Clinicopathological correlates and prognostic significance of glutathione S-transferase Pi expression in 468 patients after potentially curative resection of node-positive colonic cancer.

PubMed

Tan, King L; Jankova, Lucy; Chan, Charles; Fung, Caroline L-S; Clarke, Candice; Lin, Betty P C; Robertson, Graham; Molloy, Mark; Chapuis, Pierre H; Bokey, Les; Dent, Owen F; Clarke, Stephen J

2011-12-01

This study investigated the association between glutathione S-transferase Pi (GST Pi) expression, histopathology and overall survival in 468 patients after resection of stage C colonic adenocarcinoma. Data were drawn from a prospective hospital registry of consecutive bowel cancer resections with a minimum follow-up of 5 years. Nuclear and cytoplasmic GST Pi expression, assessed by both intensity of staining and percentage of stained cells at both the central part of the tumour and the invasive tumour front, were evaluated retrospectively by tissue microarray immunohistochemistry on archival specimens. The most effective measure of GST Pi expression was the percentage of immunostained nuclei in central tumour tissue, where >40% stained was associated significantly with high grade, invasion beyond the muscularis propria, involvement of a free serosal surface or apical node, and invasion into an adjacent organ or structure. After adjustment of other predictors, GST Pi expression remained independently prognostic for reduced overall survival (hazard ratio 1.4, P = 0.002). In patients with clinicopathological stage C colonic cancer, GST Pi expression is associated with features of tumour aggressiveness and with reduced overall survival. Further appropriately designed studies should aim to discover whether GST Pi can predict response to adjuvant chemotherapy. © 2011 Blackwell Publishing Limited.

Mangiferin prevents the growth of gastric carcinoma by blocking the PI3K-Akt signalling pathway.

PubMed

Du, Min; Wen, Gang; Jin, Juan; Chen, Yuanguang; Cao, Jun; Xu, Aman

2017-12-05

The aim of the present study was to investigate the effects of mangiferin on gastric carcinoma cells and to determine the possible mechanisms underlying such effects. The MTT assay was performed to evaluate the antiproliferative effect of mangiferin. Following treatment, apoptosis rates of SGC-7901 were established by flow cytometry and laser confocal microscopy, and western blot analysis was used to detect the expression of apoptosis-related proteins. The MTT assay showed that mangiferin inhibited the proliferation of SGC-7901 and BCG-823 cells in a dose-dependent and time-dependent manner. After SGC-7901 cells were exposed to mangiferin for 24, 48 and 72 h, the half-maximal inhibitory concentration values were 16.00, 8.63 and 4.79 µmol/l, respectively. SGC-7901 cell apoptosis induced by mangiferin was observed by Annexin V/PI doubling staining and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive staining. We found a significant decrease in Bcl-2, Bcl-xL and Mcl-1 expression and a significant increase in Bax, Bad and cleaved caspase-3 and caspase-9 expression in SGC-7901 cells by mangiferin treatment. Moreover, mangiferin significantly decreased the levels of p-PI3K, p-Akt and p-mTOR, but had no effects on those of PI3K, Akt and mTOR in epidermal growth factor-treated SGC-7901 cells. Interestingly, the proapoptotic effect of mangiferin on SGC-7901 cells was partially blocked by the Akt activator SC79, whereas LY294002 significantly increased mangiferin-induced apoptosis and growth inhibition. Taken together, our findings indicate that mangiferin effectively inhibits cell growth and induces apoptosis of gastric cancer cells through inhibiting the PI3K/Akt pathways with relative safety, and may be used as a novel chemotherapeutic agent against gastric cancer.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

PubMed

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K

2008-03-01

BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

PubMed Central

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

2010-01-01

Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability. PMID:20814586

A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

PubMed

Russell, R L; Rohrmann, G F

1990-01-01

A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

Evaluation of toxicity of essential oils palmarosa, citronella, lemongrass and vetiver in human lymphocytes.

PubMed

Sinha, Sonali; Jothiramajayam, Manivannan; Ghosh, Manosij; Mukherjee, Anita

2014-06-01

The present investigation was undertaken to study the cytotoxic and genotoxic potential of the essential oils (palmarosa, citronella, lemongrass and vetiver) and monoterpenoids (citral and geraniol) in human lymphocytes. Trypan blue dye exclusion and MTT test was used to evaluate cytotoxicity. The genotoxicity studies were carried out by comet and DNA diffusion assays. Apoptosis was confirmed by Annexin/PI double staining. In addition, generation of reactive oxygen species was evaluated by DCFH-DA staining using flow cytometry. The results demonstrated that the four essential oils and citral induced cytotoxicity and genotoxicity at higher concentrations. The essential oils were found to induce oxidative stress evidenced by the generation of reactive oxygen species. With the exception of geraniol, induction of apoptosis was confirmed at higher concentrations of the test substances. Based on the results, the four essential oils are considered safe for human consumption at low concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Double phosphorylation of the myosin regulatory light chain during rigor mortis of bovine Longissimus muscle.

PubMed

Muroya, Susumu; Ohnishi-Kameyama, Mayumi; Oe, Mika; Nakajima, Ikuyo; Shibata, Masahiro; Chikuni, Koichi

2007-05-16

To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis. Bovine; myosin regulatory light chain (RLC, MyLC2); phosphorylation; rigor mortis; skeletal muscle.

Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells.

PubMed

Da Costa, Andréia Ferreira Eduardo; Pereira, Camila Serva; Santos, Gabriela Da Silva; Carvalho, Técia Maria Ulisses; Hirata, Raphael; De Mattos-Guaraldi, Ana Luiza; Rosa, Ana Cláudia De Paula; Nagao, Prescilla Emy

2011-05-01

Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.

Quercetin suppresses HeLa cells by blocking PI3K/Akt pathway.

PubMed

Xiang, Tao; Fang, Yong; Wang, Shi-Xuan

2014-10-01

To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.

Molecular hydrogen protects human lymphocyte AHH-1 cells against 12C6+ heavy ion radiation.

PubMed

Yang, Yanyong; Gao, Fu; Zhang, Hong; Hunag, Yijuan; Zhang, Pei; Liu, Cong; Li, Bailong; Cai, Jianming

2013-12-01

To investigate the potential protective role of molecular hydrogen (H(2)) against (12)C(6+) heavy ion radiation, which is a major hazard for space travel and has been also widely used in heavy ion radiotherapy. H(2) was dissolved in Roswell Park Memorial Institute (RPMI) 1640 medium under high pressure (0.4 Mpa) to a saturated level by using an apparatus produced by our department. A 2-[6-(4'-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoate (HPF) probe and a 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFH-DA) fluorescent dye were used to measure the intracellular reactive oxygen species (ROS) level. Cell apoptosis were determined by double-staining with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) as well as a Hoechst 33342 staining method alternatively. Subsequently, cell cycle analysis was performed using a PI staining method and the expression of apoptotic protein was examined by Western blot. In this study, we demonstrated H(2) reduced ROS level in Human lymphocyte AHH-1 cells as well as in the radiolysis of water. Our data also showed H(2) attenuated (12)C(6+) radiation- induced cell apoptosis and also alleviated radiation-induced G2/M cell cycle arrest. Heavy ion radiation-induced Caspase 3 activation was also inhibited by H(2) treatment. In conclusion, these data showed that H(2) attenuated (12)C(6+) radiation-induced cell apoptosis through reducing the ROS level and modulating apoptotic molecules, thus indicating the potential of H(2) as a safe and effective radioprotectant.

Cytometric Approach for Detection of Encephalitozoon intestinalis, an Emergent Agent▿

PubMed Central

Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

2009-01-01

Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 μg/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 × 104 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 × g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health. PMID:19439525

Apoptosis and pro-death autophagy induced by a spirostanol saponin isolated from Rohdea chinensis (Baker) N. Tanaka (synonym Tupistra chinensis Baker) on HL-60 cells.

PubMed

Yi, Xiaomin; Xiang, Limin; Huang, Yuying; Wang, Yihai; He, Xiangjiu

2018-03-15

Our previous study has revealed that the spirostanol saponins isolated from the rhizomes of Rohdea chinensis (Baker) N. Tanaka (synonym Tupistra chinensis Baker) (Convallariaceae) (a reputed folk medicine) exhibited potent antiproliferative activity. However, the underlying mechanism of purified saponins remains unclear. More studies are necessary to assess the apoptosis and autophagy activities of the saponins from R. chinensis and clarify their antiproliferative mechanisms. The present study certificated the potential antiproliferative activity and mechanism of 5β-spirost-25(27)-en-1β,3β-diol-1-O-α-L-rhamnopyranosyl-(1→2)- β-D-xylopyranosyl-3-O-α-L-rhamnopyranoside (SPD), a spirostanol saponin from R. chinensis, against human acute promyelocytic leukemia cells (HL-60). The antiproliferative activity of SPD in vitro was evaluated by MTT assay compared with cis-dichlorodiammineplatinum (II). The autophagic activity was assessed using MDC staining and western blot, cell apoptosis inspection was detected by Annexin V-FITC/PI double staining and the mitochondrial membrane potential was detected by JC-1 fluorescence dye combined with flow cytometry. The potential mechanisms for protein levels of apoptosis and autophagy were evaluated by western blot. Treatment of HL-60 cells with SPD resulted in growth inhibition (IC 50 value of 2.0 ± 0.2 µM, after 48 h treatment) and induction of apoptosis and autophagy. Results from Annexin V-FITC/PI double-staining assay and mitochondrial membrane potential detection showed that apoptosis was happened after SPD treatment. The regulation of caspase-3, Bax, Bcl-2, PARP following SPD treatment contributed to the induction of mitochondria-dependent apoptosis. Meanwhile, SPD induced autophagy related with Akt/mTOR/p70S6K signaling and activated of AMPK signaling pathway. Furthermore, blocking autophagy with bafilomycin A1 reduced the cytotoxicity of SPD in HL-60 cells. The antiproliferative, apoptosis and pro-death autophagy activities of SPD suggested that spirostanol saponins from R. chinensis would be a potential cytotoxic candidate against acute promyelocytic leukemia. Copyright © 2018 Elsevier GmbH. All rights reserved.

Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

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

Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu

Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

DOE PAGES

Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu; ...

2015-06-03

Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

Reaction. pi. /sup -/p. --> pi. /sup -/. pi. /sup +/. pi. /sup -/p at 8 GeV/c

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

Kitagaki, T.; Tanaka, S.; Yuta, H.

1982-10-01

Results from a high-statistics experiment involving an exposure of the SLAC 82-in. hydrogen bubble chamber to a beam of 8-GeV/c ..pi../sup -/ yielding a final state of ..pi../sup -/..pi../sup +/..pi../sup -/p are presented. Copious production of rho, ..delta../sup + +/, and f is found. Considerable quasi-two-body production in which one particle decays to one of the above resonances is also observed. Some double-resonance production involving baryon and meson resonances is also seen. The production properties of rho, ..delta../sup + +/, and f mesons are well described by a double-Regge model.

Double immunohistochemical staining with MUC4/p53 is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray-based study.

PubMed

Bhardwaj, Atul; Marsh, William L; Nash, Jason W; Barbacioru, Catalin C; Jones, Susie; Frankel, Wendy L

2007-04-01

Immunohistochemical stains have been used for the distinction of pancreatic adenocarcinoma from chronic pancreatitis. To determine if a double stain for MUC/p53 improved specificity and sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis by comparing maspin, mucin 4 (MUC4), p53, Smad4, and the double stain MUC4/p53. Seventy-four pancreatic adenocarcinomas and 19 chronic pancreatitis cases were retrieved from archival files. Tissue cores were arrayed to create a tissue microarray of 2-mm cores. Sections were stained with antibodies against maspin, MUC4, p53, and Smad4. Additionally, a 2-color, double stain for MUC4 and p53 was developed and evaluated. Five percent or greater staining in either of the cores was considered positive. Intensity (0, 1, 2) and extent (%) of tumor cells staining was also determined. The sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis with maspin, MUC4, p53, and Smad4 was 90%, 77%, 60%, and 63%, respectively; the specificity was 67%, 78%, 88%, and 88%, respectively. When MUC4 and p53 were combined in a double stain, and positive staining for either considered a positive result, the sensitivity increased to 96% but specificity was 73%. When immunoreactivity for both antibodies was necessary for a positive result, sensitivity fell to 39% but specificity was 100%. No correlation was found between intensity or extent of staining with any of the individual stains and tumor differentiation. The double immunohistochemical stain for MUC4/p53 can be a useful diagnostic tool in conjunction with the hematoxylin-eosin-stained section for pancreatic adenocarcinoma, particularly when limited tumor is available for multiple stains.

Changes in energy metabolism in the quadriceps femoris after a single bout of acute exhaustive swimming in rats: a ³¹P-magnetic resonance spectroscopy study.

