regolith on asteroids is made from material ejected from impacts and re-accumulated on the surface and from surface rocks that are broken down by micrometeoroid impacts. However, this regolith formation process has problems to explain the regolith on km-sized and smaller asteroids: it is known that impact fragments can reach escape velocities and breaks free from the gravitational forces of these small asteroids, indicating the impact mechanism is not the dominant process for regolith creation. Other studies also reveal that there is too much regolith on small asteroids’ surfaces to have been deposited there solely by impacts over the millions of years of asteroids’ evolution.We proposed that another process is capable of gently breaking rocks at the surface of asteroids: thermal fatigue by temperature cycling. As asteroids spin about their rotation axes, their surfaces go in and out of shadow resulting in large surface temperature variations. The rapid heating and cooling creates thermal expansion and contraction in the asteroid material, initiating cracking and propagating existing cracks. As the process is repeated over and over, the crack damage increases with time, leading eventually to rock fragmentation (and production of new regolith).To study this process, in the laboratory, we subjected meteorites, used as asteroid material analogs, to 37 days of thermal cycles similar to those occurring on asteroids. We measured cracks widening at an average rate of 0.5 mm/y. Some fragments were also produced, indicating meteorite fragmentation. To scale our results to asteroid lifetime, we incorporated our measurements into a fracture model and we deduced that thermal cycling is more efficient than micrometeorite bombardment at fragmenting rock over millions of years on asteroids (see Delbo et al. 2014. Nature 508, 233-236).This work was supported by the French Agence National de la Recherche (ANR) SHOCKS,

Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo.

PubMed

Prodon, François; Dru, Philippe; Roegiers, Fabrice; Sardet, Christian

2005-06-01

The mature ascidian oocyte is a large cell containing cytoplasmic and cortical domains polarized along a primary animal-vegetal (a-v) axis. The oocyte cortex is characterized by a gradient distribution of a submembrane monolayer of cortical rough endoplasmic reticulum (cER) and associated maternal postplasmic/PEM mRNAs (cER-mRNA domain). Between fertilization and first cleavage, this cER-mRNA domain is first concentrated vegetally and then relocated towards the posterior pole via microfilament-driven cortical contractions and spermaster-microtubule-driven translocations. The cER-mRNA domain further concentrates in a macroscopic cortical structure called the centrosome attracting body (CAB), which mediates a series of asymmetric divisions starting at the eight-cell stage. This results in the segregation of determinant mRNAs and their products in posterior cells of the embryo precursors of the muscle and germ line. Using two species of ascidians (Ciona intestinalis and Phallusia mammillata), we have pursued and amplified the work initiated in Halocynthia roretzi. We have analysed the cortical reorganizations in whole cells and in cortical fragments isolated from oocytes and from synchronously developing zygotes and embryos. After fertilization, we observe that a cortical patch rich in microfilaments encircles the cER-mRNA domain, concentrated into a cortical cap at the vegetal/contraction pole (indicating the future dorsal pole). Isolated cortices also retain microtubule asters rich in cER (indicating the future posterior pole). Before mitosis, parts of the cER-mRNA domain are detected, together with short microtubules, in isolated posterior (but not anterior) cortices. At the eight-cell stage, the posteriorly located cER-mRNA domain undergoes a cell-cycle-dependant compaction into the CAB. The CAB with embedded centrosomal microtubules can be isolated with cortical fragments from eight-cell-stage embryos. These and previous observations indicate that cytoskeleton-driven repositioning and compaction of a polarized cortical domain made of rough ER is a conserved mechanism used for polarization and segregation of cortical maternal mRNAs in embryos of evolutionarily distant species of ascidians.

Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy.

PubMed

Nezis, Ioannis P; Lamark, Trond; Velentzas, Athanassios D; Rusten, Tor Erik; Bjørkøy, Geir; Johansen, Terje; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H; Stenmark, Harald; Brech, Andreas

2009-04-01

Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.

Visual and highly sensitive detection of cancer cells by a colorimetric aptasensor based on cell-triggered cyclic enzymatic signal amplification.

PubMed

Zhang, Xianxia; Xiao, Kunyi; Cheng, Liwei; Chen, Hui; Liu, Baohong; Zhang, Song; Kong, Jilie

2014-06-03

Rapid and efficient detection of cancer cells at their earliest stages is one of the central challenges in cancer diagnostics. We developed a simple, cost-effective, and highly sensitive colorimetric method for visually detecting rare cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and linker DNAs stably coexist in solution, and the linker DNA assembles DNA-AuNPs, producing a purple solution. In the presence of target cells, the specific binding of HAPs to the target cells triggers a conformational switch that results in linker DNA hybridization and cleavage by nicking endonuclease-strand scission cycles. Consequently, the cleaved fragments of linker DNA can no longer assemble into DNA-AuNPs, resulting in a red color. UV-vis spectrometry and photograph analyses demonstrated that this CTCESA-based method exhibited selective and sensitive colorimetric responses to the presence of target CCRF-CEM cells, which could be detected by the naked eye. The linear response for CCRF-CEM cells in a concentration range from 10(2) to 10(4) cells was obtained with a detection limit of 40 cells, which is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. Given the high specificity and sensitivity of CTCESA, this colorimetric method provides a sensitive, label-free, and cost-effective approach for early cancer diagnosis and point-to-care applications.

High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

NASA Astrophysics Data System (ADS)

Miller, Cynthia K.; Temin, Howard M.

1983-05-01

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

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

Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille

Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of hostmore » range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies. IMPORTANCEInfluenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and provide a well-characterized tool to further study them.« less

GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

PubMed

Shimizu, Hiroyuki; Nagamori, Ippei; Yabuta, Norikazu; Nojima, Hiroshi

2009-09-01

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.

Regulatory networks and connected components of the neutral space. A look at functional islands

NASA Astrophysics Data System (ADS)

Boldhaus, G.; Klemm, K.

2010-09-01

The functioning of a living cell is largely determined by the structure of its regulatory network, comprising non-linear interactions between regulatory genes. An important factor for the stability and evolvability of such regulatory systems is neutrality - typically a large number of alternative network structures give rise to the necessary dynamics. Here we study the discretized regulatory dynamics of the yeast cell cycle [Li et al., PNAS, 2004] and the set of networks capable of reproducing it, which we call functional. Among these, the empirical yeast wildtype network is close to optimal with respect to sparse wiring. Under point mutations, which establish or delete single interactions, the neutral space of functional networks is fragmented into ≈ 4.7 × 108 components. One of the smaller ones contains the wildtype network. On average, functional networks reachable from the wildtype by mutations are sparser, have higher noise resilience and fewer fixed point attractors as compared with networks outside of this wildtype component.

Immunoglobulin A with protease activity secreted in human milk activates PAR-2 receptors, of intestinal epithelial cells HT-29, and promotes beta-defensin-2 expression.

PubMed

Barrera, G J; Portillo, R; Mijares, A; Rocafull, M A; del Castillo, J R; Thomas, L E

2009-03-24

Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.

Long-term carbon loss in fragmented Neotropical forests.

PubMed

Pütz, Sandro; Groeneveld, Jürgen; Henle, Klaus; Knogge, Christoph; Martensen, Alexandre Camargo; Metz, Markus; Metzger, Jean Paul; Ribeiro, Milton Cezar; de Paula, Mateus Dantas; Huth, Andreas

2014-10-07

Tropical forests play an important role in the global carbon cycle, as they store a large amount of carbon (C). Tropical forest deforestation has been identified as a major source of CO2 emissions, though biomass loss due to fragmentation--the creation of additional forest edges--has been largely overlooked as an additional CO2 source. Here, through the combination of remote sensing and knowledge on ecological processes, we present long-term carbon loss estimates due to fragmentation of Neotropical forests: within 10 years the Brazilian Atlantic Forest has lost 69 (±14) Tg C, and the Amazon 599 (±120) Tg C due to fragmentation alone. For all tropical forests, we estimate emissions up to 0.2 Pg C y(-1) or 9 to 24% of the annual global C loss due to deforestation. In conclusion, tropical forest fragmentation increases carbon loss and should be accounted for when attempting to understand the role of vegetation in the global carbon balance.

Polysialic acid enters the cell nucleus attached to a fragment of the neural cell adhesion molecule NCAM to regulate the circadian rhythm in mouse brain.

PubMed

Westphal, Nina; Kleene, Ralf; Lutz, David; Theis, Thomas; Schachner, Melitta

2016-07-01

In the mammalian nervous system, the neural cell adhesion molecule NCAM is the major carrier of the glycan polymer polysialic acid (PSA) which confers important functions to NCAM's protein backbone. PSA attached to NCAM contributes not only to cell migration, neuritogenesis, synaptic plasticity, and behavior, but also to regulation of the circadian rhythm by yet unknown molecular mechanisms. Here, we show that a PSA-carrying transmembrane NCAM fragment enters the nucleus after stimulation of cultured neurons with surrogate NCAM ligands, a phenomenon that depends on the circadian rhythm. Enhanced nuclear import of the PSA-carrying NCAM fragment is associated with altered expression of clock-related genes, as shown by analysis of cultured neuronal cells deprived of PSA by specific enzymatic removal. In vivo, levels of nuclear PSA in different mouse brain regions depend on the circadian rhythm and clock-related gene expression in suprachiasmatic nucleus and cerebellum is affected by the presence of PSA-carrying NCAM in the cell nucleus. Our conceptually novel observations reveal that PSA attached to a transmembrane proteolytic NCAM fragment containing part of the extracellular domain enters the cell nucleus, where PSA-carrying NCAM contributes to the regulation of clock-related gene expression and of the circadian rhythm. Copyright © 2016 Elsevier Inc. All rights reserved.

[Effects of cucurmosin on the cell proliferation and apoptosis in human pancreatic PANC-1 cells].

PubMed

Xu, Chun-Sen; Huang, He-Guang; Chen, Ming-Huang

2012-02-01

To observe the effects of cucurmosin (CUS) on the cell proliferation and apoptosis in pancreatic PANC-1 cells. The inhibition of CUS on the PANC-1 cell growth was observed using MTT assay. The inhibition ratio of CUS on the pancreatic orthotopic transplantation was in vivo observed in the NOD/SCID mouse model. The changes of microstructure of the apoptosis-inducing effect of CUS on PANC-1 was observed under electron microscope. The cell cycle and apoptosis after CUS intervention was detected using flow cytometry. The Caspase-3 activity after CUS treatment was detected using enzyme linked immunospecific assay (ELISA). Treatment with CUS at the dose of 0.125, 0.25, and 0.5 mg/kg inhibited the growth of pancreatic carcinoma PANC-1 xenografs with the ratio of 45.2%, 50.0%, and 59.7%, respectively (P < 0.05). After exposure to 10 microg/mL CUS for 24 h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation and shrunken nucleus. The apoptotic cells increased. Some nuclear shrinkage and fragmentation, as well as the apoptotic body were observed when cells were exposed to CUS for 72 h. Being exposed to 0, 2.5, 10.0, and 40.0 microg/mL of the CUS for 72 h, the percentage of G0/G1 phase cells was 46.56% +/- 5.08%, 53.33% +/- 5.05%, 67.50% +/- 6.50%, and 77.00% +/- 6.73%, respectively (P < 0.05). The apoptosis ratio was 2.50% +/- 0.13%, 8.30% +/- 1.23%, 23.40% +/- 2.45%, and 48.50% +/- 3.65% shown by Annexin V/PI (P < 0.05). The Caspase-3 activity (unit) was 0.009 +/- 0.002, 0.011 +/- 0.003, 0.035 +/- 0.009, and 0.065 +/- 0.009, respectively (P < 0.05). These data showed that CUS induced the apoptosis of PANC-1 cells in a dose-dependent maner. Being exposed to 40.0 microg/mL of the CUS for 24, 48, and 72 h, the percentage of G0/ G1 phase cells was 56.60% +/- 6.65%, 67.83% +/- 6.76%, and 77.00% +/- 6.73%, respectively (P < 0.05), the apoptosis ratio was 16.51% +/- 2.97%, 38.51% +/- 2.38%, and 48.50% +/- 3.65% shown by Annexin V/PI (P < 0.05). These data showed that CUS induced apoptosis of PANC-1 cells in the G0/G1 phase of the cell cycle in a time-dependent maner. CUS significantly inhibited the growth of PANC-1 cells possibly through the G0/G1 cell cycle arrest and apoptosis.

Carrier-envelope phase dependence of the directional fragmentation and hydrogen migration in toluene in few-cycle laser fields.

PubMed

Li, Hui; Kling, Nora G; Förg, Benjamin; Stierle, Johannes; Kessel, Alexander; Trushin, Sergei A; Kling, Matthias F; Kaziannis, Spyros

2016-07-01

The dissociative ionization of toluene initiated by a few-cycle laser pulse as a function of the carrier envelope phase (CEP) is investigated using single-shot velocity map imaging. Several ionic fragments, CH3 (+), H2 (+), and H3 (+), originating from multiply charged toluene ions present a CEP-dependent directional emission. The formation of H2 (+) and H3 (+) involves breaking C-H bonds and forming new bonds between the hydrogen atoms within the transient structure of the multiply charged precursor. We observe appreciable intensity-dependent CEP-offsets. The experimental data are interpreted with a mechanism that involves laser-induced coupling of vibrational states, which has been found to play a role in the CEP-control of molecular processes in hydrocarbon molecules, and appears to be of general importance for such complex molecules.

Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin.

PubMed

Furlong, Suzanne J; Ridgway, Neale D; Hoskin, David W

2008-03-01

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells.

PubMed

Elmroth, Kecke; Stenerlöw, Bo

2005-04-01

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to 125IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

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

Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.

1989-01-01

Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector /lambda/pSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB/sup +/ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA/sup +/ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmidsmore » consistently transduced the markers thrB/sup +/ and lysA/sup +/. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes.« less

Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania

PubMed Central

Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

2012-01-01

Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved. PMID:22767185

HLBT-100: a highly potent anti-cancer flavanone from Tillandsia recurvata (L.) L.

PubMed

Lowe, Henry I C; Toyang, Ngeh J; Watson, Charah T; Ayeah, Kenneth N; Bryant, Joseph

2017-01-01

The incidence and mortalities from cancers remain on the rise worldwide. Despite significant efforts to discover and develop novel anticancer agents, many cancers remain in the unmet need category. As such, efforts to discover and develop new and more effective and less toxic agents against cancer remain a top global priority. Our drug discovery approach is natural products based with a focus on plants. Tillandsia recurvata (L.) L. is one of the plants selected by our research team for further studies based on previous bioactivity findings on the anticancer activity of this plant. The plant biomass was extracted using supercritical fluid extraction technology with CO 2 as the mobile phase. Bioactivity guided isolation was achieved by use of chromatographic technics combined with anti-proliferative assays to determine the active fraction and subsequently the pure compound. Following in house screening, the identified molecule was submitted to the US National Cancer Institute for screening on the NCI60 cell line panel using standard protocols. Effect of HLBT-100 on apoptosis, caspase 3/7, cell cycle and DNA fragmentation were assessed using standard protocols. Antiangiogenic activity was carried out using the ex vivo rat aortic ring assay. A flavonoid of the flavanone class was isolated from T. recurvata (L.) L. with potent anticancer activity. The molecule was code named as HLBT-100 (also referred to as HLBT-001). The compound inhibited brain cancer (U87 MG), breast cancer (MDA-MB231), leukemia (MV4-11), melanoma (A375), and neuroblastoma (IMR-32) with IC 50 concentrations of 0.054, 0.030, 0.024, 0.003 and 0.05 µM, respectively. The molecule also exhibited broad anticancer activity in the NCI60 panel inhibiting especially hematological, colon, CNS, melanoma, ovarian, breast and prostate cancers. Twenty-three of the NCI60 cell lines were inhibited with GI 50 values <0.100 µM. In terms of potential mechanisms of action, the molecule demonstrated effect on the cell cycle as evidenced by the accumulation of cells with 

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.

Endogenous protein and enzyme fragments induce immunoglobulin E-independent activation of mast cells via a G protein-coupled receptor, MRGPRX2.

PubMed

Tatemoto, K; Nozaki, Y; Tsuda, R; Kaneko, S; Tomura, K; Furuno, M; Ogasawara, H; Edamura, K; Takagi, H; Iwamura, H; Noguchi, M; Naito, T

2018-05-01

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E-dependent and E-independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas-related G protein-coupled receptors (MRGPRs). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin-10 fragment) that act as bioactive peptides and induce immunoglobulin E-independent mast cell activation via MRGPRX2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX2 responds various as-yet-unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells. © 2018 The Foundation for the Scandinavian Journal of Immunology.

Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells.

PubMed

Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

2009-08-20

Apolipoprotein E allele epsilon4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1-272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1-272) more strongly than intact apoE4(1-299). Further analysis showed that in Neuro-2a cells expressing apoE4(1-272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1-299). ApoE4(1-272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1-272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration.

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae.

PubMed

Lenz, Jonathan D; Stohl, Elizabeth A; Robertson, Rosanna M; Hackett, Kathleen T; Fisher, Kathryn; Xiong, Kalia; Lee, Mijoon; Hesek, Dusan; Mobashery, Shahriar; Seifert, H Steven; Davies, Christopher; Dillard, Joseph P

2016-05-13

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Oligosaccharins — a new class of signalling molecules in plants

NASA Astrophysics Data System (ADS)

Usov, Anatolii I.

1993-11-01

The review deals with oligosaccharins — biologically active oligosaccharides of the following structural types: the (1→6, 1→3)-β-D-glucan fragments of the fungal cell wall; the chitin and chitosan fragments; polygalacturonic acid fragments; the xyloglucan fragments of the plant cell wall; lipooligosaccharides synthesised by bacteria — symbionts of leguminous plants. The isolation, determination of the structure, and total syntheses of these complex compounds as well as the results of the study of their biological role as inductors of the immunity of plants to attack by pathogenic fungi and bacteria and as regulators of the morphogenesis in the development processes in plants and of interaction of the latter with symbiont bacteria are described. The bibliography includes 197 references.

Quality Saving Mechanisms of Mitochondria during Aging in a Fully Time-Dependent Computational Biophysical Model

PubMed Central

Mellem, Daniel; Fischer, Frank; Jaspers, Sören; Wenck, Horst; Rübhausen, Michael

2016-01-01

Mitochondria are essential for the energy production of eukaryotic cells. During aging mitochondria run through various processes which change their quality in terms of activity, health and metabolic supply. In recent years, many of these processes such as fission and fusion of mitochondria, mitophagy, mitochondrial biogenesis and energy consumption have been subject of research. Based on numerous experimental insights, it was possible to qualify mitochondrial behaviour in computational simulations. Here, we present a new biophysical model based on the approach of Figge et al. in 2012. We introduce exponential decay and growth laws for each mitochondrial process to derive its time-dependent probability during the aging of cells. All mitochondrial processes of the original model are mathematically and biophysically redefined and additional processes are implemented: Mitochondrial fission and fusion is separated into a metabolic outer-membrane part and a protein-related inner-membrane part, a quality-dependent threshold for mitophagy and mitochondrial biogenesis is introduced and processes for activity-dependent internal oxidative stress as well as mitochondrial repair mechanisms are newly included. Our findings reveal a decrease of mitochondrial quality and a fragmentation of the mitochondrial network during aging. Additionally, the model discloses a quality increasing mechanism due to the interplay of the mitophagy and biogenesis cycle and the fission and fusion cycle of mitochondria. It is revealed that decreased mitochondrial repair can be a quality saving process in aged cells. Furthermore, the model finds strategies to sustain the quality of the mitochondrial network in cells with high production rates of reactive oxygen species due to large energy demands. Hence, the model adds new insights to biophysical mechanisms of mitochondrial aging and provides novel understandings of the interdependency of mitochondrial processes. PMID:26771181

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

PubMed

Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

2014-04-01

Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. Copyright © 2014 Elsevier B.V. All rights reserved.

Hamamelitannin from Hamamelis virginiana inhibits the tumour necrosis factor-alpha (TNF)-induced endothelial cell death in vitro.