PubMed

Sun, Yingwei; Pan, Shinong; Chen, Zhian; Zhao, Heng; Ma, Ying; Zheng, Liqiang; Li, Qi; Deng, Chunbo; Fu, Xihu; Lu, Zaiming; Guo, Qiyong

2014-01-01

Little is known about the value of (31)P-magnetic resonance spectroscopy ((31)P-MRS) in in vivo assessment of exhaustive exercise-induced injury in skeletal muscle. We aimed to evaluate the value of a (31)P-MRS study using the quadriceps femoris after a single bout of acute exhaustive swimming in rats, and the correlation between (31)P-MRS and histological changes. Sixty male Sprague-Dawley rats were randomly assigned to control, half-exhaustive, and exhaustive exercise groups. (31)P-MRS of the quadriceps femoris of the right lower limb was performed immediately after swimming exercise to detect Pi, PCr, and β-ATP. The Pi/PCr, Pi/β-ATP, PCr/β-ATP, and PCr/(PCr+Pi) were calculated and pH measured. Areas under the receiver operating characteristic curve (AUCs) were calculated to evaluate the diagnostic potential of (31)P-MRS in identifying and distinguishing the three groups. HE staining, electron microscopy and desmin immunostaining after imaging of the muscle were used as a reference standard. The correlation between (31)P-MRS and the mean absorbance (A value) of desmin staining were analyzed with the Pearson correlation test. Pi, PCr, Pi/PCr, and PCr/(PCr+Pi) showed statistically significant intergroup differences (P < 0.05). AUCs of Pi, PCr, Pi/PCr, and PCr/(PCr+Pi) were 0.905, 0.848, 0.930, and 0.930 for the control and half-exhaustive groups, while sensitivity and specificity were 90%/85%, 95%/55%, 95%/80%, and 90%/85%, respectively. The AUCs of Pi, PCr, Pi/PCr and PCr/(PCr+Pi) were 0.995, 0.980, 1.000, and 1.000 for the control and exhaustive groups, while sensitivity and specificity were 95%/90%, 100%/90%, 100%/95%, and 100%/95%, respectively. The AUCs of Pi, PCr, Pi/PCr, and PCr/(PCr+Pi) were 0.735, 0.865, 0.903, and 0.903 for the half-exhaustive and exhaustive groups, while sensitivity and specificity were 80%/60%, 90%/75%, 95%/65%, and 95%/70%, respectively. In the half-exhaustive group, some muscle fibers exhibited edema in HE staining, and the unclear Z-discs and the mitochondria with vacuolar degeneration under electron microscopy. Compared with the half-exhaustive group, muscle fiber edema was increased in the exhaustive group, and the Z-discs were broken and the mitochondria exhibited marked vacuolar degeneration under electron microscopy. There were significant difference in A values of desmin staining in the right vastus lateralis among the control, half-exhaustive, and exhaustive groups with 0.58 ± 0.06, 0.30 ± 0.04, and 0.21 ± 0.02, respectively (P < 0.05). Histological examination also showed injury-induced changes in the vastus lateralis among the different intensities groups. Statistically a moderate correlation between (31)P-MRS and desmin was observed, the correlation coefficients of Pi, PCr, Pi/PCr, and PCr/(PCr+Pi) were -0.706, 0.709, -0.726, and 0.791, respectively (P < 0.01). (31)P-MRS can effectively reflect the changes in energy metabolism in the skeletal muscle after a single bout of acute exhaustive swimming in rats. Based on the significant correlation between (31)P-MRS parameters and histological changes, the changes of Pi, PCr, Pi/PCr, and PCr/(PCr+Pi) can indirectly reflect the degree of exercise-induced injury.

Virus replication cycle of white spot syndrome virus in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei.

PubMed

Li, Wenfeng; Desmarets, Lowiese M B; De Gryse, Gaëtan M A; Theuns, Sebastiaan; Van Tuan, Vo; Van Thuong, Khuong; Bossier, Peter; Nauwynck, Hans J

2015-09-01

The replication cycle of white spot syndrome virus (WSSV) was investigated in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei. The secondary cells formed a confluent monolayer at 24 h post-reseeding, and this monolayer could be maintained for 10 days with a viability of 90 %. Binding of WSSV to cells reached a maximum (73 ± 3 % of cells and 4.84 ± 0.2 virus particles per virus-binding cell) at 120 min at 4 °C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30 min post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60 min p.i. and that the uncoating of WSSV occurred in the same period. After 1 h inoculation at 27 °C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3 h p.i., and were transported into nuclei from 3 and 6 h p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50 ± 4 %) at 12 h p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6 h p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12 h p.i. and peaked at 18 h p.i. In conclusion, the secondary cell cultures from the lymphoid organ were proven to be ideal for examination of the replication cycle of WSSV.

Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage.

PubMed

Huo, Li-Jun; Ma, Xing-Hong; Yang, Zeng-Ming

2002-10-15

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.

Synthesis of a posterior indicator protein in normal embryos and double abdomens of Smittia sp. (Chironomidae, Diptera).

PubMed Central

Jäckle, H; Kalthoff, K

1980-01-01

In embryos of the chironomid midge Smittia, synthesis of a posterior indicator protein designated PI1 (Mr approximately 50,000; pI approximately 5.5) forecasts development of an abdomen as opposed to head and thorax. The protein is synthesized several hours before germ anlage formation. In normal embryos at early blastoderm stages, synthesis of PI1 is restricted to posterior embryonic fragments but not to pole cells. In "double-abdomen" embryos, a mirror-image duplication of the abdomen is formed by cells that would otherwise develop into head and thorax. Embryos were programmed for double-abdomen development by UV irradiation of the anterior pole, and half of them were reprogrammed for normal development by subsequent exposure to visible light (photoreversal). Correspondingly, PI1 was synthesized in anterior fragments of UV-irradiated embryos but not after photoreversal. In a control experiment, UV irradiation of the posterior pole caused neither double-abdomen formation nor PI1 synthesis in anterior fragments. The identity of PI1 formed in anterior fragments of prospective double abdomens with the protein found in posterior fragments was revealed by two-dimensional gel electrophoresis and limited proteolysis. Suppression of PI1 synthesis in anterior fragments of normal embryos is ascribed to the activity of cytoplasmic ribonucleoprotein particles thought to act as anterior determinants. Images PMID:6935679

[Oxidative stress promotes hepatocyte apoptosis mediated by glycogen synthase kinase 3β].

PubMed

Zhang, Xiangying; Guo, Yuanyuan; Zhang, Li; Wen, Tao; Piao, Zhengfu; Shi, Hongbo; Chen, Dexi; Duan, Zhongping; Ren, Feng

2015-01-01

To analyze the role of glycogen synthase kinase 3β (GSK3β) in hepatocyte apoptosis induced by oxidative stress. Human HL-7702 hepatoma cells were induced by H₂O₂/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H₂O₂/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide (PI) double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase (LDH) assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved caspase-3, c-Jun N-terminal kinase (JNK) and cytochrome C (CytC) proteins were examined using Western blotting. Oxidative stress triggered by H₂O₂/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased. GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

PubMed

Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

2016-09-01

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Involvement of PI3K/Akt, ERK and p38 signaling pathways in emodin-mediated extrinsic and intrinsic human hepatoblastoma cell apoptosis.

PubMed

Cui, Yuting; Lu, Peiran; Song, Ge; Liu, Qian; Zhu, Di; Liu, Xuebo

2016-06-01

As a natural anthraquinone derivative, 1,3,8-trihydroxy-6-methylanthraquinone, known as emodin, has recently been reported to possess potential chemopreventive capacity, but the underlying molecular mechanism of its hepatocyte toxicity remains poorly clarified. The present research indicated that emodin targeted HepG2 cells without being cytotoxic to primary human hepatocyte cells in comparison with chrysophanol and rhein. The anti-proliferative effect of emodin was ascribed to occurrence of apoptosis, which characterized by higher ethidium bromide signal, brighter DAPI fluorescence, cleavages of procaspase-3 and poly (ADP-ribose) polymerase as well as quantitative result from Annexin V-FITC/PI double staining. Furthermore, emodin improved Bax/Bcl-2 ratio, elicited disruption of mitochondrial membrane potential and promoted efflux of cytochrome c to cytosol, indicative of features of mitochondria-dependent apoptotic signals. Emodin concurrently led to activations of Fas, Fas-L, caspase-8 and tBid, which provoked death receptor apoptotic signals. Notably, activated tBid relayed the Fas apoptotic signal to the mitochondrial pathway. Besides, emodin effectively attenuated phosphorylations of Akt and ERK and promoted phosphorylation of p38. Inhibitions of PI3K/Akt and ERK and activation of p38 mediated emodin-induced apoptosis through modulating the mitochondrial pathway and/or death receptor pathway. Additionally, there was a cross-talk between PI3K/Akt and MAPKs pathways in emodin-induced apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

PubMed Central

Zuo, Hongyan; Lin, Tao; Wang, Dewen; Peng, Ruiyun; Wang, Shuiming; Gao, Yabing; Xu, Xinping; Li, Yang; Wang, Shaoxia; Zhao, Li; Wang, Lifeng; Zhou, Hongmei

2014-01-01

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm2. JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation. PMID:24688304

Immunoreactivities for glutathione S-transferases and glutathione peroxidase in the lateral wall of pigmented and albino guinea pig cochlea.

PubMed

Fujimura, Takeyuki; Suzuki, Hideaki; Udaka, Tsuyoshi; Shiomori, Teruo; Mori, Takanori; Inaba, Tsuyoshi; Hiraki, Nobuaki; Kayashima, Kotaro; Doi, Yoshiaki

2008-09-01

Dark-skinned people are known to be more tolerant of ototraumatic noise than are light-skinned people, and pigmented animals are more tolerant of ototraumatic noise and aminoglycoside ototoxicity than are albino animals. Such tolerance may be dependent on the local ability of detoxification and antioxidant enzymes, including glutathione S-transferase (GST) and glutathione peroxidase (GSPx). In the present study, we examined the difference in GST/GSPx expression in the lateral wall of the cochlea between pigmented and albino guinea pigs. Eight-week-old male pigmented and albino guinea pigs were killed by transcardiac perfusion with 2% paraformaldehyde. The cochlear ducts were isolated, further fixed with 4% paraformaldehyde, decalcified, and then embedded in paraffin. Sections prepared at 5-microm thickness were incubated with anti-GST-alpha,-mu,-pi, or anti-GSPx antibody, reacted with Alexa Fluorconjugated secondary antibody, and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The cochlea ducts were also subjected to immunoelectron microscopy for GST-pi by the postembedment method. The stria vascularis of pigmented guinea pigs was strongly immunoreactive for GST-alpha,-mu,-pi, and GSPx, whereas no or only weak immunoreactivities were seen in the stria vascularis of albino guinea pigs. The spiral ligament showed positive but different immunoreactivities for these enzymes between the strains. Double-stained immunofluorescence micrographs for GST-pi and GSPx showed a close resemblance of localization between the two enzymes in both pigmented and albino guinea pigs. At the ultrastructural level, immunoreactivity for GST-pi was localized preferentially in the melanin cells of pigmented guinea pigs. These results suggest that correlation between pigmentation and inner ear susceptibility is, at least partially, attributed to the different distribution of GST/GSPx in the stria vascularis.

A double-labeling immunohistochemical study of tau exon 10 in Alzheimer's disease, progressive supranuclear palsy and Pick's disease.

PubMed

Ishizawa, K; Ksiezak-Reding, H; Davies, P; Delacourte, A; Tiseo, P; Yen, S H; Dickson, D W

2000-09-01

Neurofibrillary tangles (NFT), one of the histopathological hallmarks of Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), and Pick bodies in Pick's disease (PiD) are composed of microtubule-associated protein tau, which is the product of alternative splicing of a gene on chromosome 17. Alternative expression of exon 10 leads to formation of three- or four-repeat tau isoforms. To study the differential expression of exon 10, we performed double-labeling immunohistochemistry of the hippocampal formation in nine AD, four PSP and three PiD cases. Cryostat sections were processed with and without formic acid (FA) treatment, and double-stained with anti-tau (Alz-50 or PHF-1) or anti-amyloid P component antibodies and one of two specific anti-exon 10 antibodies (E-10). The effect of proteinase-K treatment was also evaluated. The results suggest the following. First, in AD, E-10 immunoreactivity is present in most intracellular NFT, but not in most dystrophic neurites and neuropil threads, suggesting differential expression of tau isoforms in specific cellular domains. Second, in AD, E-10 immunoreactivity is lost or blocked in most extracellular NFT, possibly due to proteolysis. Third, in PSP, E-10 immunoreactivity is hidden or blocked in NFT and tau-positive glial inclusions, but FA treatment exposes the epitope consistent with the hypothesis that PSP inclusions contain four-repeat tau. Fourth, E-10 immunoreactivity is present in dentate fascia NFT in AD and PSP, but not in Pick bodies in the dentate fascia or other areas. The results suggest that expression of exon 10 in tau is specific for cellular domains in a disease-specific manner.

Cryptotanshinone induces cell cycle arrest and apoptosis through the JAK2/STAT3 and PI3K/Akt/NFκB pathways in cholangiocarcinoma cells.