PubMed

Habtemariam, Solomon

2002-01-01

The tumour necrosis factor-alpha (TNF) inhibitory activity of hamamelitannin from Hamamelis virginiana was investigated by assessing the TNF-mediated EAhy926 endothelial cell death and adhesiveness to monocytes. Treatment of the cells by TNF (25 ng/ml) and actinomycin D (0.1ng/ml) resulted in significant DNA fragmentation (34+/-0.6, n=4) and cytotoxicity (97+/-4.5%, n=6) following treatment for 8 and 24h, respectively. One to 100 microM concentrations of hamamelitannin inhibited the TNF-mediated endothelial cell death and DNA fragmentation in a dose-dependent manner. One hundred % protection against TNF-induced DNA fragmentation and cytotoxicity was obtained for hamamelitannin concentrations higher than 10 microM. The protective effect of hamamelitannin was comparable with that of a related compound epigallocatechin gallate while gallic acid was a weak protective agent (<40% protection). EAhy926 endothelial cells upregulated (by 4- to 7-fold) the surface expression of intercellular adhesion molecule-1 (ICAM-1) and adhesiveness to monocytic U937 cells after treatment with TNF (0.5ng/ml) for 6 or 24h. Concentrations (1-100 microM) of hamamelitannin that inhibited the TNF-mediated cell death and DNA fragmentation, however, failed to inhibit the TNF-induced ICAM-1 expression and EAhy926 cell adhesiveness to U937 cells. Thus, hamamelitannin inhibits the TNF-mediated endothelial cell death without altering the TNF-induced upregulation of endothelial adhesiveness. The observed anti-TNF activity of hamamelitannin may explain the antihamorrhaegic use of H. virginiana in traditional medicine and its claimed use as a protective agent for UV radiation.

Glycyrrhizic acid alters Kaposi sarcoma–associated herpesvirus latency, triggering p53-mediated apoptosis in transformed B lymphocytes

PubMed Central

Curreli, Francesca; Friedman-Kien, Alvin E.; Flore, Ornella

2005-01-01

Kaposi sarcoma–associated herpesvirus (KSHV) is linked with all clinical forms of Kaposi sarcoma and several lymphoproliferative disorders. Like other herpesviruses, KSHV becomes latent in the infected cells, expressing only a few genes that are essential for the establishment and maintenance of its latency and for the survival of the infected cells. Inhibiting the expression of these latent genes should lead to eradication of herpesvirus infection. All currently available drugs are ineffective against latent infection. Here we show, for the first time to our knowledge, that latent infection with KSHV in B lymphocytes can be terminated by glycyrrhizic acid (GA), a triterpenoid compound earlier shown to inhibit the lytic replication of other herpesviruses. We demonstrate that GA disrupts latent KSHV infection by downregulating the expression of latency-associated nuclear antigen (LANA) and upregulating the expression of viral cyclin and selectively induces cell death of KSHV-infected cells. We show that reduced levels of LANA lead to p53 reactivation, an increase in ROS, and mitochondrial dysfunction, which result in G1 cell cycle arrest, DNA fragmentation, and oxidative stress–mediated apoptosis. Latent genes are involved in KSHV-induced oncogenesis, and strategies to interfere with their expression might prove useful for eradicating latent KSHV infection and have future therapeutic implications. PMID:15765147

Glycyrrhizic acid alters Kaposi sarcoma-associated herpesvirus latency, triggering p53-mediated apoptosis in transformed B lymphocytes.

PubMed

Curreli, Francesca; Friedman-Kien, Alvin E; Flore, Ornella

2005-03-01

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with all clinical forms of Kaposi sarcoma and several lymphoproliferative disorders. Like other herpesviruses, KSHV becomes latent in the infected cells, expressing only a few genes that are essential for the establishment and maintenance of its latency and for the survival of the infected cells. Inhibiting the expression of these latent genes should lead to eradication of herpesvirus infection. All currently available drugs are ineffective against latent infection. Here we show, for the first time to our knowledge, that latent infection with KSHV in B lymphocytes can be terminated by glycyrrhizic acid (GA), a triterpenoid compound earlier shown to inhibit the lytic replication of other herpesviruses. We demonstrate that GA disrupts latent KSHV infection by downregulating the expression of latency-associated nuclear antigen (LANA) and upregulating the expression of viral cyclin and selectively induces cell death of KSHV-infected cells. We show that reduced levels of LANA lead to p53 reactivation, an increase in ROS, and mitochondrial dysfunction, which result in G1 cell cycle arrest, DNA fragmentation, and oxidative stress-mediated apoptosis. Latent genes are involved in KSHV-induced oncogenesis, and strategies to interfere with their expression might prove useful for eradicating latent KSHV infection and have future therapeutic implications.

An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1.

PubMed

Singh, Neeloo; Kaur, Jaspreet; Kumar, Pranav; Gupta, Swati; Singh, Nasib; Ghosal, Angana; Dutta, Avijit; Kumar, Ashutosh; Tripathi, Ramapati; Siddiqi, Mohammad Imran; Mandal, Chitra; Dube, Anuradha

2009-10-01

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

Targeting gastrin-releasing peptide as a new approach to treat aggressive refractory neuroblastomas.

PubMed

Paul, Pritha; Gillory, Lauren A; Kang, JungHee; Qiao, Jingbo; Chung, Dai H

2011-03-01

The overall survival for neuroblastoma remains dismal, in part due to the emergence of resistance to chemotherapeutic drugs. We have demonstrated that gastrin-releasing peptide (GRP), a gut peptide secreted by neuroblastoma, acts as an autocrine growth factor. We hypothesized that knockdown of GRP will induce apoptosis in neuroblastoma cells and potentiate the cytotoxic effects of chemotherapeutic agents. The human neuroblastoma cell lines (JF, SK-N-SH) were transfected with small interfering (si) RNA targeted at GRP. Apoptosis was assessed by DNA fragmentation assay. Immunoblotting was used to confirm molecular markers of apoptosis, and flow cytometry was performed to determine cell cycle arrest after GRP knockdown. siGRP resulted in an increase in apoptosis in the absence of chemotherapeutic interventions. A combination of GRP silencing and chemotherapeutic drugs resulted in enhanced apoptosis when compared to either of the treatments alone. GRP silencing led to increased expression of proapoptotic proteins, p53 and p21. Silencing of GRP induces apoptosis in neuroblastoma cells; it acts synergistically with chemotherapeutic effects of etoposide and vincristine. GRP knockdown-mediated apoptosis appears to be associated with upregulation of p53 in neuroblastoma cells. Targeting GRP may be postulated as a potential novel agent for combinational treatment to treat aggressive neuroblastomas. Copyright © 2011 Mosby, Inc. All rights reserved.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

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

Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan

Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lungmore » cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.« less

Differential Control of Growth, Apoptotic Activity, and Gene Expression in Human Breast Cancer Cells by Extracts Derived from Medicinal Herbs Zingiber officinale

PubMed Central

Elkady, Ayman I.; Abuzinadah, Osama A.; Baeshen, Nabih A.; Rahmy, Tarek R.

2012-01-01

The present study aimed to examine the antiproliferative potentiality of an extract derived from the medicinal plant ginger (Zingiber officinale) on growth of breast cancer cells. Ginger treatment suppressed the proliferation and colony formation in breast cancer cell lines, MCF-7 and MDA-MB-231. Meanwhile, it did not significantly affect viability of nontumorigenic normal mammary epithelial cell line (MCF-10A). Treatment of MCF-7 and MDA-MB-231 with ginger resulted in sequences of events marked by apoptosis, accompanied by loss of cell viability, chromatin condensation, DNA fragmentation, activation of caspase 3, and cleavage of poly(ADP-ribose) polymerase. At the molecular level, the apoptotic cell death mediated by ginger could be attributed in part to upregulation of Bax and downregulation of Bcl-2 proteins. Ginger treatment downregulated expression of prosurvival genes, such as NF-κB, Bcl-X, Mcl-1, and Survivin, and cell cycle-regulating proteins, including cyclin D1 and cyclin-dependent kinase-4 (CDK-4). On the other hand, it increased expression of CDK inhibitor, p21. It also inhibited the expression of the two prominent molecular targets of cancer, c-Myc and the human telomerase reverse transcriptase (hTERT). These findings suggested that the ginger may be a promising candidate for the treatment of breast carcinomas. PMID:22969274

Immunohistochemical localization of the NH(2)-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth.

PubMed

Yuan, Guohua; Yang, Guobin; Song, Guangtai; Chen, Zhi; Chen, Shuo

2012-08-01

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH(2)-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH(2)-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH(2) antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH(2)-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.

Immunohistochemical localization of the NH2-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth

PubMed Central

Yuan, Guohua; Yang, Guobin; Song, Guangtai

2013-01-01

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH2-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH2-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH2 antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH2-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN. PMID:22581382

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

PubMed Central

Birone, Claire; Brown, Maria; Hernandez, Jesus; Neff, Sherry; Williams, Daniel; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

2014-01-01

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size. PMID:24988328

A linear relationship between crystal size and fragment binding time observed crystallographically: implications for fragment library screening using acoustic droplet ejection.

PubMed

Cole, Krystal; Roessler, Christian G; Mulé, Elizabeth A; Benson-Xu, Emma J; Mullen, Jeffrey D; Le, Benjamin A; Tieman, Alanna M; Birone, Claire; Brown, Maria; Hernandez, Jesus; Neff, Sherry; Williams, Daniel; Allaire, Marc; Orville, Allen M; Sweet, Robert M; Soares, Alexei S

2014-01-01

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.

Forests on the edge: Microenvironmental drivers of carbon cycle response to edge effects

NASA Astrophysics Data System (ADS)

Reinmann, A.; Hutyra, L.; Smith, I. A.; Thompson, J.

2017-12-01

Twenty percent of the world's forest is within 100 m of a forest edge, but much of our understanding of forest carbon (C) cycling comes from large, intact ecosystems, which creates an important mismatch between the landscapes we study and those we aim to characterize. The temperate broadleaf forest is the most heavily fragmented forest biome in the world and its growth and carbon storage responses to forest edge effects appear to be the opposite of those in the tropical and boreal regions. We used field measurements to quantify the drivers of temperate forest C cycling response to edge effects, characterizing vegetative growth, respiration, and forest structure. We find large gradients in air and soil temperature from the forest interior to edge (up to 4 and 10° C, respectively) and the magnitude of this gradient is inversely correlated to the size of the forest edge growth enhancement. Further, leaf area index increases with proximity to the forest edge. While we also find increases in soil respiration between the forest interior and edge, this flux is small relative to aboveground growth enhancement near the edge. These findings represent an important advancement in our understanding of forest C cycle response to edge effects and will greatly improve our capacity to constrain biogenic C fluxes in fragmented and heterogeneous landscapes.

Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

PubMed

Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

2014-12-01

The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Protection against β-amyloid neurotoxicity by a non-toxic endogenous N-terminal β-amyloid fragment and its active hexapeptide core sequence.

PubMed

Forest, Kelly H; Alfulaij, Naghum; Arora, Komal; Taketa, Ruth; Sherrin, Tessi; Todorovic, Cedomir; Lawrence, James L M; Yoshikawa, Gene T; Ng, Ho-Leung; Hruby, Victor J; Nichols, Robert A

2018-01-01

High levels (μM) of beta amyloid (Aβ) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aβ, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aβ at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aβ. An N-terminal Aβ fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aβ-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aβ fragment and a shorter hexapeptide core sequence in the Aβ fragment (Aβcore: 10-15) to protect or reverse Aβ-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aβ fragment and Aβcore on Aβ-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aβ fragment and Aβcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aβcore were also shown to be fully protective against Aβ-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aβ fragment, while active stabilized N-terminal Aβcore derivatives offer the potential for therapeutic application. © 2017 International Society for Neurochemistry.

17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

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

Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok

2008-09-15

A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56more » phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.« less

Surface antigen in early differentiation.

PubMed Central

Kemler, R; Babinet, C; Eisen, H; Jacob, F

1977-01-01

Addition of Fab fragments from rabbit antiserum to surface antigen F9 to 2-cell stage mouse embryos in culture does not alter cleavage; however, the addition prevents culture does not alter cleavage; however, the addition prevents the formation of compact morulae and blastocysts. A similar effect is observed when Fab fragments are added to already compact 8-cell stage or even older morulae, but disappears at the beginning of blastocoel formation. This effect is reversible: uncompact 30-cell embryos washed free of Fab become compact in a few hours, produce blastocysts, and upon reimplantation into pseudopregnant mothers can produce mice. Development is not altered by divalent anti-F9 antibodies, by Fab fragments from sera directed against other embryo surface antigens, or by succinyl concanavalin A. Images PMID:270688

Targeting of a chlamydial protease impedes intracellular bacterial growth.

PubMed

Christian, Jan G; Heymann, Julia; Paschen, Stefan A; Vier, Juliane; Schauenburg, Linda; Rupp, Jan; Meyer, Thomas F; Häcker, Georg; Heuer, Dagmar

2011-09-01

Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target.

[Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

PubMed

Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

2017-03-01

Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

PubMed

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity.

PubMed

Méndez-Olvera, Estela T; Bustos-Martínez, Jaime A; López-Vidal, Yolanda; Verdugo-Rodríguez, Antonio; Martínez-Gómez, Daniel

2016-10-01

Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). The results obtained showed that the eight strains of C. jejuni , including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA , cdtB and cdtC genes.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity

PubMed Central

Méndez-Olvera, Estela T.; Bustos-Martínez, Jaime A.; López-Vidal, Yolanda; Verdugo-Rodríguez, Antonio; Martínez-Gómez, Daniel

2016-01-01

Background Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. Objectives The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. Methods Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). Results The results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. Conclusions This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes. PMID:27942359

C-terminal substance P fragments elicit histamine release from a murine mast cell line.

PubMed

Krumins, S A; Broomfield, C A

1993-01-01

Incubation of mouse mast cells with C-terminal substance P fragments in the micromolar range caused a release of histamine. Maximum release was observed with the tetrapeptide SP(8-11), followed by the tripeptide SP(9-11). SP(6-11) and SP(5-11) were nearly equipotent, while SP(4-11) caused only a slight histamine release. The substance P parent molecule and the N-terminal substance P fragments SP(1-4), SP(1-6) and SP(1-7) evoked no release of histamine. In confirmation of our previous findings, incubation with neurokinin A caused a release comparable to that of SP(8-11). Whereas neurokinin A-induced release was partially preventable by pretreating the cells with the NK2 receptor-selective antagonist cyclo(Gln-Trp-Phe-(R)Gly[ANC-2]Leu-Met), SP(8-11)-induced release was completely abolished by such treatment. The results provide the first evidence for the involvement of NK2 tachykinin receptors in the release of histamine by C-terminal substance P fragments.

Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

PubMed

Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

2014-01-01

Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

Multiple melanocortin receptors are expressed in bone cells

NASA Technical Reports Server (NTRS)

Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

2005-01-01

Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

Voronoi Cell Patterns: theoretical model and application to submonolayer growth

NASA Astrophysics Data System (ADS)

González, Diego Luis; Einstein, T. L.

2012-02-01

We use a simple fragmentation model to describe the statistical behavior of the Voronoi cell patterns generated by a homogeneous and isotropic set of points in 1D and in 2D. In particular, we are interested in the distribution of sizes of these Voronoi cells. Our model is completely defined by two probability distributions in 1D and again in 2D, the probability to add a new point inside an existing cell and the probability that this new point is at a particular position relative to the preexisting point inside this cell. In 1D the first distribution depends on a single parameter while the second distribution is defined through a fragmentation kernel; in 2D both distributions depend on a single parameter. The fragmentation kernel and the control parameters are closely related to the physical properties of the specific system under study. We apply our model to describe the Voronoi cell patterns of island nucleation for critical island sizes i=0,1,2,3. Experimental results for the Voronoi cells of InAs/GaAs quantum dots are also described by our model.

The mechanism of thioacetamide-induced apoptosis in the L37 albumin-SV40 T-antigen transgenic rat hepatocyte-derived cell line occurs without DNA fragmentation.

PubMed

Bulera, S J; Sattler, C A; Gast, W L; Heath, S; Festerling, T A; Pitot, H C

1998-10-01

The hepatotoxicant thioacetamide (TH) has classically been used as a model to study hepatic necrosis; however, recent studies have shown that TH can also induce apoptosis. In this report we demonstrate that 2.68+/-0.54% of the albumin-SV40 T-antigen transgenic rat hepatocytes undergo TH-induced apoptosis, a level comparable to other in vivo models of liver apoptosis. In addition, TH could induce apoptosis and necrosis in the L37 albumin-SV40 T-antigen transgenic rat liver-derived cell line. Examination of dying L37 cells treated with 100 mM TH by electron microscopy revealed distinct morphological characteristics that could be attributed to apoptosis. Quantitation of apoptosis by FACS analysis 24 h after treatment with 100 mM TH revealed that 81.3+/-1.6% of the cells were undergoing apoptosis. In contrast, when L37 cells were treated with 250 mM TH, cells exhibited characteristics consistent with necrotic cell death. DNA fragmentation ladders were produced by growth factor withdrawal-induced apoptosis; however, in 100 mM TH-induced apoptosis, DNA fragmentation ladders were not observed. Analysis of endonuclease activity in L37 cells revealed that the enzymes were not inactivated in the presence of 100 mM TH. The data presented in this report indicate that the L37 cell line could be used to study the mechanism of TH-induced apoptosis that was not mediated through a mechanism requiring DNA fragmentation.

Protein expression profiling in head fragments during planarian regeneration after amputation.

PubMed

Chen, Xiaoguang; Xu, Cunshuan

2015-04-01

Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various cell types. Taken together, our study demonstrated that proteomic analysis approach used by us is a powerful tool in understanding molecular process related to head regeneration of planarian.

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders.

PubMed

Pérez-Llano, Begoña; Enciso, María; García-Casado, Pedro; Sala, Rubén; Gosálvez, Jaime

2006-12-01

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.

Towards optimized methods to study viral impacts on soil microbial carbon cycling

NASA Astrophysics Data System (ADS)

Trubl, G. G.; Roux, S.; Jang, H. B.; Solonenko, N.; Sullivan, M. B.; Rich, V. I.

2016-12-01

Permafrost contains 50% of global soil carbon and is rapidly thawing. While the fate of this carbon is currently unknown, it will undoubtedly be shaped by microbes and their associated viruses, which modulate host activities via mortality and metabolic control. However, little is known about soil viruses generally and their impact on terrestrial biogeochemistry; this is partially due to the presence of inhibitory substances (e.g. humic acids) in soils that interfere with sample processing and sequence-based metagenomics surveys. To address this problem, we examined viral populations in three different peat soils along a permafrost thaw gradient. These samples yielded low viral DNA recoveries, and shallow metagenomic sequencing, but still resulted in the recovery of 40 viral genome fragments. Genome- and network-based classification suggested that these new references represented 11 viral clusters, and ecological patterns (based upon non-redundant fragment recruitment) showed that viral populations were distinct in each habitat. Although only 31% of the genes could be functionally classified, pairwise genome comparisons classified 63% of the viruses taxonomically. Additionally, comparison of the 40 viral genome fragments to 53 previously recovered fragments from the same site showed no overlap, suggesting only a small portion of the resident viral community has been sampled. A follow-up experiment was performed to remove more humics during extraction and thereby obtain better viral metagenomes. Three DNA extraction protocols were tested (CTAB, PowerSoil, and Wizard columns) and the DNA was further purified with an AMPure clean-up. The PowerSoil kit maximized DNA yield (3x CTAB and 6x Wizard), and yielded the purest DNA (based on NanoDrop 260:230 ratio). Given the important roles of viruses in biogeochemical cycles in better-studied systems, further research and humic-removal optimization on these thawing permafrost-associated viral communities is needed to clarify their involvement in carbon cycle feedbacks.

Antitumor effect and toxicity of free rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles in mice bearing breast cancer

PubMed Central

2013-01-01

Background Magnetic fluids containing superparamagnetic iron oxide nanoparticles represent an attractive platform as nanocarriers in chemotherapy. Recently, we developed a formulation of maghemite nanoparticles coated with rhodium (II) citrate, which resulted in in vitro cytotoxicity enhanced up to 4.6 times when compared to free rhodium (II) citrate formulation on breast carcinoma cells. In this work, we evaluate the antitumor activity and toxicity induced by these formulations in Balb/c mice bearing orthotopic 4T1 breast carcinoma. Methods Mice were evaluated with regard to the treatments’ toxicity through analyses of hemogram, serum levels of alanine aminotransferase, iron, and creatinine; DNA fragmentation and cell cycle of bone marrow cells; and liver, kidney and lung histology. In addition, the antitumor activity of rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate was verified by tumor volume reduction, histology and immunohistochemistry. Results Regarding the treatments’ toxicity, no experimental groups had alterations in levels of serum ALT or creatinine, and this suggestion was corroborated by the histopathologic examination of liver and kidney of mice. Moreover, DNA fragmentation frequency of bone marrow cells was lower than 15% in all experimental groups. On the other hand, the complexes rhodium (II) citrate-functionalized maghemite and free rhodium (II) citrate led to a marked growth inhibition of tumor and decrease in CD31 and Ki-67 staining. Conclusions In summary, we demonstrated that both rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate formulations exhibited antitumor effects against 4T1 metastatic breast cancer cell line following intratumoral administration. This antitumor effect was followed by inhibition of both cell proliferation and microvascularization and by tumor tissue injury characterized as necrosis and fibrosis. Remarkably, this is the first published report demonstrating the therapeutic efficacy of maghemite nanoparticles coated with rhodium (II) citrate. This treatment prolonged the survival period of treated mice without inducing apparent systemic toxicity, which strengthens its use for future breast cancer therapeutic applications. PMID:23414068

Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

PubMed

Valente, Sabrina; Rossi, Roberta; Resta, Leonardo; Pasquinelli, Gianandrea

2015-04-01

Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

A map of protein dynamics during cell-cycle progression and cell-cycle exit

PubMed Central

Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

2017-01-01

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491

Cell division cycle 45 promotes papillary thyroid cancer progression via regulating cell cycle.