PubMed

Ke, Fayong; Wang, Zheng; Song, Xiaoling; Ma, Qiang; Hu, Yunping; Jiang, Lin; Zhang, Yijian; Liu, Yingbin; Zhang, Yong; Gong, Wei

2017-01-01

Cholangiocarcinoma (CCA) is the most common biliary tract malignancy in the world with high resistance to current chemotherapies and extremely poor prognosis. The main objective of this study was to investigate the inhibitory effects of cryptotanshinone (CTS), a natural compound isolated from Salvia miltiorrhiza Bunge , on CCA both in vitro and in vivo and to explore the underlying mechanisms of CTS-induced apoptosis and cell cycle arrest. The anti-tumor activity of CTS on HCCC-9810 and RBE cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and Hoechst 33342 staining assays. The efficacy of CTS in vivo was evaluated using a HCCC-9810 xenograft model in athymic nude mice. The expression of key proteins involved in cell apoptosis and signaling pathway in vitro was analyzed by Western blot analysis. CTS induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in HCCC-9810 and RBE cells in a dose-dependent manner. Intraperitoneal injection of CTS (0, 10, or 25 mg/kg) for 4 weeks significantly inhibited the growth of HCCC-9810 xenografts in athymic nude mice. CTS treatment induced S-phase arrest with a decrease of cyclin A1 and an increase of cyclin D1 protein level. Bcl-2 expression was downregulated remarkably, while Bax expression was increased after apoptosis occurred. Additionally, the activation of JAK2/STAT3 and PI3K/Akt/NFκB was significantly inhibited in CTS-treated CCA cells. CTS induced CCA cell apoptosis by suppressing both the JAK2/STAT3 and PI3K/Akt/NFκB signaling pathways and altering the expression of Bcl-2/Bax family, which was regulated by these two signaling pathways. CTS may serve as a potential therapeutic agent for CCA.

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.

PubMed

Blank, Norbert; Burger, Renate; Duerr, Birgit; Bakker, Frank; Wohlfarth, Anika; Dumitriu, Ingrid; Kalden, Joachim R; Herrmann, Martin

2002-08-01

Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered. Copyright 2002 Wiley-Liss, Inc.

Anti-proliferative effect of biogenic gold nanoparticles against breast cancer cell lines (MDA-MB-231 & MCF-7)

NASA Astrophysics Data System (ADS)

K. S., Uma Suganya; Govindaraju, K.; Ganesh Kumar, V.; Prabhu, D.; Arulvasu, C.; Stalin Dhas, T.; Karthick, V.; Changmai, Niranjan

2016-05-01

Breast cancer is a major complication in women and numerous approaches are being developed to overcome this problem. In conventional treatments such as chemotherapy and radiotherapy the post side effects cause an unsuitable effect in treatment of cancer. Hence, it is essential to develop a novel strategy for the treatment of this disease. In the present investigation, a possible route for green synthesis of gold nanoparticles (AuNPs) using leaf extract of Mimosa pudica and its anticancer efficacy in the treatment of breast cancer cell lines is studied. The synthesized nanoparticles were found to be effective in killing cancer cells (MDA-MB-231 & MCF-7) which were studied using various anticancer assays (MTT assay, cell morphology determination, cell cycle analysis, comet assay, Annexin V-FITC/PI staining and DAPI staining). Cell morphological analysis showed the changes occurred in cancer cells during the treatment with AuNPs. Cell cycle analysis revealed apoptosis in G0/G1 to S phase. Similarly in Comet assay, there was an increase in tail length in treated cells in comparison with the control. Annexin V-FITC/PI staining assay showed prompt fluorescence in treated cells indicating the translocation of phosphatidylserine from the inner membrane. PI and DAPI staining showed the DNA damage in treated cells.

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

PubMed Central

Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

2002-01-01

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

Expression of thymidylate synthase and glutathione-s-transferase pi in patients with esophageal squamous cell carcinoma.

PubMed

Huang, Jun-Xing; Li, Feng-Yue; Xiao, Wei; Song, Zheng-Xiang; Qian, Rong-Yu; Chen, Ping; Salminen, Eeva

2009-09-14

To investigate the expression of thymidylate synthase (TS) and glutathione-s-transferase pi (GST-pi) in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. Immunohistochemical methods were used to detect the expression of TS and GST-pi in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma (ESCC) tissue sections from 102 patients (median age, 58 years) and in 28 normal esophageal mucosa (NEM) samples. The relationship between TS and GST-pi expression and clinicopathologic factors was examined. The expression of TS and GST-pi was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples (P < 0.05). The expression level of TS was not only significantly lower in well-differentiated (21.88%) than in poorly-differentiated carcinomas (51.43%, P < 0.05), but was also significantly higher in samples from male patients (46.51%) than from female patients (18.75%, P < 0.05). GST-pi was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples (P < 0.05). The expression level of GST-pi was also significantly higher in well-differentiated carcinomas (65.63%) than in poorly-differentiated carcinomas (35.00%, P < 0.05). The expression of TS and of GST-pi may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of TS and reduced expression of GST-pi.

[Effect of P38MAPK signal transduction pathway on apoptosis of THP-1 induced by allicin].

PubMed

Liao, Yang; Chen, Jianbin; Tang, Weixue; Ge, Qunfang; Lu, Qianwei; Yang, Zesong

2009-06-01

The objective of this paper was to study the change of P38MAPK and Fas in the apoptosis of THP-1 cells induced by allicin. The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay. Apoptosis rate was determined with Annexin V- FITC/PI double staining by flow cytometry. The expression and distribution change of the phosphorylation p38MAPK (P-p38MAPK) were detected by immunohistochemical staining. The changes of P-p38 MAPK and Fas proteins were detected by Western blot. The proliferations of leukemia cell line THP-1 are inhibited by allicin. MTT assay showed that allicin can inhibit the proliferation of the THP-1 cell, and the inhibition was dependent on both dose and time. The IC50 of 72 hours was 12.8 mg x L(-1). Apoptosis rate detected by Annexin V-FITC/PI was proportional to the concentration of the allicin. After the immunohistochemical staining test, the P-p38MAPK was located in the cell nucleus and plasma, showing deep brown, when adding allicin to THP-1 cell. Western blot test showed that the P-p38MAPK proteins expression was proportional to the concentration of Allicin and was also dose dependent. The levels of P-p38MAPK in negative control group, 1/2 IC50 of 72 hours group and IC50 of 72 hours group were 0.259 8 +/- 0.013 2, 0.61 2 +/- 0.008 3 and 0.505 6 +/- 0.005 5 respectively, and the levels of Fas proteins were 0.287 4 +/- 0.008 9, 0.426 8 +/- 0.007 9 and 0.597 1 +/- 0.010 9 respectively. The difference was statistically significant when compared with the negative control group (P < 0.01). Allicin can significantly induce THP-1 cells apoptosis, and its mechanism may be related to the activation of P38MAPK/Fas.

Preventive effects of 125I seeds on benign restenosis following esophageal stent implantation in a dog model

PubMed Central

GAN, ZHEN; JING, JIAN; ZHU, GUANGYU; QIN, YONGLIN; TENG, GAOJUN; GUO, JINHE

2015-01-01

The present study aimed to evaluate the effects of iodine-125 (125I) seeds on the proliferation of primary esophageal fibroblasts in dogs, and to assess the safety and preventive efficacy of 125I seed-pre-loaded esophageal stents in benign restenosis following implantation. Primary fibroblasts were cultured with various 125I seed activities, which were then evaluated using cell proliferation and apoptosis assays as well as cell cycle analysis using Annexin V/propidium iodide (PI) double staining and PI staining. Prior to sacrification, animals were submitted to esophageal radiography under digital subtraction angiography. Esophageal tissues were collected and examined for macroscopic, microscopic and pathological alterations. The results demonstrated a significant and dose-dependent inhibition of fibroblast proliferation and increased apoptosis following exposure to 125I seeds. G0/G1 fibroblast populations increased in a dose-dependent manner following treatment with 125I seeds, in contrast to cells in S phase. Four weeks following implantation, α-smooth muscle actin and proliferating cell nuclear antigen expression levels in the experimental group were significantly lower compared with those in the control group; in addition, eight weeks following implantation, esophageal inner diameters were increased in the experimental group. 125I seeds inhibited proliferation of dog esophageal fibroblasts via cell cycle arrest and apoptosis. In conclusion, 125I seed-pre-loaded esophageal stents inhibited benign hyperplasia in the upper edge of the stent to a certain extent, which relieved benign restenosis following implantation with a good safety profile. PMID:25543838

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

PubMed

Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method. Copyright © 2012 Elsevier B.V. All rights reserved.

Brilliant Blue G double staining enhances successful internal limiting membrane peeling with minimal adverse effect by low cellular permeability into live cells.

PubMed

Hisatomi, Toshio; Notomi, Shoji; Tachibana, Takashi; Oishi, Seiichiro; Asato, Ryo; Yamashita, Takehiro; Murakami, Yusuke; Ikeda, Yasuhiro; Enaida, Hiroshi; Sakamoto, Taiji; Ishibashi, Tatsuro

2015-02-01

Brilliant Blue G is used as a surgical adjuvant for retinal surgery. Although BBG double or multiple staining was reported, the effectiveness and safety of repeated staining is still elusive. To further examine the effectiveness and safety, we examined BBG in clinical cases in vivo, primary cell culture in vitro, and surgically resected specimen ex vivo. A retrospective interventional case series with in vitro and ex vivo studies were performed. Vitrectomy was performed in 28 cases of epiretinal membrane with BBG single to multiple staining. The surgically resected membranes were stained by BBG with or without cellular fixation. Primary cell cultures were examined with BBG and live/death cell markers, such as Calcein AM and TUNEL. Single staining provided satisfactory staining in seven cases. Double or multiple staining substantially visualized internal limiting membrane (21 cases), especially the edges of remaining internal limiting membrane (11 cases). Adverse retinal staining was not noted and the final visual acuity showed no difference with multiple staining. The live cells barely stained with BBG, while some dead cells were stained. Brilliant Blue G multiple staining substantially enhanced the visualization of internal limiting membrane. The absence of abnormal staining supports the safety of repeated BBG staining.

Multiple parton interactions and forward double pion production in pp and dA scattering

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

Strikman, M.; Vogelsang, W.

2011-02-01

We estimate the contributions by double-parton interactions to the cross sections for pp{yields}{pi}{sup 0}{pi}{sup 0}X and dA{yields}{pi}{sup 0}{pi}{sup 0}X at the Relativistic Heavy Ion Collider (RHIC). We find that such contributions become important at large forward rapidities of the produced pions. This is, in particular, the case for dA scattering, where they strongly enhance the azimuthal-angular independent pedestal component of the cross section, providing a natural explanation of this feature of the RHIC dA data. We argue that the discussed processes open a window to studies of double quark distributions in nucleons. We also briefly address the roles of shadowingmore » and energy loss in dA scattering, which we show to affect the double-inclusive pion cross section much more strongly than the single-inclusive one. We discuss the implications of our results for the interpretation of pion azimuthal correlations.« less

Anti-proliferative and apoptosis inducing potential of hydroalcoholic Achillea wilhelmsii C. Koch extract on human breast adenocarcinoma cell lines MCF-7 and MDA-Mb-468.

PubMed

Galavi, Hamid Reza; Saravani, Ramin; Shahraki, Ali; Ashtiani, Mojtaba

2016-11-01

Achillea wilhelmsii C. Koch contains a variety of components such as flavonoid. The previous studies showed that flavonoid has anti-cancer properties. The aim of the present study was to determine the anti-proliferative and apoptosis-inducing potential of hydroalcoholic Achillea wilhelmsii C. Koch extract (HAWE) on MCF-7 and MDA-Mb-468 human breast carcinoma cell lines. The anti-proliferative activity of HAWE was evaluated using MTT, flowcytometry by annexin V/PI double staining, and caspase-3 activity. The results of MTT showed that the ED50 of MCF-7 and MDA-Mb-468 was 25μg/ml of HAWE, 48h after treatment. Flowcytometry by annexin V/PI showed that HAWE induced late apoptosis in MCF-7 and early apoptosis in MDA-Mb-468. In addition, the caspase-3 colorimetric method showed that caspase-3 increased in the MDA-Mb-468 after treatment with HAWE. This study found that the hydroalcoholic extract of Achillea wilhelmsii C. Koch induced apoptosis in both the MCF-7 and MDA-Mb-468 human breast carcinoma cell lines.

Expression and distribution of the duck enteritis virus UL51 protein in experimentally infected ducks.

PubMed

Shen, Chanjuan; Cheng, Anchun; Wang, Mingshu; Xu, Chao; Jia, Renyong; Chen, Xiaoyue; Zhu, Dekang; Luo, Qihui; Cui, Hengmin; Zhou, Yi; Wang, Yin; Xu, Zhiwen; Chen, Zhengli; Wang, Xiaoyu

2010-06-01

To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Throughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120-144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types of cells, especially most abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of UL51p expression and distribution in vivo but also for a greater understanding of the pathogenesis of this DVE.

Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

PubMed

Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

2016-07-01

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

Glycans coated silver nanoparticles induces autophagy and necrosis in HeLa cells

NASA Astrophysics Data System (ADS)

Panzarini, Elisa; Mariano, Stefania; Dini, Luciana

2015-06-01

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

Apoptosis in liver cancer (HepG2) cells induced by functionalized gold nanoparticles.