PubMed

Sun, Jing; Shi, Run; Zhao, Sha; Li, Xiaona; Lu, Shan; Bu, Hemei; Ma, Xianghua

2017-05-01

Cell division cycle 45 was reported to be overexpressed in some cancer-derived cell lines and was predicted to be a candidate oncogene in cervical cancer. However, the clinical and biological significance of cell division cycle 45 in papillary thyroid cancer has never been investigated. We determined the expression level and clinical significance of cell division cycle 45 using The Cancer Genome Atlas, quantitative real-time polymerase chain reaction, and immunohistochemistry. A great upregulation of cell division cycle 45 was observed in papillary thyroid cancer tissues compared with adjacent normal tissues. Furthermore, overexpression of cell division cycle 45 positively correlates with more advanced clinical characteristics. Silence of cell division cycle 45 suppressed proliferation of papillary thyroid cancer cells via G1-phase arrest and inducing apoptosis. The oncogenic activity of cell division cycle 45 was also confirmed in vivo. In conclusion, cell division cycle 45 may serve as a novel biomarker and a potential therapeutic target for papillary thyroid cancer.

Combining EL4-B5-based B-cell stimulation and phage display technology for the successful isolation of human anti-Scl-70 autoantibody fragments.

PubMed

Weber, Malte; Weiss, Etienne; Engel, Alfred M

2003-07-01

Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.

Sleep homeostasis in the female rat during the estrous cycle.

PubMed

Schwierin, B; Borbély, A A; Tobler, I

1998-11-16

To investigate whether sleep homeostasis in the female rat is modulated by the estrous cycle, the vigilance states, EEG power spectra and cortical temperature (TCRT) were assessed on the basis of 4-day continuous recordings. A regulatory response was elicited by 6-h sleep deprivation (SD) during the proestrous (PRO) and the estrous (EST) day and compared to the baseline recordings. The vigilance states varied across the estrous cycle. In the PRO dark period the amount of sleep was reduced. The decrease in rapid-eye-movement (REM) sleep was already evident towards the end of the preceding light period, and an increased fragmentation of sleep was present throughout PRO. Compared to the other days of the estrous cycle, slow-wave activity (SWA; EEG power density 0.75-4.75 Hz) in nonREM (NREM) sleep was lower in PRO at the end of the light period and in the beginning of the dark period. High-frequency activity (HFA; EEG power density 10.25-25.0 Hz) was increased in the dark period of PRO. The SD performed during the first 6 h of the light period of PRO and EST enhanced SWA in NREM sleep and reduced sleep fragmentation during the subsequent 6 h. The extent and time course of the response to SD did not differ between the two phases of the estrous cycle. It is concluded that despite the marked baseline variations of the vigilance states and the EEG, homeostatic regulation is little affected by the estrous cycle. Copyright 1998 Elsevier Science B.V.

Landscape and flux reveal a new global view and physical quantification of mammalian cell cycle

PubMed Central

Li, Chunhe; Wang, Jin

2014-01-01

Cell cycles, essential for biological function, have been investigated extensively. However, enabling a global understanding and defining a physical quantification of the stability and function of the cell cycle remains challenging. Based upon a mammalian cell cycle gene network, we uncovered the underlying Mexican hat landscape of the cell cycle. We found the emergence of three local basins of attraction and two major potential barriers along the cell cycle trajectory. The three local basins of attraction characterize the G1, S/G2, and M phases. The barriers characterize the G1 and S/G2 checkpoints, respectively, of the cell cycle, thus providing an explanation of the checkpoint mechanism for the cell cycle from the physical perspective. We found that the progression of a cell cycle is determined by two driving forces: curl flux for acceleration and potential barriers for deceleration along the cycle path. Therefore, the cell cycle can be promoted (suppressed), either by enhancing (suppressing) the flux (representing the energy input) or by lowering (increasing) the barrier along the cell cycle path. We found that both the entropy production rate and energy per cell cycle increase as the growth factor increases. This reflects that cell growth and division are driven by energy or nutrition supply. More energy input increases flux and decreases barrier along the cell cycle path, leading to faster oscillations. We also identified certain key genes and regulations for stability and progression of the cell cycle. Some of these findings were evidenced from experiments whereas others lead to predictions and potential anticancer strategies. PMID:25228772

Identification of Cell Cycle-Regulated Genes by Convolutional Neural Network.

PubMed

Liu, Chenglin; Cui, Peng; Huang, Tao

2017-01-01

The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype are analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight to the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors

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

Wang, Le; Pratt, John K.; Soltwedel, Todd

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 μM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumormore » growth inhibition efficacy in mouse flank xenograft models.« less

Microbials for the production of monoclonal antibodies and antibody fragments.

PubMed

Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

2014-01-01

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transcriptional activity across the Epstein-Barr virus genome in Raji cells during latency and after induction of an abortive lytic cycle.

PubMed

Kirchner, E A; Bornkamm, G W; Polack, A

1991-10-01

We have studied the relative rate of transcription across the Epstein-Barr virus genome in the Burkitt's lymphoma cell line Raji by nuclear run-on analysis during latency and after induction of an abortive lytic cycle with 12-0-tetradecanoylphorbol 13-acetate (TPA) and 5-iodo-2'-deoxyuridine (IUdR). During latency the entire, or almost the entire, viral genome was found to be transcriptionally active to a low or intermediate extent, with some variation in activity along the genome. The fragment with the highest transcriptional activity was EcoRI J, which contains the genes encoding the small nuclear RNAs EBER1 and -2, transcribed predominantly by RNA polymerase III. An intermediate level of transcription was observed between positions 10 and 138 (kb), with areas of slightly higher activity on the large internal repeats and the left duplicated region (DL). The remaining part of the viral genome, between position 138 and the termini, and the termini and position 10 (kb) (with the exception of the EcoRI J fragment), showed very little transcriptional activity, except for the intermediately active regions carrying the righthand oriLyt (DR) and the terminal repeats. Upon induction of the viral genome with TPA and IUdR, the viral genome was transcriptionally active at a rate at least tenfold that seen during latency. Polymerases were not equally distributed along the genome after induction; the highest density was found in regions 48 to 58 kb, 82 to 84 kb, 102 to 104 kb, 118 to 122 kb and 142 to 145 kb of the viral genome. High transcriptional activity correlated with distinct transcription units in some cases, i.e. BamHI H1LF1 (DL), BamHI MLF1, BamHI ZLF1/BamHI RLF1 and BamHI X (thymidine kinase), but not in others (BamHI H2). Besides initiation of transcription, other regulatory processes such as stabilization and processing of primary transcripts may also contribute to regulation of virus gene expression. Addition of cycloheximide completely abolished the transcriptional activation of the genome mediated by TPA and IUdR.

Effect of quercetin on apoptosis of PANC-1 cells

PubMed Central

Lee, Joo Hyun; Lee, Han-Beom; Jung, Gum O; Oh, Jung Taek; Park, Dong Eun

2013-01-01

Purpose To investigate the chemotherapeutic effect of quercetin against cancer cells, signaling pathway of apoptosis was explored in human pancreatic cells. Methods Various anticancer drugs including adriamycin, cisplatin, 5-fluorouracil (5-FU) and gemcitabine were used. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetra zolium bromide assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole nuclei staining and flow cytometry in PANC-1 cells treated with 50 µg/mL quercetin for 24 hours. Expression of endoplas mic reticulum (ER) stress mediators including, Grp78/Bip, p-PERK, PERK, ATF4, ATF6 and GADD153/CHOP proteins were measured by Western blot analysis. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. Quercetin induced the apoptosis of PANC-1, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G0/G1 fraction of cell cycle increase. But not adriamycin, cisplatin, gemcitabine, and 5-FU. PANC-1 cells were markedly sensitive to quercetin. Results Treatment with quercetin resulted in the increased accumulation of intracellular Ca2+ ion. Treatment with quercetin also increased the expression of Grp78/Bip and GADD153/CHOP protein and induced mitochondrial dysfunction. Quercetin exerted cytotoxicity against human pancreatic cancer cells via ER stress-mediated apoptotic signaling including reactive oxygen species production and mitochondrial dysfunction. Conclusion These data suggest that quercetin may be an important modulator of chemosensitivity of cancer cells against anticancer chemotherapeutic agents. PMID:24368982

Effect of quercetin on apoptosis of PANC-1 cells.

PubMed

Lee, Joo Hyun; Lee, Han-Beom; Jung, Gum O; Oh, Jung Taek; Park, Dong Eun; Chae, Kwon Mook

2013-12-01

To investigate the chemotherapeutic effect of quercetin against cancer cells, signaling pathway of apoptosis was explored in human pancreatic cells. Various anticancer drugs including adriamycin, cisplatin, 5-fluorouracil (5-FU) and gemcitabine were used. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetra zolium bromide assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole nuclei staining and flow cytometry in PANC-1 cells treated with 50 µg/mL quercetin for 24 hours. Expression of endoplas mic reticulum (ER) stress mediators including, Grp78/Bip, p-PERK, PERK, ATF4, ATF6 and GADD153/CHOP proteins were measured by Western blot analysis. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. Quercetin induced the apoptosis of PANC-1, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G0/G1 fraction of cell cycle increase. But not adriamycin, cisplatin, gemcitabine, and 5-FU. PANC-1 cells were markedly sensitive to quercetin. Treatment with quercetin resulted in the increased accumulation of intracellular Ca(2+) ion. Treatment with quercetin also increased the expression of Grp78/Bip and GADD153/CHOP protein and induced mitochondrial dysfunction. Quercetin exerted cytotoxicity against human pancreatic cancer cells via ER stress-mediated apoptotic signaling including reactive oxygen species production and mitochondrial dysfunction. These data suggest that quercetin may be an important modulator of chemosensitivity of cancer cells against anticancer chemotherapeutic agents.

Effect of platinum nanoparticles on cell death induced by ultrasound in human lymphoma U937 cells.

PubMed

Jawaid, Paras; Rehman, Mati Ur; Hassan, Mariame Ali; Zhao, Qing Li; Li, Peng; Miyamoto, Yusei; Misawa, Masaki; Ogawa, Ryohei; Shimizu, Tadamichi; Kondo, Takashi

2016-07-01

In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of the RhoA GTPase Activity Increases Sensitivity of Melanoma Cells to UV Radiation Effects

PubMed Central

Espinha, Gisele; Osaki, Juliana Harumi; Costa, Erico Tosoni; Forti, Fabio Luis

2016-01-01

Ultraviolet radiation is the main cause of DNA damage to melanocytes and development of melanoma, one of the most lethal human cancers, which leads to metastasis due to uncontrolled cell proliferation and migration. These phenotypes are mediated by RhoA, a GTPase overexpressed or overactivated in highly aggressive metastatic tumors that plays regulatory roles in cell cycle progression and cytoskeleton remodeling. This work explores whether the effects of UV on DNA damage, motility, proliferation, and survival of human metastatic melanoma cells are mediated by the RhoA pathway. Mutant cells expressing dominant-negative (MeWo-RhoA-N19) or constitutively active RhoA (MeWo-RhoA-V14) were generated and subjected to UV radiation. A slight reduction in migration and invasion was observed in MeWo and MeWo-RhoA-V14 cells but not in MeWo-RhoA-N19 cells, which presented inefficient motility and invasiveness associated with stress fibers fragmentation. Proliferation and survival of RhoA-deficient cells were drastically reduced by UV compared to cells displaying normal or high RhoA activity, suggesting increased sensitivity to UV. Loss of RhoA activity also caused less efficient DNA repair, with elevated levels of DNA lesions such as strand breaks and cyclobutane pyrimidine dimers (CPDs). Thus, RhoA mediates genomic stability and represents a potential target for sensitizing metastatic tumors to genotoxic agents. PMID:26823948

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

PubMed

Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

1984-11-01

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Annonacin Exerts Antitumor Activity through Induction of Apoptosis and Extracellular Signal-regulated Kinase Inhibition

PubMed Central

Yap, Chee Voon; Subramaniam, Kavita S.; Khor, Sik Wey; Chung, Ivy

2017-01-01

Background: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Annonacin, a natural pure compound extracted from the seeds of Annona muricata, is a potential alternative therapeutic agent to treat EC. Objective: To study the antitumor activity of annonacin and its mechanism of action in EC cells (ECCs). Materials and Methods: Viability of ECCs treated with annonacin for 72 h was determined using methyl thiazolyl tetrazolium assay. The induction of cell cycle arrest and apoptotic cell death was evaluated using propidium iodide and annexin V-PE/7-AAD assay, respectively. DNA strand breaks were visualized using transferase dUTP nick end labeling assay, and the effects of annonacin on survival signaling were determined using western blotting. Results: Annonacin exhibited antiproliferative effects on EC cell lines (ECC-1 and HEC-1A) and primary cells (EC6-ept and EC14-ept) with EC50values ranging from 4.62 to 4.92 μg/ml. EC cells were shown arrested at G2/M phase after treated with 4 μg/ml of annonacin for 72 h. This led to a significant increase in apoptotic cell death (65.7%) in these cells when compared to vehicle-treated cells (P < 0.005). We further showed that annonacin-mediated apoptotic cell death was associated with an increase in caspase-3 cleavage and DNA fragmentation. Cell apoptosis was accompanied with downregulation of extracellular signal-regulated kinase survival protein expression and induction of G2/M cell cycle arrest. Conclusion: Annonacin may be a potential novel therapeutic agent for EC patients. SUMMARY We aimed to study the antitumor activity of annonacin and its mechanism of action in endometrial cancer cells. Annonacin exerted antiproliferation effects on both endometrial cancer cell lines and primary cells via induction of apoptosis and inhibition of extracellular signal-regulated kinase. Our data represented that annonacin could be an alternative therapeutic treatment to combat endometrial cancer. Abbreviations Used: 7-AAD: 7-Amino-Actinomycin, ATP: Adenosine diphosphate, BSA: Bovine serum albumin, DNA: Deoxyribonucleic acid, EC: Endometrial cancer, ECC-1: Endometrial cancer cell-1, EC50: Half maximal effective concentration, Ept: Epithelial, FBS: Fetal bovine serum, HEC-1A: Human endometrial carcinoma-1A, MTT: Methyl thiazolyl tetrazolium, NaCl: Sodium chloride, NADH: Nicotinamide adenine dinucleotide, RPMI 1640: Roswell Park Memorial Institute Medium, SDS: Sodium dodecyl sulfate PMID:29263632

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

PubMed Central

1984-01-01

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells. PMID:6725397

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

PubMed

Smith, A F; Bigsby, R M; Word, R A; Herring, B P

1998-05-01

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

Carrier-envelope phase dependence of the directional fragmentation and hydrogen migration in toluene in few-cycle laser fields

PubMed Central

Li, Hui; Kling, Nora G.; Förg, Benjamin; Stierle, Johannes; Kessel, Alexander; Trushin, Sergei A.; Kling, Matthias F.; Kaziannis, Spyros

2016-01-01

The dissociative ionization of toluene initiated by a few-cycle laser pulse as a function of the carrier envelope phase (CEP) is investigated using single-shot velocity map imaging. Several ionic fragments, CH3+, H2+, and H3+, originating from multiply charged toluene ions present a CEP-dependent directional emission. The formation of H2+ and H3+ involves breaking C-H bonds and forming new bonds between the hydrogen atoms within the transient structure of the multiply charged precursor. We observe appreciable intensity-dependent CEP-offsets. The experimental data are interpreted with a mechanism that involves laser-induced coupling of vibrational states, which has been found to play a role in the CEP-control of molecular processes in hydrocarbon molecules, and appears to be of general importance for such complex molecules. PMID:26958589

Recalcitrance and structural analysis by water-only flowthrough pretreatment of 13C enriched corn stover stem

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

Foston, Marcus; Trajano, Heather L.; Samuel, Reichel

This article presents high temperature water-only continuous flowthrough pretreatment coupled with nuclear magnetic resonance (NMR) as a promising analytical tool to examine the plant cell wall, to understand its recalcitrance (i.e., cell wall resistance to deconstruction), and to probe the chemistry occurring during batch pretreatment of biomass. 13C-enriched corn stover stems were pretreated at 170 °C for 60 minutes with a hot-water flow rate of 20 mL/min to control fractionation of the cell wall. This approach helped elucidate the nature of plant cell wall chemical recalcitrance and biomass pretreatment chemistry by tracking cell wall fragmentation as a function of time.more » Fractions of the reactor effluent were collected in a time-resolved fashion and characterized by various NMR techniques to determine the degree and sequence of fragments released, as well as, the chemical composition, molecular structure, and relative molecular weight of those released fragments.« less

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Recalcitrance and structural analysis by water-only flowthrough pretreatment of 13C enriched corn stover stem

DOE PAGES

Foston, Marcus; Trajano, Heather L.; Samuel, Reichel; ...

2015-08-28

This article presents high temperature water-only continuous flowthrough pretreatment coupled with nuclear magnetic resonance (NMR) as a promising analytical tool to examine the plant cell wall, to understand its recalcitrance (i.e., cell wall resistance to deconstruction), and to probe the chemistry occurring during batch pretreatment of biomass. 13C-enriched corn stover stems were pretreated at 170 °C for 60 minutes with a hot-water flow rate of 20 mL/min to control fractionation of the cell wall. This approach helped elucidate the nature of plant cell wall chemical recalcitrance and biomass pretreatment chemistry by tracking cell wall fragmentation as a function of time.more » Fractions of the reactor effluent were collected in a time-resolved fashion and characterized by various NMR techniques to determine the degree and sequence of fragments released, as well as, the chemical composition, molecular structure, and relative molecular weight of those released fragments.« less

Structural Dissection of Crotalicidin, a Rattlesnake Venom Cathelicidin, Retrieves a Fragment with Antimicrobial and Antitumor Activity.

PubMed

Falcao, Claudio Borges; Pérez-Peinado, Clara; de la Torre, Beatriz G; Mayol, Xavier; Zamora-Carreras, Héctor; Jiménez, M Ángeles; Rádis-Baptista, Gandhi; Andreu, David

2015-11-12

In silico dissection of crotalicidin (Ctn), a cathelicidin from a South American pit viper, yielded fragments Ctn[1-14] and Ctn[15-34], which were tested to ascertain to what extent they reproduced the structure and activity of the parent peptide. NMR data showing Ctn to be α-helical at the N-terminus and unstructured at the C-terminus were matched by similar data from the fragments. The peptides were tested against Gram-positive and -negative bacteria and for toxicity against both tumor and healthy cells. Despite its amphipathic α-helical structure, Ctn[1-14] was totally inert toward bacteria or eukaryotic cells. In contrast, unstructured Ctn[15-34] replicated the activity of parent Ctn against Gram-negative bacteria and tumor cells while being significantly less toxic toward eukaryotic cells. This selectivity for bacteria and tumor cells, plus a stability to serum well above that of Ctn, portrays Ctn[15-34] as an appealing candidate for further development as an anti-infective or antitumor lead.

The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

PubMed Central

Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173

Indirect-fired gas turbine dual fuel cell power cycle

DOEpatents

Micheli, Paul L.; Williams, Mark C.; Sudhoff, Frederick A.

1996-01-01

A fuel cell and gas turbine combined cycle system which includes dual fuel cell cycles combined with a gas turbine cycle wherein a solid oxide fuel cell cycle operated at a pressure of between 6 to 15 atms tops the turbine cycle and is used to produce CO.sub.2 for a molten carbonate fuel cell cycle which bottoms the turbine and is operated at essentially atmospheric pressure. A high pressure combustor is used to combust the excess fuel from the topping fuel cell cycle to further heat the pressurized gas driving the turbine. A low pressure combustor is used to combust the excess fuel from the bottoming fuel cell to reheat the gas stream passing out of the turbine which is used to preheat the pressurized air stream entering the topping fuel cell before passing into the bottoming fuel cell cathode. The CO.sub.2 generated in the solid oxide fuel cell cycle cascades through the system to the molten carbonate fuel cell cycle cathode.