PubMed

Ashokkumar, Thirunavukkarasu; Prabhu, Durai; Geetha, Ravi; Govindaraju, Kasivelu; Manikandan, Ramar; Arulvasu, Chinnasamy; Singaravelu, Ganesan

2014-11-01

An ethnopharmacological approach for biosynthesis of gold nanoparticles is being demonstrated using seed coat of Cajanus cajan. Medicinal value of capping molecule investigated for anticancer activity and results disclose its greater potential. The active principle of the seed coat [3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate] is elucidated. Rapid one-step synthesis yields highly stable, monodisperse (spherical) gold nanoparticles in the size ranging from 9 to 41 nm. Anticancer activity has been studied using liver cancer cells and cytotoxic mechanism has been evaluated using MTT, Annexin-V/PI Double-Staining Assay, Cell cycle, Comet assay and Flow cytometric analysis for apoptosis. The present investigation will open up a new possibility of functionalizing gold nanoparticles for apoptosis studies in liver cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Feng, Yi; Wan, Mingxi

2017-03-01

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

Simulated microgravity induce apoptosis and down-regulation of erythropoietin receptor of UT-7/EPO cells

NASA Astrophysics Data System (ADS)

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

2010-11-01

Hematopoietic progenitor cell proliferation can be alternated on either spaceflight or under simulated microgravity experiments on the ground; however, the underlying mechanism remains largely unknown. In the present study, we have demonstrated that exposure of human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO cells to conditions of simulated microgravity with a rotary culture instrument significantly inhibited the cellular proliferation rate. Adding higher concentrations of EPO to the culture medium failed to improve the inhibitory status. Cell apoptosis was detected by fluorescence staining of cell nuclei and a flow cytometry assay using Annexin V/PI double staining. This microgravity-induced apoptosis in UT-7/EPO cells could be blocked by a pancaspase inhibitor Z-VAD-FMK. Immunoblotting demonstrated that rotary culture resulted in a reduction of the expression of Bcl-xL, an anti-apoptotic protein, and the cleavage of caspase-3. Furthermore, rotary culture reduced surface localization and protein content, as well as the mRNA expression of erythropoietin receptor (EPOR) of UT-7/EPO. Take together, the findings indicated that simulated microgravity may induce mitochondrial related apoptosis of UT-7/EPO cell through depressing the EPO-EPOR pathway.

Design, synthesis, antiproliferative activity and docking studies of quinazoline derivatives bearing 2,3-dihydro-indole or 1,2,3,4-tetrahydroquinoline as potential EGFR inhibitors.

PubMed

OuYang, Yiqiang; Zou, Wensheng; Peng, Liang; Yang, Zunhua; Tang, Qidong; Chen, Mengzi; Jia, Shuang; Zhang, Hong; Lan, Zhou; Zheng, Pengwu; Zhu, Wufu

2018-05-09

Eight series of quinazoline derivatives bearing 2,3-dihydro-indole or 1,2,3,4-tetrahydroquinoline were designed, synthesized and evaluated for the IC 50 values against three cancer cell lines (A549, MCF-7 and PC-3). Most of the forty nine target compounds showed excellent antiproliferative activity against one or several cancer cell lines. The compound 13a showed the best activity against A549, MCF-7 and PC-3 cancer cell lines, with the IC 50 values of 1.09 ± 0.04 μM, 1.34 ± 0.13 μM and 1.23 ± 0.09 μM, respectively. Eight selected compounds were further selected to evaluated for the inhibitory activity against EGFR kinase. Three of them showed equal activity against EGFR kinase to positive control afatinib. AnnexinV-FITC, propidium iodide (PI) double staining and acridine orange single staining results indicated that the compound 13a could induce apoptosis of human lung cancer A549 cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway.

PubMed

Zhang, Qin; Huang, Wei-Dong; Lv, Xue-Ying; Yang, Yun-Mei

2012-05-01

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.

Anthocyanin inhibits propidium iodide DNA fluorescence in Euphorbia pulcherrima: implications for genome size variation and flow cytometry.

PubMed

Bennett, Michael D; Price, H James; Johnston, J Spencer

2008-04-01

Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics. DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia). There were large differences in PI staining (35-70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2.8-6.9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1.69-1.76 pg) than green leaves (1.81 pg). Chopping pea or poinsettia tissue in buffer with 0-200 microm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin. Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or correlated with environmental factors known to induce variation in pigmentation.

A Study of Double-Charm and Charm-Strange Baryons inElectron-Positron Annihilations

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

Edwards, Adam J.; /SLAC

2007-10-15

In this dissertation I describe a study of double-charm and charm-strange baryons based on data collected with the BABAR Detector at the Stanford Linear Accelerator Center. In this study I search for new baryons and make precise measurements of their properties and decay modes. I seek to verify and expand upon double-charm and charm-strange baryon observations made by other experiments. The BABAR Detector is used to measure subatomic particles that are produced at the PEP-II storage rings. I analyze approximately 300 million e+e- {yields} c{bar c} events in a search for the production of double-charm baryons. I search for themore » double-charm baryons {Xi}{sup +}{sub cc} (containing the quarks ccd) and {Xi}{sup ++}{sub cc} (ccu) in decays to {Lambda}{sup +}{sub c}K{sup -}{pi}{sup +} and {Lambda}{sup +}{sub c}K{sup -}{pi}{sup +}{pi}{sup +}, respectively. No statistically significant signals for their production are found, and upper limits on their production are determined. Statistically significant signals for excited charm-strange baryons are observed with my analysis of approximately 500 million e+e- {yields} c{bar c} events. The charged charm-strange baryons {Xi}{sub c}(2970){sup +}, {Xi}{sub c}(3055){sup +}, {Xi}{sub c}(3123){sup +} are found in decays to {Lambda}{sup +}{sub c}K{sup -}{pi}{sup +}, the same decay mode used in the {Xi}{sup +}{sub cc} search. The neutral charm-strange baryon {Xi}{sub c}(3077){sup 0} is observed in decays to {Lambda}{sup +}{sub c}K{sub 8}{pi}{sup -}. I also search for excited charm-strange baryon decays to {Lambda}{sup +}{sub c}K{sub 8}, {Lambda}{sup +}{sub c}K{sup -}, {Lambda}{sup +}{sub c}K{sub 8}{pi}{sup -}{pi}{sup +}, and {Lambda}{sup +}{sub c}K{sup -}{pi}{sup -}{pi}{sup +}. No significant charm-strange baryon signals a f h these decay modes. For each excited charm-strange baryon state that I observe, I measure its mass, natural width (lifetime), and production rate. The properties of these excited charm-strange baryons and their decay modes provide constraints for phenomenological models of quark interactions through quantum chromodynamics. My discovery of the two new charm-strange baryons {Xi}{sub c}(3055){sup +} and {Xi}{sub c}(3123){sup +} influences our theoretical understanding of charm-strange baryon states.« less

Cryptotanshinone induces cell cycle arrest and apoptosis through the JAK2/STAT3 and PI3K/Akt/NFκB pathways in cholangiocarcinoma cells

PubMed Central

Ke, Fayong; Wang, Zheng; Song, Xiaoling; Ma, Qiang; Hu, Yunping; Jiang, Lin; Zhang, Yijian; Liu, Yingbin; Zhang, Yong; Gong, Wei

2017-01-01

Background Cholangiocarcinoma (CCA) is the most common biliary tract malignancy in the world with high resistance to current chemotherapies and extremely poor prognosis. The main objective of this study was to investigate the inhibitory effects of cryptotanshinone (CTS), a natural compound isolated from Salvia miltiorrhiza Bunge, on CCA both in vitro and in vivo and to explore the underlying mechanisms of CTS-induced apoptosis and cell cycle arrest. Methods The anti-tumor activity of CTS on HCCC-9810 and RBE cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and Hoechst 33342 staining assays. The efficacy of CTS in vivo was evaluated using a HCCC-9810 xenograft model in athymic nude mice. The expression of key proteins involved in cell apoptosis and signaling pathway in vitro was analyzed by Western blot analysis. Results CTS induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in HCCC-9810 and RBE cells in a dose-dependent manner. Intraperitoneal injection of CTS (0, 10, or 25 mg/kg) for 4 weeks significantly inhibited the growth of HCCC-9810 xenografts in athymic nude mice. CTS treatment induced S-phase arrest with a decrease of cyclin A1 and an increase of cyclin D1 protein level. Bcl-2 expression was downregulated remarkably, while Bax expression was increased after apoptosis occurred. Additionally, the activation of JAK2/STAT3 and PI3K/Akt/NFκB was significantly inhibited in CTS-treated CCA cells. Conclusion CTS induced CCA cell apoptosis by suppressing both the JAK2/STAT3 and PI3K/Akt/NFκB signaling pathways and altering the expression of Bcl-2/Bax family, which was regulated by these two signaling pathways. CTS may serve as a potential therapeutic agent for CCA. PMID:28670110

Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.

PubMed Central

Dzandu, J K; Deh, M E; Barratt, D L; Wise, G E

1984-01-01

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes. Images PMID:6200882

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

PubMed

Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

2017-12-01

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

Improved recovery of Listeria monocytogenes from stainless steel and polytetrafluoroethylene surfaces using air/water ablation.

PubMed

Gião, M S; Blanc, S; Porta, S; Belenguer, J; Keevil, C W

2015-07-01

To develop a gentle ablation technique to recover Listeria monocytogenes biofilms from stainless steel (SS) and polytetrafluoroethylene (PTFE) surfaces by using compressed air and water injection. Biofilms were grown for 4, 24 and 48 h or 7 days and a compressed air and water flow at 2, 3 and 4 bars was applied for cell removal. Collected cells were quantified for total/dead by staining with SYTO 9/PI double staining and cultivable populations were determined by plating onto brain heart infusion (BHI) agar, while coupon surfaces also were stained with DAPI to quantify in situ the remaining cells. The recovery efficiency was compared to that of conventional swabbing. Results showed that the air/water ablation is able to collect up to 98·6% of cells from SS surfaces while swabbing only recovered 11·2% of biofilm. Moreover, air/water ablation recovered 99·9% of cells from PTFE surfaces. The high recovery rate achieved by this technique, along with the fact that cells were able to retain membrane integrity and cultivability, indicate that this device is suitable for the gentle recovery of viable L. monocytogenes biofilm cells. This work presents a highly efficient technique to remove, collect and quantify L. monocytogenes from surfaces commonly used in the food industry, which can thus serve as an important aid in verifying cleaning and sanitation as well as in reducing the likelihood of cross-contamination events. © 2015 The Society for Applied Microbiology.

Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)

NASA Astrophysics Data System (ADS)

Mittag, Anja; Marecka, Monika; Pierzchalski, Arkadiusz; Malkusch, Wolf; Bocsi, József; Tárnok, Attila

2009-02-01

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Peritumoral stromal remodeling, pattern of invasion and expression of c-met/HGF in advanced squamous cell carcinoma of the cervix uteri, FIGO stages III and IV.

PubMed

Horn, L-C; Hommel, N; Roschlau, U; Bilek, K; Hentschel, B; Einenkel, J

2012-07-01

Different patterns of invasion (PIs) have prognostic impact in several types of cancer and are associated with different grades of peritumoral stromal remodeling, characterized by the desmoplastic stromal response (DSR). One key regulator influencing cellular motility and peritumoral stromal response is c-met/HGF. This study evaluates the association between different PI, peritumoral DSR and its correlation to the expression of c-met/HGF in squamous cell carcinomas of the uterine cervix (CX). 131 advanced stage CX (FIGO III/IV) were re-evaluated histologically regarding PI, using a two-level scoring system. The tumor grows in solid cords/trabeculae in finger-like PI and in very small groups or single cells in spray-like PI. DSR was categorized as none/weak and moderate/strong. The tumors were stained with antibodies against c-met and HGF. The staining of >30% of tumor cells was defined as overexpression. The PI was correlated to the prognostic outcome, different categories of DSR and expression status of c-met and HGF. 66.4% of the tumors showed a finger-like, and 33.6% a spray-like PI. The spray-like PI showed a reduced two-year overall survival when compared to the finger-like PI (14.0% vs. 29.1%, respectively; p=0.012), and was associated with moderate/strong DSR. The majority of the tumors showed overexpression of c-met (85.4%) and HGF (74.8%). There was no correlation between the expression status of c-met/HGF and the FIGO stage, peritumoral DSR or the prognostic outcome. Spray-like PI is of prognostic impact in cervical carcinoma FIGO III/IV and is associated with strong peritumoral stromal remodeling. There is no prognostic impact of the immunohistochemical expression of c-met/HGF in advanced stage cervical carcinomas. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Levels of cyclic-2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum during phosphate limitation.

PubMed Central

Seely, R J; Fahrney, D E

1984-01-01

Batch-grown Methanobacterium thermoautotrophicum cells grew nonexponentially in the absence of exogenous Pi until intracellular cyclic-2,3-diphosphoglycerate (cyclic DPG) had fallen below 2 mumol/g (dry weight), the limit of detection. Growth resumed immediately upon transfer to medium containing Pi Cyclic DPG levels were also below detection in Pi-limited chemostat cultures operating at a dilution rate of 0.173 h-1 (4-h doubling time), with reservoir Pi concentrations below 200 microM. At this dilution rate, the Pi concentration in the culture was 4 microM. An H2-limited steady state was achieved with 400 microM Pi in the inflowing medium (67 microM in the culture). The cyclic DPG content of these cells was 72 to 74 mumol/g, about one-third the amount in batch-grown cells. The specific growth rate accelerated immediately to 0.36 h-1 (1.9-h doubling time) under washout conditions at high dilution rate. The cellular content of cyclic DPG declined over a 2-h period, and then increased rapidly as the Pi level in the medium approached 200 microM. Expansion of the cyclic DPG pool coincided with a marked increase in Pi assimilation. These results indicated that M. thermoautotrophicum accumulated cyclic DPG only when Pi and H2 were readily available. PMID:6480564

Levels of cyclic-2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum during phosphate limitation.