The global regulatory architecture of transcription during the Caulobacter cell cycle.

PubMed

Zhou, Bo; Schrader, Jared M; Kalogeraki, Virginia S; Abeliuk, Eduardo; Dinh, Cong B; Pham, James Q; Cui, Zhongying Z; Dill, David L; McAdams, Harley H; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

PubMed

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

PubMed Central

Johard, Helena; Mahdessian, Diana; Fedr, Radek; Marks, Carolyn; Medalová, Jiřina; Souček, Karel; Lundberg, Emma; Linnarsson, Sten; Bryja, Vítězslav; Sekyrova, Petra; Altun, Mikael; Andäng, Michael

2017-01-01

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. PMID:29228002

Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-β1 and G-CSF.

PubMed

Marmotti, A; Bonasia, D E; Bruzzone, M; Rossi, R; Castoldi, F; Collo, G; Realmuto, C; Tarella, C; Peretti, G M

2013-08-01

Minced chondral fragments are becoming popular as a source of cells for cartilage repair, as a growing interest is developing towards one-stage procedures to treat cartilage lesions. The purpose of this study is to (A) compare cell outgrowth from cartilage fragments of adult and young donors using two different types of scaffolds and (B) evaluate the influence of transforming-growth-factor-β1 (TGF-β1) and granulocyte colony-stimulating factor (G-CSF) on chondrocyte behaviour. In part (A) cartilage fragments from adult and young donors were either loaded onto an HA-derivative injectable paste scaffold or onto an HA-derivative membrane scaffold. Construct sections were then examined for cell counting after 1, 2 and 3 months. In part (B) only membrane scaffolds were prepared using cartilage fragments from young donors. Constructs were cultured either in standard growth medium or in the presence of specific growth factors, such as TGF-β1 or G-CSF or TGF-β1 + G-CSF. After 1 month, construct sections were examined for cell counting. Expression of chondrocyte markers (SOX9, CD151, CD49c) and proliferative markers (β-catenin, PCNA) was assessed using immunofluorescence techniques, both in unstimulated construct sections and in cells from unstimulated and stimulated construct cultures. Part (A): histological analysis showed age-dependent and time-dependent chondrocyte migration. A significant difference (p < 0.05) was observed between young and older donors at the same time point. No difference was detected between the two types of scaffolds within the same group at the same time point. Part (B): after 1 month, the number of migrating cells/area significantly increased due to exposure to TGF-β1 and/or G-CSF (p < 0.05). Immunofluorescence revealed that outgrowing cells from unstimulated scaffold sections were positive for SOX9, CD151, CD49c and G-CSF receptor. Immunofluorescence of cells from construct cultures showed an increase in β-catenin in all stimulated groups and an increased PCNA expression in G-CSF-exposed cultures (p < 0.05). Outgrowing cells may represent a subset of chondrocytes undergoing a phenotypic shift towards a proliferative state. TGF-β1, and to a greater extent G-CSF, may accelerate this outgrowth. The clinical relevance of this study may involve a potential future clinical application of scaffolds preloaded with growth factors as an additional coating for chondral fragments. Indeed, a controlled delivery of G-CSF, widely employed in various clinical settings, might improve the repair process driven by minced human cartilage fragments during one-stage cartilage repair.

RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

PubMed Central

Spiering, Michelle M.; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J.

2017-01-01

The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes. PMID:28507156

Fragment-based approaches to anti-HIV drug discovery: state of the art and future opportunities.

PubMed

Huang, Boshi; Kang, Dongwei; Zhan, Peng; Liu, Xinyong

2015-12-01

The search for additional drugs to treat HIV infection is a continuing effort due to the emergence and spread of HIV strains resistant to nearly all current drugs. The recent literature reveals that fragment-based drug design/discovery (FBDD) has become an effective alternative to conventional high-throughput screening strategies for drug discovery. In this critical review, the authors describe the state of the art in FBDD strategies for the discovery of anti-HIV drug-like compounds. The article focuses on fragment screening techniques, direct fragment-based design and early hit-to-lead progress. Rapid progress in biophysical detection and in silico techniques has greatly aided the application of FBDD to discover candidate agents directed at a variety of anti-HIV targets. Growing evidence suggests that structural insights on key proteins in the HIV life cycle can be applied in the early phase of drug discovery campaigns, providing valuable information on the binding modes and efficiently prompting fragment hit-to-lead progression. The combination of structural insights with improved methodologies for FBDD, including the privileged fragment-based reconstruction approach, fragment hybridization based on crystallographic overlays, fragment growth exploiting dynamic combinatorial chemistry, and high-speed fragment assembly via diversity-oriented synthesis followed by in situ screening, offers the possibility of more efficient and rapid discovery of novel drugs for HIV-1 prevention or treatment. Though the use of FBDD in anti-HIV drug discovery is still in its infancy, it is anticipated that anti-HIV agents developed via fragment-based strategies will be introduced into the clinic in the future.

RNA primer-primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication.

PubMed

Spiering, Michelle M; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J

2017-05-30

The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior-that is, the signaling mechanism-have not been definitively identified. We examined the role of RNA primer-primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer-primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer-primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer-primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cell - Update II

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1992-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV) nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles, compared to 3500 cycles for cells containing 31 percent KOH. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2X normal rate). The depth-of-discharge was 80 percent. Six 48-Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16,000 cycles during the continuing test.

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

PubMed

Hayashi-Takanaka, Yoko; Stasevich, Timothy J; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

2014-01-01

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria.

PubMed

Moreira, Wilfried; Lim, Jia Jie; Yeo, Si Ying; Ramanujulu, Pondy M; Dymock, Brian W; Dick, Thomas

2016-01-01

Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate.

The cell cycle.

PubMed

Singh, N; Lim, R B; Sawyer, M A

2000-07-01

The cell cycle and the cell cycle control system are the engines that drive life. They allow for the processes of cell renewal and the growth of organisms, under controlled conditions. The control system is essential for the monitoring of normal cell growth and replication of genetic material and to ensure that normal, functional daughter cells are produced at completion of each cell cycle. Although certain clinical applications exist which take advantage of the events of the cell cycle, our understanding of its mechanisms and how to manipulate them is infantile. The next decades will continue to see the effort of many researchers focused upon unlocking the mysteries of the cell cycle and the cell cycle control system.

Soluble fragments of e-cadherin cell-adhesion molecule increase in urinary-excretion of cancer-patients, potentially indicating its shedding from epithelial tumor-cells.

PubMed

Katayama, M; Hirai, S; Yasumoto, M; Nishikawa, K; Nagata, S; Otsuka, M; Kamihagi, K; Kato, I

1994-11-01

E-cadherin (Ecad) is well known to be a calcium-ion-dependent cell-cell adhesion molecule expressed mostly in epithelial tissues. Previous immunohistochemical studies suggested that this cell adhesion molecule acts as an invasion suppressor and is negligibly detected in cancer metastatic regions. Soluble Ecad fragments derived from the proteolysed membrane-associated form were detected in culture supernatants of two cell lines, COLO 205 and A-431, with normal distribution of cell surface Ecad. Soluble Ecad levels released into culture of COLO 205 exhibiting reduced cell-cell adhesion were apparently elevated above those of A-431 with tight cell-cell adhesion. Furthermore, human circulation and urine continuously contain soluble Ecad which consists mainly of homogeneous 75-85 kDa extracellular domains. Soluble Ecad urinary level per urinary creatinine level was found to be significantly elevated in 53% of patients suffering from various types of cancers including lung, liver, stomach, colon and rectal cancers, as compared with those in the age-matched healthy subjects. These results suggest that dysfunction of cell surface Ecad is responsible for its enhanced proteolytic shedding in tumorigenesis, which may lead to the decrease of cell surface Ecads. Furthermore, excretion of high levels of soluble Ecad fragments potentially indicates the progression of epithelial tumors excessively degrading cell surface Ecad in clinical subjects.

Does habitat fragmentation cause stress in the agile antechinus? A haematological approach.

PubMed

Johnstone, Christopher P; Lill, Alan; Reina, Richard D

2012-01-01

Although the vertebrate stress response is essential for survival, frequent or prolonged stress responses can result in chronic physiological stress, which is associated with a suite of conditions that can impair survivorship and reproductive output. Anthropogenic habitat fragmentation and degradation are potential stressors of free-living vertebrates, and in theory could result in chronic stress. To address this issue, we compared haematological indicators of stress and condition in agile antechinus (Antechinus agilis) populations in 30 forest fragments and 30 undisturbed, continuous forest sites (pseudofragments) in south-eastern Australia over 2 years. In peripheral blood, the total leucocyte count was lower and the neutrophil/lymphocyte ratio and percentage of eosinophils in the total leucocyte population was higher in fragment than pseudofragment populations, indicating that fragment populations were probably experiencing higher levels of stress hormone-mediated and/or parasite infection-related chronic physiological stress. The total erythrocyte count and haematocrit were higher and mean erythrocyte haemoglobin content was lower in fragment than pseudofragment populations. This suggests that fragment populations showed possible signs of regenerative anaemia, a syndrome associated with elevated hypothalamus-pituitary-adrenal axis mediated stress. However, mean erythrocyte volume was also lower in fragments, and red blood cell distribution width did not differ between the study populations, findings which were not consistent with this diagnosis. Whole blood and mean cell haemoglobin concentrations were similar in fragment and pseudofragment populations. We suggest that where anthropogenic activity results in habitat fragmentation and degradation, chronic stress could contribute to a decline in agile antechinus populations. The broader implication is that chronic stress could be both symptomatic of, and contributing to, decline of some vertebrate populations in anthropogenically fragmented and degraded habitats.

Discovery of and Use of Fragments of DOC1 as Antiangiogenic and Antitumor Therapy | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes small cDNA fragments of the coding region for wild type filamin A interacting protein 1-like (FILIP1L) and variant 2 of FILIP1L genes that encode proteins inhibit cell migration and motility, induce cell apoptosis and inhibit cell proliferation. The significance of this invention is that it could provide for a series of new anti-cancer therapeutics and for the diagnostic means to follow their expression levels.