PubMed

Seely, R J; Fahrney, D E

1984-10-01

Batch-grown Methanobacterium thermoautotrophicum cells grew nonexponentially in the absence of exogenous Pi until intracellular cyclic-2,3-diphosphoglycerate (cyclic DPG) had fallen below 2 mumol/g (dry weight), the limit of detection. Growth resumed immediately upon transfer to medium containing Pi Cyclic DPG levels were also below detection in Pi-limited chemostat cultures operating at a dilution rate of 0.173 h-1 (4-h doubling time), with reservoir Pi concentrations below 200 microM. At this dilution rate, the Pi concentration in the culture was 4 microM. An H2-limited steady state was achieved with 400 microM Pi in the inflowing medium (67 microM in the culture). The cyclic DPG content of these cells was 72 to 74 mumol/g, about one-third the amount in batch-grown cells. The specific growth rate accelerated immediately to 0.36 h-1 (1.9-h doubling time) under washout conditions at high dilution rate. The cellular content of cyclic DPG declined over a 2-h period, and then increased rapidly as the Pi level in the medium approached 200 microM. Expansion of the cyclic DPG pool coincided with a marked increase in Pi assimilation. These results indicated that M. thermoautotrophicum accumulated cyclic DPG only when Pi and H2 were readily available.

PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

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

Kan-o, Keiko; Matsumoto, Koichiro, E-mail: koichi@kokyu.med.kyushu-u.ac.jp; Asai-Tajiri, Yukari

Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (polymore » IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.« less

Effects of small interfering RNA inhibit Class I phosphoinositide 3-kinase on human gastric cancer cells

PubMed Central

Zhu, Bao-Song; Yu, Li-Yan; Zhao, Kui; Wu, Yong-You; Cheng, Xiao-Li; Wu, Yong; Zhong, Feng-Yun; Gong, Wei; Chen, Qiang; Xing, Chun-Gen

2013-01-01

AIM: To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class I phosphoinositide 3-kinase (Class I PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells. METHODS: We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant. RESULTS: The viability of gastric cancer cells was inhibited after siRNA targeting to the Class I PI3K blocked Class I PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P < 0.05 compared to control group). It was determined that when 50 MOI, the transfection efficiency was 95% ± 2.4%. Adenovirus PI3K(I)-RNAi-GFP (50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells, and the results described here prove that RNAi of Class I PI3K induced apoptosis in SGC7901 cells. The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity, indicating increased formation of autophagosomes. The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low. After incubating with adenovirus PI3K(I)-RNAi-GFP (50 MOI), Beclin-1, LC3, and p53 protein expression was significantly increased from 24 to 72 h. We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP (50 MOI). A number of isolated membranes, possibly derived from ribosome-free endoplasmic reticulum, were seen. These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)-RNAi-GFP (50 MOI) treatment. Control cells showed a round shape and contained normal-looking organelles, nucleus, and chromatin, while adenovirus PI3K(I)-RNAi-GFP (50 MOI)-treated cells exhibited the typical signs of autophagy. CONCLUSION: After the Class I PI3K signaling pathway has been blocked by siRNA, the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced. PMID:23555164

Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy

PubMed Central

Li, Jiao; Ahn, Juhee; Liu, Donghong; Chen, Shiguo; Ye, Xingqian

2016-01-01

As a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry. PMID:26746712

Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

PubMed

Yu, Fei; Zeng, Hui; Lei, Ming; Xiao, De-Ming; Li, Wei; Yuan, Hao; Lin, Jian-Jing

2016-10-01

This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1 +/+ control group (group A, n=6); SIRT1 +/+ osteoarthritis group (group B, n=6); SIRT1 -/- control group (group C, n=6); SIRT1 -/- osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1 -/- osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1 +/+ osteoarthritis group and SIRT1 -/- control group, SIRT1 protein expression was not obviously changed in the SIRT1 -/- osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

Combinatorial Study of a Novel Poly (ADP-ribose) Polymerase Inhibitor and an HDAC Inhibitor, SAHA, in Leukemic Cell Lines.

PubMed

Hegde, Mahesh; Mantelingu, Kempegowda; Pandey, Monica; Pavankumar, Chottanahalli S; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2016-10-01

Cancer is a multifactorial disease, which makes it difficult to cure. Since more than one defective cellular component is often involved during oncogenesis, combination therapy is gaining prominence in the field of cancer therapeutics. The purpose of this study was to investigate the combinatorial effects of a novel PARP inhibitor, P10, and HDAC inhibitor, SAHA, in leukemic cells. Combinatorial effects of P10 and SAHA were tested using propidium iodide staining in different leukemic cells. Further, flowcytometry-based assays such as calcein-AM/ethidium homodimer staining, annexin-FITC/PI staining, and JC-1 staining were carried out to elucidate the mechanism of cell death. In addition, cell-cycle analysis, immunocytochemistry studies, and western blotting analysis were conducted to check the combinatorial effect in Nalm6 cells. Propidium iodide staining showed that P10 in combination with SAHA induced cell death in Nalm6 cells, in which PARP expression and activity is high with a combination index of <0.2. Annexin-FITC/PI staining, JC-1 staining, and other biochemical assays revealed that P10 in combination with SAHA induced apoptosis by causing a change in mitochondrial membrane potential in >65 % cells. Importantly, combinatorial treatment induced S phase arrest in 40-45 % cells due to DNA damage and plausible replicative stress. Finally, we demonstrated that treatment with P10 led to DNA strand breaks, which were further potentiated by SAHA (p < 0.01), leading to activation of apoptosis and increased cell death in PARP-positive leukemic cells. Our study reveals that coadministration of PARP inhibitor with SAHA could be used as a combination therapy against leukemic cells that possess high levels of intrinsic PARP activity.

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation.

PubMed

Taghi-Kilani, R; Gyürék, L L; Millard, P J; Finch, G R; Belosevic, M

1996-06-01

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

Flow cytomeric measurement of DNA and incorporated nucleoside analogs

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1989-01-01

A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

1996-01-01

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

In Vitro Morphology and Maturation of Lymphocystis Virus 1

PubMed Central

Midlige, F. H.; Malsberger, R. G.

1968-01-01

The temporal sequence of development of lymphocystis disease virus (LDV) was studied by electron microscopy of thin sections of infected tissue-culture monolayers. Neither the typical cytoplasmic inclusion nor virus was detected at 4 days postinfection (PI). Inclusions, but no viruses, were detected at 8 days PI. Inclusions and associated virions were detected at 15 days PI, and by 28 days PI the undisrupted cells were filled with the typical virions. No release mechanism was detected, and severe clumping of particles was noted. Negatively stained preparations revealed particles 200 nm in diameter with no capsomere structure and apparent spikes associated with the particle. The relationship of LDV to the well-defined deoxyribonucleic acid virus groups is discussed. Images PMID:5701821

Effects of activin A and its downstream ERK1/2 in oxygen and glucose deprivation after isoflurane-induced postconditioning.

PubMed

Wang, Qin; Yin, Jiangwen; Wang, Sheng; Cui, Di; Lin, Hong; Ge, Mingyue; Dai, Zhigang; Xie, Liping; Si, Junqiang; Ma, Ketao; Li, Li; Zhao, Lei

2016-12-01

Isoflurane postconditioning (ISPOC) plays a neuroprotection role in the brain. Previous studies confirmed that isoflurane postconditioning can provide better protection than preconditioning in acute hypoxic-ischemic brain damage, such as acute craniocerebral trauma and ischemic stroke. Numerous studies have reported that activin A can protect rat's brain from cell injury. However, whether activin A and its downstream ERK1/2 were involved in isoflurane postconditioning-induced neuroprotection is unknown. A total of 80 healthy Sprague-Dawley rats weighing 50-70g were randomly divided into 10 groups of 8: normal control, oxygen and glucose deprivation (OGD), 1.5% ISPOC, 3.0% ISPOC, 4.5% ISPOC, blocker of activin A (SB431542), blocker of ERK1/2 (U0126), 3.0% ISPOC+SB431542, 3.0% ISPOC+U0126, and vehicle (dimethyl sulfoxide(DMSO)) group. Blockers (SB431542 and U0126) were used in each concentration of isoflurane before OGD. Hematoxylin-eosin staining, 2,3,5-triphenyl tetrazolium chloride staining, and propidium iodide (PI) staining were conducted to assess the reliability in the brain slices. Immunofluorescence, Western blot, and quantitative real-time PCR(Q-PCR) were performed to validate the protein expression levels of activin A, Smad2/3, P-Smad2/3, ERK1/2, and phosphorylation ERK1/2 (P-ERK1/2). The number of damaged neurons and mean fluorescence intensity(MFI) of PI staining increased, but formazan generation, expression levels of activin A and P-ERK1/2 protein, and mRNA synthesis level of activin A decreased in the OGD group compared with the normal control group (p<0.05). The number of damaged neurons and MFI of PI staining decreased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A increased significantly in the 1.5% ISPOC and 3.0% ISPOC groups (p<0.05) compared with the OGD group. The result in the 4.5% ISPOC group, was completely opposite to the 1.5% ISPOC and 3.0% ISPOC groups. The number of damage neuron and MFI of PI staining increased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A decreased in the 4.5% ISPOC group. However, the expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A in the 4.5% ISPOC group were higher than the OGD group (p<0.05). The other results were compared between the SB431542 group/the U0126 group and 3.0% ISPOC group. The MFI of PI staining increased, but the expression levels of activin A, P-Smad2/3, and P-ERK1/2 decreased (p<0.05). The expression level of ERK1/2 protein in all groups exhibited no change (p>0.05). Results of this study showed that 3.0% concentration of isoflurane postconditioning provided better neuroprotection than 1.5% and 4.5% concentrations of isoflurane. Activin A/Smad 2/3 and activin A/ERK1/2 signaling pathway may be involved in ISPOC-induced neuroprotection. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

PI3K/AKT/mTOR-mediated autophagy in the development of autism spectrum disorder.

PubMed

Zhang, Jun; Zhang, Ji-Xiang; Zhang, Qin-Liang

2016-07-01

To investigate the association between PI3K/AKT/mTOR-mediated autophagy and the pathogenesis of autism spectrum disorder (ASD). A sodium valproate (VPA)-induced baby rat model of ASD was built. Nine pregnant rats were randomly assigned into three groups, with three rats for each group: healthy control group, VPA group and mTOR inhibition group, receiving different drug administrations. Baby rats were grouped according to the maternal rats. Social interaction of baby rats (35days after birth) was observed and their bilateral hippocampes were sliced. We used electron microscope analysis for observation of autophagosome formation, double immunofluorescence staining for location of LC3 II, TUNEL assay for observation of cell apoptosis, Western Blot assay was used for measurement of LC3 II, P62, p53, Bcl-2, PI3K/AKT/mTOR-related proteins and p-S6. VPA group had significantly lowered ability of social interaction than the control group and mTOR inhibition group (both P<0.05). The control group and the mTOTR inhibition group presented the visual of autophagosomes, while VPA group seldom had autophagosomes. By comparison with VPA group, mTOR group had a remarkable green fluorescence in the hippocampal CA1 (P<0.05). Western Blot assay revealved that mTOR inhibition group had a significantly higher LC3 II expression, higher LC3 II/LC3 I ratio, higher Bcl-2 expression and lower p53 than VPA group (all P<0.05). TUNEL assay showed that mTOR inhibition group had a significant smaller number of apoptotic cells in the hippocampal CA1. Besides, lowered expressions of p-PI3K, p-AKT and p-S6 were identified in the baby rats in mTOR inhibition group compared with VPA group (all P<0.05). mTOR inhibition can increase PI3K/AKT/mTOR-mediated autophagic activity and improve social interaction in VPA-induced ASD, providing a novel target and direction for the treatment of ASD. Copyright © 2016 Elsevier Inc. All rights reserved.

PHP14 regulates hepatic stellate cells migration in liver fibrosis via mediating TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway.

PubMed

Xu, Anjian; Li, Yanmeng; Zhao, Wenshan; Hou, Fei; Li, Xiaojin; Sun, Lan; Chen, Wei; Yang, Aiting; Wu, Shanna; Zhang, Bei; Yao, Jingyi; Wang, Huan; Huang, Jian

2018-02-01

Hepatic fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Migration of the activated HSCs to the site of injury is one of the key characteristics during the wound healing process. We have previously demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) is involved in migration and lamellipodia formation of HSCs. However, the role of PHP14 in liver fibrosis remains unknown. In this study, we first assessed PHP14 expression and distribution in liver fibrotic tissues using western blot, immunohistochemistry, and double immunofluorescence staining. Next, we investigated the role of PHP14 in liver fibrosis and, more specifically, the migration of HSCs by Transwell assay and 3D collagen matrices assay. Finally, we explored the possible molecular mechanisms of the effects of PHP14 on these processes. Our results show that the PHP14 expression is up-regulated in fibrotic liver and mainly in HSCs. Importantly, TGF-β1 can induce PHP14 expression in HSCs accompanied with the activation of HSCs. Consistent with the previous study, PHP14 promotes HSCs migration, especially, promotes 3D floating collagen matrices contraction but inhibits stressed-released matrices contraction. Mechanistically, the PI3Kγ/AKT/Rac1 pathway is involved in migration regulated by PHP14. Moreover, PHP14 specifically mediates the TGF-β1 signaling to PI3Kγ/AKT pathway and regulates HSC migration, and thus participates in liver fibrosis. Our study identified the role of PHP14 in liver fibrosis, particularly HSC migration, and suggested a novel mediator of transducting TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway. PHP14 is up-regulated in fibrotic liver and activated hepatic stellate cells. The expression of PHP14 is induced by TGF-β1. The migration of hepatic stellate cells is regulated by PHP14. PHP14 is a mediator of TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway in hepatic stellate cells.

Doxycycline synergizes with doxorubicin to inhibit the proliferation of castration-resistant prostate cancer cells.

PubMed

Zhu, Chao; Yan, Xueting; Yu, Ao; Wang, Yongjian

2017-11-01

Castration-resistant prostate cancer (CRPC) is fatal and there is currently no effective clinical treatment. The antibiotic doxycycline has shown anti-cancer effect in several kinds of solid tumors including prostate cancer. In this study, a combination of doxycycline and doxorubicin was used to investigate the synergistic effect on CRPC cells. MTT assay was employed to determine the viability of cells in two-dimensional (2D) cultures. Apoptosis was determined by Annexin V/propidium iodide (PI) double staining assay. Cell cycle was analyzed by PI staining, and reverse transcription-PCR (RT-PCR) was used to determine the expressions of apoptosis-related genes at mRNA level. Western blot analysis was used to analyze the expressions of Bcl-2, Bax, and Poly (ADP-ribose) polymerase proteins. Cytotoxicity assay and morphological observation of PC3 cells in three-dimensional (3D) cultures were used to determine the effect of combination treatment. Results showed that doxycycline combined with doxorubicin significantly inhibited PC3 cells in both 2D and 3D cultures, enhanced apoptosis, and increased the accumulation of cells in G2/M phase. RT-PCR showed down-regulation of Bcl-2 and up-regulation of Bax mRNA after combination treatment. Meanwhile, western blot analysis showed that combination treatment resulted in down-regulation of Bcl-2 protein and up-regulation of Bax protein, and that PARP cleavage was obviously exhibited after combination treatment. Confocal imaging analysis indicated that doxorubicin penetrated deeply into the core of spheroids when combined with doxycycline. These data indicated that doxycycline in combination with doxorubicin had a synergistic effect on PC3 cells and may provide a potential novel strategy for the treatment of CRPC. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

1082-39, an analogue of sorafenib, inhibited human cancer cell growth more potently than sorafenib.

PubMed

Chu, Jia-Hui; Zhao, Cui-Rong; Song, Zhi-Yu; Wang, Rui-Qi; Qin, Yi-Zhuo; Li, Wen-Bao; Qu, Xian-Jun

2014-04-01

1082-39, an analogue of sorafenib, is a derivative of indazole diarylurea. We evaluated the activity of 1082-39 against human cancer cell growth. Its effects and mechanisms of action were then compared with those of sorafenib. The experiments were performed in human melanoma M21 cells. Cell viability was estimated by using the colorimetric assay. Annexin V-FITC/PI staining assay was used to recognize the apoptotic cells. Further analysis of the mitochondria membrane potential (MMP) was performed by the JC-1 fluorescence probe staining. The levels of apoptotic proteins and kinases related to cancer proliferation were determined by western blotting assay. 1082-39 possessed the activity against cancer cell proliferation with time- and dose-dependent manner. 1082-39 induced M21 cell to apoptosis, showing the increase of annexin V-FITC/PI staining cells, the MMP collapse and releasing cytochrome c from mitochondria. Western blotting analysis showed the activation of the mitochondria-mediated intrinsic pathway, showing the increase of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. Statistical analysis suggested that 1082-39 possessed greater activities than sorafenib in the inhibition of M21 proliferation and induction of apoptosis. These effects of 1082-39 might arise from its activity of regulation the PI3K/Akt and Wnt/β-catenin signaling pathways. 1082-39 is a promising candidate compound which could develop as a potent anticancer agent. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

PubMed

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-05-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining

PubMed Central

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-01-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke. PMID:21245871

Hydroxyl X2Pi pure rotational transitions

NASA Astrophysics Data System (ADS)

Goorvitch, D.; Goldman, A.; Dothe, Hoang; Tipping, R. H.; Chackerian, C., Jr.

1992-12-01

We present a list of frequencies, term values, Einstein A values, and assignments for the pure rotational transitions of the X2Pi state of the OH molecule. This list includes transitions from 3 to 2015/cm for Delta-v = 0, v-double-prime = 0-4, and J-double-prime = 0.5-49.5. The A values were computed using recent advances in calculating wave functions for a coupled system and an experimentally derived electric dipole moment function (Nelson et al., 1990) which exhibits curvature.

[Role of PI3K/Akt pathway in endoplasmic reticulum stress and apoptosis induced by saturated fatty acid in human steatotic hepatocytes].

PubMed

Qu, Mei; Shen, Wei

2015-03-01

To investigate the roles of PI3K/Akt signaling in the unfolded protein response (UPR) and non-UPR signaling pathways of endoplasmic reticulum stress and apoptosis in hepatocytes under conditions of saturated fatty acid-induced steatosis. A steatosis model of hepatocytes (L02 cell and HepG2 cell line) was induced by palmitate sodium saturated fatty acids.The hepatocytes were divided into normal control group,experimental group (treated with palmitate sodium) and intervention group (treated with palmitate sodium and LY294002, a PI3K/Akt inhibitor). Cell apoptosis was detected by flow cytometry with Annexin V/PI double-staining.Western blot analysis was used to examine the protein expression of GRP78, PI3K, P-PI3K,Akt, P-Akt, CHOP and Bax.The F test and t-test were used in statistical analyses. Flow cytometry showed that palmitate sodium induced cell apoptosis in steatotic hepatocytes;moreover, a significant increase in cell apoptosis was observed in the palmitate sodium-induced steatotic hepatocytes in the presence of LY294002.For the normal control group, the experimental group and the intervention group, the apoptosis ratios of L02 cells were 4.41 ± 0.78% vs. 6.01 ± 1.49% vs. 19.50 ± 2.53% after 24 hours of treatment,and 12.56 ± 2.78% vs. 29.72 ± 6.39% vs. 44.60 ± 4.17% after 48 hours of treatment in respectively (all P < 0.05),and of HepG2 cells were 11.16 ± 1.15% vs. 17.50 ± 6.83% vs. 30.41 ± 3.62% after 24 hours of treatment, and 22.37 ± 1.24% vs. 33.85 ± 5.79% vs. 48.56 ± 4.21% after 48 hours of treatment (all P < 0.05). Western blot analysis showed that expression of GRP78 was significantly upregulated in the palmitate sodium-induced steatosis hepatocytes, indicating activation of endoplasmic reticulum stress. In addition, the palmitate sodium treatment also activated the PI3K/Akt pathway,induced expression of CHOP and Bax of the UPR and non-UPR signaling pathways respectively. Moreover, Pretreatment with LY294002 inhibited the palmitate sodium induced-phosphorylation of PI3K and Akt, and promoted upregulation of CHOP and Bax induced by palmitate sodium. The PI3K/Akt pathway may be involved in regulation of the UPR and non-UPR signaling pathways of endoplasmic reticulum stress and may promote apoptosis of hepatocytes by enhancing the expression of CHOP and Bax protein in saturated fatty acid-induced steatotic hepatocytes.

Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer.

PubMed

Kessel, Sarah; Cribbes, Scott; Bonasu, Surekha; Rice, William; Qiu, Jean; Chan, Leo Li-Ying

2017-09-01

The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better identify more qualified cancer drug candidates for drug discovery research. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Bioinspired double polysaccharides-based nanohybrid scaffold for bone tissue engineering.

PubMed

Fan, Tiantang; Chen, Jingdi; Pan, Panpan; Zhang, Yujue; Hu, Yimin; Liu, Xiaocui; Shi, Xuetao; Zhang, Qiqing

2016-11-01

The fabrication of bone scaffolds with interconnected porous structure, adequate mechanical properties and excellent biocompatibility presents a great challenge. Herein, a hybrid nanostructured chitosan/chondroitin sulfate/hydroxyapatite (ChS/CSA/HAP) in situ composite scaffold was prepared by in situ fabrication and freeze-drying technique. The composition and morphology of scaffold were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). It proved that the low crystallinity of HAP crystals were uniformly distributed in ChS/CSA organic matrix and the nanostructured hybrid scaffold exhibited good mechanical property. The biocompatibility and in vitro bioactivity were detected by MTT-assay, maturation (alkaline phosphatase (ALP) activity), Hoechst 33258 and PI fluorescence staining. In vitro tests indicated that the hybrid scaffold not only promoted the adhesion and proliferation of osteoblasts, but also improved the growth of the osteoblasts. Therefore, it is promising for bone repair application in bone tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

Interlayer tunneling in double-layer quantum hall pseudoferromagnets.

PubMed

Balents, L; Radzihovsky, L

2001-02-26

We show that the interlayer tunneling I-V in double-layer quantum Hall states displays a rich behavior which depends on the relative magnitude of sample size, voltage length scale, current screening, disorder, and thermal lengths. For weak tunneling, we predict a negative differential conductance of a power-law shape crossing over to a sharp zero-bias peak. An in-plane magnetic field splits this zero-bias peak, leading instead to a "derivative" feature at V(B)(B(parallel)) = 2 pi Planck's over 2 pi upsilon B(parallel)d/e phi(0), which gives a direct measurement of the dispersion of the Goldstone mode corresponding to the spontaneous symmetry breaking of the double-layer Hall state.

Expression of c-Jun and Bcl-2 family proteins in apoptotic photoreceptors of RCS rats.

PubMed

Katai, Naomichi; Yanagidaira, Tomoko; Senda, Nami; Murata, Toshinori; Yoshimura, Nagahisa

2006-01-01

To determine if c-Jun and Bcl-2 family proteins play a role in photoreceptor apoptosis in Royal College of Surgeons (RCS) rats. RCS and Sprague-Dawley rats were used. Cryosections of retinas harvested at various postnatal periods were immunostained with antibodies against c-Jun, Bcl-2, and Bax. Double staining with TdT-dUTP nick-end labeling (TUNEL) or propidium iodide (PI) and antibodies was also done. To study the time course of gene and protein expression, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting analyses were carried out. TUNEL-positive photoreceptors of RCS rats were stained strongly with antibodies against c-Jun and Bax. The number of immunoreactive cells increased on days 21 and 28 after birth (P21 and P28) and decreased on P45. Semiquantitative RT-PCR analysis showed that mRNAs for c-Jun and Bax were upregulated at P21 and P28, but those for Bcl-2 were unchanged. On immunoblotting, a 43-kDa band was revealed by the anti-c-Jun antibody and a 21-kDa band, by the anti-Bax antibody. Protein expression of c-Jun and Bax were increased at both P21 and P28. The temporal profiles of immunoreactivity, protein expression, and mRNA expression were similar. c-Jun and Bax may play a role in photoreceptor apoptosis in RCS rats.

Central Exclusive $$\\pi^{+}\\pi^{-}$$ Production in $$p\\bar{p}$$ Collisions at $$\\sqrt{s} = 0.9$$ and 1.96 TeV at the Tevatron

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

Žurek, Maria; Albrow, Michael

2014-01-01

Exclusivemore » $$\\pi^{+}\\pi^{-}$$ production in proton-antiproton collisions at $$\\sqrt{s} = 0.9$$ and 1.96 TeV in the Collider Detector at Fermilab has been measured. We selected events with exactly two particles with opposite charge, in $$|\\eta|<1.3$$, with no other particles detected in $$|\\eta| < 5.9$$. We require the central $$\\pi^{+}\\pi^{-}$$ to have rapidity $|y|<1$. Since these events are dominated by double pomeron exchange, the quantum numbers of the central state are constrained. The data show resonance structures attributed to the $$f_0$$ and $$f_2$$ mesons.« less

Brucella dissociation is essential for macrophage egress and bacterial dissemination.

PubMed

Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.

Brucella dissociation is essential for macrophage egress and bacterial dissemination

PubMed Central

Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A.

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4–5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination. PMID:24634889

Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway.

PubMed

Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

2018-01-01

Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata , was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately.

Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway

PubMed Central

Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

2018-01-01

Background Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata, was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. Materials and methods U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Results Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. Conclusion The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately. PMID:29785137

Turnover of cyclic 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum. Phosphate flux in P1- and H2-limited chemostat cultures.

PubMed

Krueger, R D; Campbell, J W; Fahrney, D E

1986-09-15

The archaebacterium Methanobacterium thermoautotrophicum was grown at 65 degrees C in H2- and Pi-limited chemostat cultures at dilution rates corresponding to 3- and 4-h doubling times, respectively. Under these conditions the steady state concentration of cyclic 2,3-diphosphoglycerate was 44 mM in the H2-limited cells and 13 mM in the cells grown under Pi limitation. Flux of Pi into the cyclic pyrophosphate pool was estimated by two 32P-labeling procedures: approach to isotopic equilibrium and replacement of prelabeled cyclic diphosphoglycerate with unlabeled compound. The results unequivocally demonstrate turnover of the phosphoryl groups; either both phosphoryl groups of the cyclic pyrophosphate leave together or the second leaves at a faster rate. The half-life of the rate-determining step for loss of the phosphoryl groups was approximately equal to the culture doubling time. The Pi flowing into the cyclic diphosphoglycerate pool accounted for 19% of the total Pi flux into Pi-limited cells and 43% of the total for H2-limited cells. The high phosphate flux through the large cyclic diphosphoglycerate pool suggests that this molecule plays an important role in the phosphorus metabolism of this methanogen.