Pathological implications of cell cycle re-entry in Alzheimer disease.

PubMed

Bonda, David J; Lee, Hyun-pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

2010-06-29

The complex neurodegeneration underlying Alzheimer disease (AD), although incompletely understood, is characterised by an aberrant re-entry into the cell cycle in neurons. Pathological evidence, in the form of cell cycle markers and regulatory proteins, suggests that cell cycle re-entry is an early event in AD, which precedes the formation of amyloid-beta plaques and neurofibrillary tangles (NFTs). Although the exact mechanisms that induce and mediate these cell cycle events in AD are not clear, significant advances have been made in further understanding the pathological role of cell cycle re-entry in AD. Importantly, recent studies indicate that cell cycle re-entry is not a consequence, but rather a cause, of neurodegeneration, suggesting that targeting of cell cycle re-entry may provide an opportunity for therapeutic intervention. Moreover, multiple inducers of cell cycle re-entry and their interactions in AD have been proposed. Here, we review the most recent advances in understanding the pathological implications of cell cycle re-entry in AD.

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

PubMed

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-08-17

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

The Effect of Sleep Continuity on Pain in Adults with Sickle Cell Disease

PubMed Central

Moscou-Jackson, Gyasi; Finan, Patrick H.; Campbell, Claudia M.; Smyth, Joshua M.; Haythornthwaite, Jennifer A.

2015-01-01

This analysis examined the influence of quantifiable parameters of daily sleep continuity, primarily sleep duration and sleep fragmentation, on daily pain in adults with Sickle Cell Disease (SCD). Seventy-five adults with SCD completed baseline psychosocial measures and daily morning (sleep) and evening (pain) diaries over a three-month period. Mixed-effect modeling was used to examine daily between- and within-subjects effects of sleep continuity parameters on pain, as well as the synergistic effect of sleep fragmentation and sleep duration on pain. Results revealed nights of shorter sleep duration and time in bed, increased fragmentation, and less efficient sleep (relative to one’s own mean) were followed by days of greater pain severity. Further, the analgesic benefit of longer sleep duration was attenuated when sleep fragmentation was elevated. These results suggest that both the separate and combined effects of sleep duration and fragmentation should be considered in evaluating pain in adults with SCD. PMID:25842346

Different cell death pathways induced by drugs in Trypanosoma cruzi: an ultrastructural study.

PubMed

Menna-Barreto, Rubem F S; Salomão, Kelly; Dantas, Andréia P; Santa-Rita, Ricardo M; Soares, Maurilio J; Barbosa, Helene S; de Castro, Solange L

2009-02-01

Electron microscopy has proven to be a reliable and essential tool to determine morphological alterations and target organelles in the investigation of new drugs for Chagas disease. In this review, we focused on evaluating different agents that induce death of Trypanosoma cruzi, i.e. lysophospholipids analogues, naphthoquinones and derivatives, cytoskeletal inhibitors and natural products. Apoptosis-like presents as morphological characteristics DNA fragmentation, membrane blebbing and apoptotic body formation. Autophagy involves autophagosome formation, with the appearance of membranes surrounding organelles and cytosolic structures. Necrosis causes the loss of osmotic balance, an increase of cytoplasmic vacuolization and plasma membrane disruption. Mitochondrion appears as a central checkpoint in both apoptosis and necrosis. Our evidences of ultrastructural changes to T. cruzi treated with the different classes of compounds point to dramatic mitochondrial alterations and similar autophagic phenotypes. Lysophospholipid analogues interfere in the lipid biosynthesis in epimastigotes, altering the amount of both phospholipids and sterols, and consequently the physical properties of the membrane. Naphthoquinone derivatives led to a strong DNA fragmentation in trypomastigotes and to the release of cysteine proteases from reservosomes to cytosol in epimastigotes, starting a proteolytic process which results in parasite death. The susceptibility of reservosomes was also observed in parasites treated with propolis, suggesting impairment of lipid metabolism, compromising membrane fluidity and leading to lysis. The cytoskeletal agents blocked mitosis of epimastigotes, arresting cell cycle and impairing the parasite proliferation. The variety of drug stimuli converge to the same pathway of death suggests an intense cross-talking between the three types of PCD in the protozoa.

Symmetrical choline-derived dications display strong anti-kinetoplastid activity

PubMed Central

Ibrahim, Hasan M. S.; Al-Salabi, Mohammed I.; El Sabbagh, Nasser; Quashie, Neils B.; Alkhaldi, Abdulsalam A. M.; Escale, Roger; Smith, Terry K.; Vial, Henri J.; de Koning, Harry P.

2011-01-01

Objectives To investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. Methods From an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. Results The activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC50 values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24–48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. Conclusions The choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation. PMID:21078603

Voronoi cell patterns: Theoretical model and applications

NASA Astrophysics Data System (ADS)

González, Diego Luis; Einstein, T. L.

2011-11-01

We use a simple fragmentation model to describe the statistical behavior of the Voronoi cell patterns generated by a homogeneous and isotropic set of points in 1D and in 2D. In particular, we are interested in the distribution of sizes of these Voronoi cells. Our model is completely defined by two probability distributions in 1D and again in 2D, the probability to add a new point inside an existing cell and the probability that this new point is at a particular position relative to the preexisting point inside this cell. In 1D the first distribution depends on a single parameter while the second distribution is defined through a fragmentation kernel; in 2D both distributions depend on a single parameter. The fragmentation kernel and the control parameters are closely related to the physical properties of the specific system under study. We use our model to describe the Voronoi cell patterns of several systems. Specifically, we study the island nucleation with irreversible attachment, the 1D car-parking problem, the formation of second-level administrative divisions, and the pattern formed by the Paris Métro stations.

Salt Weathering on Mars

NASA Astrophysics Data System (ADS)

Jagoutz, E.

2006-12-01

Large well rounded boulders and angular rock fragments characterizes the Martian landscape as seen on the recent excellent quality photos. Analyzing the different rock-shapes indicates a time sequence of emplacement, fragmentation and transport of different rocks on Mars, which might give interesting insight into transport and weathering processes. Larger commonly well rounded boulders were emplaced onto gravel plains. After emplacement, these rocks were fragmented and disassembled. Nests of angular rock fragments are marking the locations of preexisting larger rocks. Frequently it is possible to reconstruct larger rounded rocks from smaller angular fragments. In other cases transport after fragmentation obscured the relationship of the fragments. However, a strewn field of fragments is still reminiscent of the preexisting rock. Mechanical salt weathering could be a plausible explanation for the insitu fragmentation of larger rounded blocks into angular fragments. Impact or secondary air fall induced fragmentation produces very different patterns, as observed around impact crates on Earth. Salt weathering of rocks is a common process in terrestrial environments. Salt crystallization in capillaries causes fragmentation of rocks, irrespective of the process of salt transportation and concentration. On Earth significant salt weathering can be observed in different climatic environments: in the transition zone of alluvial aprons and salt playas in desserts and in dry valleys of Antarctica. In terrestrial semi-arid areas the salt is transported by salt solution, which is progressively concentrated by evaporation. In Antarctic dry valleys freeze-thaw cycles causes salt transportation and crystallization resulting in rock fragmentation. This salt induced process can lead to complete destruction of rocks and converts rocks to fine sand. The efficient breakdown of rocks is dominating the landscape in some dry valleys of the Earth but possibly also on Mars. (Malin, 1974). However, irrespectively of the climatic environment a liquid brine is a necessity for salt induced fragmentation of rocks.M. C. Malin (1974) JGR Vol 79,26 p 3888-3894

Episodic Aging and End States of Comets

NASA Technical Reports Server (NTRS)

Sekanina, Zdenek

2008-01-01

It is known that comets are aging very rapidly on cosmic scales, because they rapidly shed mass. The processes involved are (i) normal activity - sublimation of ices and expulsion of dust from discrete emission sources on and/or below the surface of a comet's nucleus, and (ii) nuclear fragmentation. Both modes are episodic in nature, the latter includes major steps in the comet's life cycle. The role and history of dynamical techniques used are described and results on mass losses due to sublimation and dust expulsion are reviewed. Studies of split comets, Holmes-like exploding comets, and cataclysmically fragmenting comets show that masses of 10 to 100 million tons are involved in the fragmentation process. This and other information is used to investigate the nature of comets' episodic aging. Based on recent advances in understanding the surface morphology of cometary nuclei by close-up imaging, a possible mechanism for large-scale fragmentation events is proposed and shown to be consistent with evidence available from observations. Strongly flattened pancake-like shapes appear to be required for comet fragments by conceptual constraints. Possible end states are briefly examined.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

PubMed

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

PubMed Central

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R.

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase. PMID:19729042

Chromosomal mosaicism in mouse two-cell embryos after paternal exposure to acrylamide

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

Marchetti, Francesco; Bishop, Jack; Lowe, Xiu

2008-10-14

Chromosomal mosaicism in human preimplantation embryos is a common cause ofspontaneous abortions, however, our knowledge of its etiology is limited. We used multicolor fluorescence in situ hybridization (FISH) painting to investigate whether paternally-transmitted chromosomal aberrations result in mosaicism in mouse 2-cell embryos. Paternal exposure to acrylamide, an important industrial chemical also found in tobacco smoke and generated during the cooking process of starchy foods, produced significant increases in chromosomally defective 2-cell embryos, however, the effects were transient primarily affecting the postmeiotic stages of spermatogenesis. Comparisons with our previous study of zygotes demonstrated similar frequencies of chromosomally abnormal zygotes and 2-cellmore » embryos suggesting that there was no apparent selection against numerical or structural chromosomal aberrations. However, the majority of affected 2-cell embryos were mosaics showing different chromosomal abnormalities in the two blastomeric metaphases. Analyses of chromosomal aberrations in zygotes and 2-cell embryos showed a tendency for loss of acentric fragments during the first mitotic division ofembryogenesis, while both dicentrics and translocations apparently underwent propersegregation. These results suggest that embryonic development can proceed up to the end of the second cell cycle of development in the presence of abnormal paternal chromosomes and that even dicentrics can persist through cell division. The high incidence of chromosomally mosaic 2-cell embryos suggests that the first mitotic division of embryogenesis is prone to missegregation errors and that paternally-transmitted chromosomal abnromalities increase the risk of missegregation leading to embryonic mosaicism.« less

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

PubMed

Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

2016-07-01

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.