4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Expression of phospho-ERK1/2 and PI3-K in benign and malignant gallbladder lesions and its clinical and pathological correlations

PubMed Central

Li, Qinglong; Yang, Zhulin

2009-01-01

Background An increasing number of studies have shown that ERK and PI3-K/AKT signaling pathways are involved in various human cancers including hepatocellular carcinoma and cholangiocarcinoma. However, few studies have examined gallbladder cancer specimens, and little is known about the clinical and pathological significance of ERK1/2 and PI3-K/AKT signaling changes in gallbladder adenocarcinoma. In this study, we examined phospho-ERK1/2 (p-ERK1/2) and PI3K expression and analyzed its clinicopathological impact in gallbladder adenocarcinoma. Methods Immunohistochemistry was used to detect and compare the frequency of p-ERK1/2 and PI3-K expression in gallbladder adenocarcinoma, peri-tumor tissues, adenomatous polyps, and chronic cholecystitis specimens. Results The positive staining for p-EKR1/2 and PI3-K were 63/108 (58.3%) and 55/108 (50.9%) in gallbladder adenocarcinoma; 14/46 (30.4%) and 5/46 (10.1%) in peri-tumor tissues; 3/15 (20%) and 3/15 (20%) in adenomatous polyps; and 4/35 (11.4%) and 3/35 (8.6%) in chronic cholecystitis. The positive rate of p-ERK1/2 or PI3-K in gallbladder adenocarcinoma was significantly higher than that in peri-tumor tissue (both, P < 0.01), adenomatous polyps (p-ERK1/2, P < 0.01; PI3-K, P < 0.05), and chronic cholecystitis (both, P < 0.01). The positive staining for p-ERK1/2 or PI3-K was significantly lower in well/highly-differentiated adenocancinomas with maximal diameter < 2.0 cm, no metastasis to lymph node, and no infiltration of regional tissues or organs compared to poorly-differentiated adenocarcinomas which are characterized by a maximal diameter ≥ 2.0 cm, with metastasis to lymph node and infiltration of regional tissues or organs (P < 0.05 or P < 0.01). Moreover, the frequency of p-ERK1/2 expression in gallbladder adenocarcinomas without gallstone was significantly lower than those with gallstones. Increased expression of p-ERK1/2 (P < 0.05) and PI3K (P = 0.062) was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased p-ERK1/2 expression was an independent prognostic predictor in gallbladder carcinoma (P = 0.028). Conclusion Increased expression of p-ERK1/2 and PI3K might contribute to gallbladder carcinogenesis. p-ERK1/2 over-expression is correlated with decreased survival and therefore may serve as an important biological marker in development of gallbladder adenocarcinoma. PMID:19445727

High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

PubMed

Nguyen, Suong T T; McCurdy, David W

2015-04-23

Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level. The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation. Use of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.

Using an AMACR (P504S)/34betaE12/p63 cocktail for the detection of small focal prostate carcinoma in needle biopsy specimens.

PubMed

Jiang, Zhong; Li, Cuizhen; Fischer, Andrew; Dresser, Karen; Woda, Bruce A

2005-02-01

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (alpha-methylacyl coenzyme A racemase [AMACR, or P504S], 34betaE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34betaE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.

Astragalus polysaccharide suppresses palmitate-induced apoptosis in human cardiac myocytes: the role of Nrf1 and antioxidant response

PubMed Central

Zhang, Ji; Gu, Jian-Yun; Chen, Zhi-Song; Xing, Kai-Chen; Sun, Bing

2015-01-01

Objective: Previous studies have shown that Astragalus polysaccharides (APS) can be used to ameliorate cardiotoxicity due to chemotherapy and improve the cardiac function. However, the mechanism by which APS mediate this effect is unclear. In the present study, the effects of APS, which suppressed ROS-mediated apoptosis through Nrf1 accumulation in human cardiac myocytes (HCMs), was investigated. Methods: The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining. Expression of genes and proteins were analyzed by real-time PCR and western blotting respectively. Nrf1 gene was overexpressed using a lentiviral expression vector in HCMs in vitro, in order to explore the mechanism by which the Nrf1 promoted cell growth. Results: CCK8 and Annexin V-PI double-labeling showed that PAL induced cell death in a concentration-dependent manner, and suppressed HCMs proliferation. The combination PAL with APS was significantly decreased the percentage of the early phase of apoptosis cells. ROS levels were increased in HCMs by exposure to PAL. APS treatment significantly inhibited generation of ROS in response to palmitate. Moreover, PAL administration significantly decreased the mRNA and proteins expression of Bcl-2 as well as increased the mRNA expression of BAX and the protein expression of caspase-3 and caspase-8 as compare to those of control group, but APS treatment could reverse PA-induced HCMs apoptosis. The levels of reactive oxygen species (ROS), which was an oxidative stress marker, was significantly increased in cardiomyocytes by exposure to PAL, but overexpressing Nrf1 could ameliorate ROS-induced cardiomyocyte toxicity and increase the expression of SOD1 and SOD2 in HCMs by overexpressing Nrf1. Conclusions: This study demonstrated that the PAL could induce HCMs apoptosis. However, APS could reverse PAL-induced cardiomyocyte toxicity, at least partially, through suppression ROS and Nrf1 accumulation in HCMs. PMID:26045757

Astragalus polysaccharide suppresses palmitate-induced apoptosis in human cardiac myocytes: the role of Nrf1 and antioxidant response.

PubMed

Zhang, Ji; Gu, Jian-Yun; Chen, Zhi-Song; Xing, Kai-Chen; Sun, Bing

2015-01-01

Previous studies have shown that Astragalus polysaccharides (APS) can be used to ameliorate cardiotoxicity due to chemotherapy and improve the cardiac function. However, the mechanism by which APS mediate this effect is unclear. In the present study, the effects of APS, which suppressed ROS-mediated apoptosis through Nrf1 accumulation in human cardiac myocytes (HCMs), was investigated. The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining. Expression of genes and proteins were analyzed by real-time PCR and western blotting respectively. Nrf1 gene was overexpressed using a lentiviral expression vector in HCMs in vitro, in order to explore the mechanism by which the Nrf1 promoted cell growth. CCK8 and Annexin V-PI double-labeling showed that PAL induced cell death in a concentration-dependent manner, and suppressed HCMs proliferation. The combination PAL with APS was significantly decreased the percentage of the early phase of apoptosis cells. ROS levels were increased in HCMs by exposure to PAL. APS treatment significantly inhibited generation of ROS in response to palmitate. Moreover, PAL administration significantly decreased the mRNA and proteins expression of Bcl-2 as well as increased the mRNA expression of BAX and the protein expression of caspase-3 and caspase-8 as compare to those of control group, but APS treatment could reverse PA-induced HCMs apoptosis. The levels of reactive oxygen species (ROS), which was an oxidative stress marker, was significantly increased in cardiomyocytes by exposure to PAL, but overexpressing Nrf1 could ameliorate ROS-induced cardiomyocyte toxicity and increase the expression of SOD1 and SOD2 in HCMs by overexpressing Nrf1. This study demonstrated that the PAL could induce HCMs apoptosis. However, APS could reverse PAL-induced cardiomyocyte toxicity, at least partially, through suppression ROS and Nrf1 accumulation in HCMs.

Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

PubMed

Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

2015-10-01

Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Histochemical Examination on Periodontal Tissues of Klotho-Deficient Mice Fed With Phosphate-Insufficient Diet

PubMed Central

Hikone, Kumiko; Hasegawa, Tomoka; Tsuchiya, Erika; Hongo, Hiromi; Sasaki, Muneteru; Yamamoto, Tomomaya; Kudo, Ai; Oda, Kimimitsu; Haraguchi, Mai; de Freitas, Paulo Henrique Luiz; Li, Minqi; Iida, Junichiro; Amizuka, Norio

2017-01-01

To elucidate which of elevated serum concentration of inorganic phosphate (Pi) or disrupted signaling linked to αklotho/fibroblast growth factor 23 (FGF23) is a predominant regulator for senescence-related degeneration seen in αKlotho-deficient mice, we have examined histological alteration of the periodontal tissues in the mandibular interalveolar septum of αKlotho-deficient mice fed with Pi-insufficient diet. We prepared six groups of mice: wild-type, kl/kl, and αKlotho−/− mice with normal diet or low-Pi diet. As a consequence, kl/klnorPi and αKlotho−/−norPi mice showed the same abnormalities in periodontal tissues: intensely stained areas with hematoxylin in the interalveolar septum, dispersed localization of alkaline phosphatase–positive osteoblasts and tartrate-resistant acid phosphatase–reactive osteoclasts, and accumulation of dentin matrix protein 1 in the osteocytic lacunae. Although kl/kllowPi mice improved these histological abnormalities, αKlotho−/− lowPi mice failed to normalize those. Gene expression of αKlotho was shown to be increased in kl/kl lowPi specimens. It seems likely that histological abnormalities of kl/kl mice have been improved by the rescued expression of αKlotho, rather than low concentration of serum Pi. Thus, the histological malformation in periodontal tissues in αKlotho-deficient mice appears to be due to not only increased concentration of Pi but also disrupted αklotho/FGF23 signaling. PMID:28122194

Activation of the PI3K-Akt pathway promotes neuroprotection of the δ-opioid receptor agonist against cerebral ischemia-reperfusion injury in rat models.

PubMed

Lv, Mei-Rong; Li, Bin; Wang, Ming-Guang; Meng, Fan-Guo; Yu, Jian-Jun; Guo, Feng; Li, Ye

2017-09-01

The central objective was to identify the role of the PI3K-Akt activation pathway on the neuroprotection of δ-opioid receptor agonist (DADLE) against cerebral ischemia-reperfusion (I/R) injury in a rat model. Fifty-five male Sprague-Dawley (SD) rats were included to establish a middle cerebral artery occlusion (MCAO) model which were then divided into the sham, MCAO, LY294002 (MCAO+DADLE+LY294002 [inhibitor of PI3K-Akt pathway]), DADLE (MCAO+DADLE) and DMSO (MCAO+DADLE+DMSO [dimethyl sulphoxide]) groups. The cerebral infarction (CI) volume and nerve cell apoptosis was determined using TTC and TUNEL staining. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry staining were applied for the expressions of Bad, Bax, Bcl-2 and cleaved caspase-3. The MCAO group showed higher CI volume, nerve cell apoptosis and cleaved caspase-3 expressions than the DADLE and DMSO groups, which were also higher in the LY294002 group than the DADLE group. Compared with the MCAO group, the mRNA and protein expressions of PI3K and Bcl-2, and the protein expressions of p-Akt and p-Bad were elevated, while the mRNA and protein expressions of Bax were decreased in the DADLE and DMSO groups. Decreased mRNA and protein expressions of PI3K and Bcl-2, reduced protein expressions of p-Akt and p-Bad and elevated mRNA and protein expressions of Bax exhibited in the LY294002 group than the DADLE group. These results indicate that activation of PI3K-Akt pathway promotes the neuroprotection of DADLE against cerebral I/R injury in a rat model by decreasing nerve cells apoptosis. Copyright © 2017. Published by Elsevier Masson SAS.

Neuroanatomy of pars intercerebralis neurons with special reference to their connections with neurons immunoreactive for pigment-dispersing factor in the blow fly Protophormia terraenovae.

PubMed

Yasuyama, Kouji; Hase, Hiroaki; Shiga, Sakiko

2015-10-01

Input regions of pars intercerebralis (PI) neurons are examined by confocal and electron microscopies with special reference to their connections with neurons immunoreactive for pigment-dispersing factor (PDF) in the blow fly, Protophormia terraenovae. PI neurons are a prerequisite for ovarian development under long-day conditions. Backfills from the cardiac recurrent nerve after severance of the posterior lateral tracts labeled thin fibers derived from the PI neurons in the superior medial protocerebrum. These PI fibers were mainly synapsin-negative and postsynaptic to unknown varicose profiles containing dense-core vesicles. Backfilled fibers in the periesophageal neuropils, derived from the PI neurons or neurons with somata in the subesophageal zone, were varicose and some were synapsin-positive. Electron microscopy revealed the presence of both presynaptic and postsynaptic sites in backfilled fibers in the periesophageal neuropils. Many PDF-immunoreactive varicosities were found in the superior medial and lateral protocerebrum and double-labeling showed that 60-88 % of PDF-immunoreactive varicosities were also synapsin-immunoreactive. Double-labeling with the backfills and PDF immunocytochemistry showed that the PI fibers and PDF-immunoreactive varicosities were located close to each other in the superior medial protocerebrum. Results of triple-labeling of PI neurons, PDF-immunoreactive neurons and synapsin-immunoreactive terminals demonstrated that the synapsin-positive PDF-immunoreactive varicosities contacted the PI fibers. These data suggest that PI neurons receive synaptic contacts from PDF-immunoreactive fibers, which are derived from circadian clock neurons, of small ventral lateral neurons (previously called OL2) or posterior dorsal (PD) neurons with somata in the pars lateralis.

The relationship between apoptosis and the expression of proliferating cell nuclear antigen and the clinical stages in gastric carcinoma.

PubMed

Tao, K; Chen, D; Tian, Y; Lu, X; Yang, X

2000-01-01

The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5 +/- 3.7)% and (49.8 +/- 15.9)% respectively, and the rate of AI/PI was 0.13 +/- 0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P < 0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage II to IV (P < 0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

NF45 inhibits cardiomyocyte apoptosis following myocardial ischemia-reperfusion injury.

PubMed

Liu, Xiaojuan; Zhang, Chi; Qian, Long; Zhang, Chao; Wu, Kunpeng; Yang, Chen; Yan, Daliang; Wu, Xiang; Shi, Jiahai

2015-12-01

Cardiomyocyte apoptosis, which occurs during ischemia and reperfusion injury, can cause irreversible damage to cardiac function. There is accumulating evidence that nuclear factor 45 (NF45) and regulatory pathways are important in understanding reparative processes in the myocardium. NF45 is a multifunctional regulator of gene expression that participates in the regulation of DNA break repair. Recently, NF45 has been proved to be associated with tumor cell apoptosis in various human malignancies. However, the underlying mechanism of NF45 regulating myocardial ischemia-reperfusion (I/R) injury remains unclear. In this study, western blot showed that NF45 expression decreased after myocardial I/R in vivo. Double immunofluorescent staining revealed that NF45, located in the nucleus of cardiomyocyes, was correlated with cardiomyocyte apoptosis. Furthermore, NF45 expression decreased in H9c2 cells after hypoxia-reoxygenation (H/R) treatment in vitro, which was in line with the results in vivo. Overexpression of NF45 in H9c2 cells reduced cell apoptosis, as evidenced by increased Bcl-2 level, as well as decreased cleaved caspase-3, p53 and p21 expression. The expression of NF45 was reduced by LY294002 (a PI3K/Akt inhibitor), but not SB203580 (a p38 inhibitor), suggesting that NF45 prevented H/R-induced H9c2 cell apoptosis via PI3K/Akt pathway. Our data may supply a novel molecular target for acute myocardial infarction (AMI) therapy. Copyright © 2015 Elsevier GmbH. All rights reserved.

Atypical localization of the oligodendrocytic isoform (PI) of glutathione-S-transferase in astrocytes during cuprizone intoxication.

PubMed

Cammer, W; Zhang, H

1993-10-01

Immunocytochemical staining for the Pi and Mu isoforms of glutathione-S-transferase was used to investigate changes in the glial cells in the mouse forebrain. During early development in mouse forebrains the localizations of carbonic anhydrase, Pi and Mu were similar to the respective cellular localizations that had been observed in neonatal rat brain. That is, Pi was found in oligodendrocyte precursors, Mu in astrocytes, and carbonic anhydrase in both oligodendrocyte precursors and astrocytes. In forebrains of 6-week-old mice the neurotoxicant, cuprizone, induced oligodendrocyte degeneration, gliosis, and partial demyelination. Degeneration, gliosis, and partial demyelination. Degeneration of oligodendrocytes, and astrocytosis, began during the initial week of cuprizone feeding, and by the end of the eighth week some demyelination was observed. After mice were fed cuprizone for 4 to 7 weeks, Pi appeared in some of the reactive astrocytes, and Pi-positive astrocytes were present for at least 7 additional weeks. Normally, Pi appeared only in oligodendrocytes. Very few Pi-positive oligodendrocytes remained after the second week. During the eighth week healthy-looking carbonic anhydrase-positive oligodendrocytes reappeared and began to accumulate, and a few small patches of Pi-positive oligodendrocytes were also observed. In summary, some novel findings about glial cells were the observation of an enzyme (Pi) that is lost earlier from oligodendrocytes than is carbonic anhydrase, the apparently unique shift in Pi expression from oligodendrocytes to astrocytes and the greater temporal dissociation between loss of oligodendrocytes and demyelination in the older mice.

Charged bottomoniumlike states Z{sub b}(10610) and Z{sub b}(10650) and the {Upsilon}(5S){yields}{Upsilon}(2S){pi}{sup +}{pi}{sup -} decay

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

Chen Dianyong; Nuclear Theory Group, Institute of Modern Physics of CAS, Lanzhou 730000; Liu Xiang

2011-10-01

Inspired by the newly observed two charged bottomoniumlike states, we consider the possible contribution from the intermediate Z{sub b}(10610) and Z{sub b}(10650) states to the {Upsilon}(5S){yields}{Upsilon}(2S){pi}{sup +}{pi}{sup -} decay process, which naturally explains Belle's previous observation of the anomalous {Upsilon}(2S){pi}{sup +}{pi}{sup -} production near the peak of {Upsilon}(5S) at {radical}(s)=10.87 GeV [K. F. Chen et al. (Belle Collaboration), Phys. Rev. Lett. 100, 112001 (2008)]. The resulting d{Gamma}({Upsilon}(5S){yields}{Upsilon}(2S){pi}{sup +}{pi}{sup -})/dm{sub {pi}}{sup +}{sub {pi}}{sup -} and d{Gamma}({Upsilon}(5S){yields}{Upsilon}(2S){pi}{sup +}{pi}{sup -})/dcos{theta} distributions agree with Belle's measurement after inclusion of these Z{sub b} states. This formalism also reproduces the Belle observation of the double-peak structuremore » and its reflection in the {Upsilon}(2S){pi}{sup +} invariant mass spectrum of the {Upsilon}(5S){yields}{Upsilon}(2S){pi}{sup +}{pi}{sup -} decay.« less

The ζ Toxin Induces a Set of Protective Responses and Dormancy

PubMed Central

Tabone, Mariangela; Gonzalez-Pastor, José E.; Daugelavicius, Rimantas; Ayora, Silvia; Alonso, Juan C.

2012-01-01

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity. PMID:22295078

Sperm membrane functionality in the dog assessed by flow cytometry.

PubMed

Cheuquemán, C; Bravo, P; Treulén, F; Giojalas, Lc; Villegas, J; Sánchez, R; Risopatrón, J

2012-02-01

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa. © 2011 Blackwell Verlag GmbH.

The ζ toxin induces a set of protective responses and dormancy.

PubMed

Lioy, Virginia S; Machon, Cristina; Tabone, Mariangela; Gonzalez-Pastor, José E; Daugelavicius, Rimantas; Ayora, Silvia; Alonso, Juan C

2012-01-01

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε(2)) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20-30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1-5×10(-5)). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K(+). We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε(2) antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε(2) antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity.

Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line.

PubMed

Gao, Lin-Lin; Feng, Lei; Yao, Shu-Tong; Jiao, Peng; Qin, Shu-Cun; Zhang, Wei; Zhang, Ya-Bin; Li, Fu-Rong

2011-01-01

Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.

Targeting property and toxicity of a novel ultrasound contrast agent microbubble carrying the targeting and drug-loaded complex FA-CNTs-PTX on MCF7 cells.

PubMed

Zhang, Jie; Zhang, Yu; Liu, Junxi; Li, Guozhong; Wen, Zhaohui; Zhao, Yue; Zhang, Xiangyu; Liu, Fenghua

2017-10-01

The application of ultrasound contrast agents not only is confined to the enhancement of ultrasound imaging but also has started to be used as a drug system for diagnosis and treatment. In this paper, Span60 and PEG1500 were used as membrane materials, and a new targeting and drug-loading multifunctional ultrasound contrast agent microbubble enveloping the FA-CNTs-PTX complex was successfully prepared by acoustic cavitation. With the breast cancer cell line MCF7 as the research target, the effects of the microbubble with FA-CNTs-PTX on the proliferation and toxicity of MCF7 cells were studied using a CCK-8 and AO/EB double-staining method. The influences of the microbubbles with FA-CNTs-PTX on the cellular morphology and apoptosis period of the MCF7 cells were detected using an inverted fluorescence microscope. The apoptosis of MCF7 cells induced by the microbubbles with FA-CNTs-PTX was investigated with flow cytometry and an annexin and PI double staining fluorescence quantitative analysis. The results indicated that the ultrasound contrast agent microbubble with FA-CNTs-PTX remarkably inhibited the proliferation of MCF7 cells, which was mainly controlled by the drug loading rate and the nanometer size of the microbubbles. Moreover, the proliferative inhibition rate of the microbubbles with FA-CNTs-PTX was related to the cell apoptosis period of MCF7 cells. Its inhibition degree on the proliferation of MCF7 cells was higher than that of the hepatoma HepG2 cells. The apoptosis rate of MCF7 cells induced by the microbubbles with FA-CNTs-PTX was higher than that of normal human umbilical vein endothelial cells (HUVECs), and the microbubbles with FA-CNTs-PTX could target the MCF7 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Measurement of Single and Double Spin Asymmetries in p(e, e' pi(+/-,0))X Semi-Inclusive Deep-Inelastic Scattering

NASA Astrophysics Data System (ADS)

Jawalkar, Sucheta Shrikant

Measurements in the late 1980s at CERN revealed that quark spins account for a small fraction of the proton's spin. This so-called spin crisis spurred a number of new experiments to identify the proton's silent spin contributors, namely, the spin of the gluons, which hold the quarks together, and the orbital angular momentum of both quarks and gluons. One such experiment was eg1-dvcs at the Thomas Jefferson National Accelerator Facility in Newport News, Va., which ran in 2009 and collected approximately 19 billion electron triggers for hydrogen. I will present new measurements of the single and double-spin asymmetries ALU, AUL and ALL for pi+, pi - and pi0, measured as a function of Bjorken xB, squared momentum transfer Q2, hadron energy fraction z, and hadron transverse momentum Ph ⊥. These asymmetries, which are convolutions of transverse-momentum-dependent parton distributions and fragmentation functions, correlate with the transverse momentum, and therefore with the orbital motion, of the struck quark.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

PubMed

Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai-Kei; Zhang, Wei

2015-07-26

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.

Propidium iodide competes with Ca(2+) to label pectin in pollen tubes and Arabidopsis root hairs.

PubMed

Rounds, Caleb M; Lubeck, Eric; Hepler, Peter K; Winship, Lawrence J

2011-09-01

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca(2+) binding. We first show that Ca(2+) is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca(2+) in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca(2+)-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca(2+) gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca(2+) in tip growth.

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

KoraMagazi, Arouna; Wang, Dandan; Yousef, Bashir

Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-likemore » ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells. - Highlights: • Rhein triggers apoptotic cell death on primary human hepatic HL-7702 cells. • Rhein leads to caspase-4 activation in HL-7702 cells. • Rhein induces endoplasmic reticulum stress pathways in HL-7702 cells. • Rhein causes elevation of intracellular calcium concentrations in HL-7702 cells.« less

Gold Nanoparticles of Diameter 13 nm Induce Apoptosis in Rabbit Articular Chondrocytes

NASA Astrophysics Data System (ADS)

Huang, Hao; Quan, Ying-yao; Wang, Xiao-ping; Chen, Tong-sheng

2016-05-01

Gold nanoparticles (AuNPs) have been widely used in biomedical science including antiarthritic agents, drug loading, and photothermal therapy. In this report, we studied the effects of AuNPs with diameters of 3, 13, and 45 nm, respectively, on rabbit articular chondrocytes. AuNPs were capped with citrate and their diameter and zeta potential were measured by dynamic light scattering (DLS). Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay after the rabbit articular chondrocytes were pre-incubated with 3, 13, and 45 nm AuNPs, respectively, for 24 h. Flow cytometry (FCM) analysis with annexin V/propidium iodide (PI) double staining and fluorescence imaging with Hoechst 33258 staining were used to determine the fashion of AuNPs-induced chondrocyte death. Further, 13 nm AuNPs (2 nM) significantly induced chondrocyte death accompanying apoptotic characteristics including mitochondrial damage, externalization of phosphatidylserine and nuclear concentration. However, 3 nm AuNPs (2 nM) and 45 nm (0.02 nM) AuNPs did not induce cytotoxicity in chondrocytes. Although 13 nm AuNPs (2 nM) increased the intracellular reactive oxygen species (ROS) level, pretreatment with Nacetyl cysteine (NAC), a ROS scavenger, did not prevent the cytotoxicity induced by 13 nm AuNPs, indicating that 13 nm AuNPs (2 nM) induced ROS-independent apoptosis in chondrocytes. These results demonstrate the size-dependent cytotoxicity of AuNPs in chondrocytes, which must be seriously considered when using AuNPs for treatment of osteoarthritis (OA).

In-vitro antitumor activity evaluation of hyperforin derivatives.

PubMed

Sun, Feng; Liu, Jin-Yun; He, Feng; Liu, Zhong; Wang, Rui; Wang, Dong-Mei; Wang, Yi-Fei; Yang, De-Po

2011-08-01

The derivatives of hyperforin, namely hyperforin acetate (2), 17,18,22,23,27,28,32,33-octahydrohyperforin acetate (3), and N,N-dicyclohexylamine salt of hyperforin (4), have been investigated for their antitumor properties. In-vitro studies demonstrated that 2 and 4 were active against HeLa (human cervical cancer), A375 (human malignant melanoma), HepG2 (human hepatocellular carcinoma), MCF-7 (human breast cancer), A549 (human nonsmall cell lung cancer), K562 (human chronic myeloid leukemia), and K562/ADR (human adriamycin-resistant K562) cell lines with IC(50) values in the range of 3.2-64.1 μM. The energy differences between highest occupied molecular orbital and lowest unoccupied molecular orbital of 2-4 were calculated to be 0.39778, 0.43106, and 0.30900 a.u., respectively, using the Gaussian 03 software package and ab initio method with the HF/6-311 G* basis set. The result indicated that the biological activity of 4 might be the strongest and that of 3 might be the weakest, which was in accordance with their corresponding antiproliferative effects against the tested tumor cell lines. Compound 4 caused cell cycle arrest at G2/M phase in flow cytometry experiment and induced apoptosis by 4',6-diamidino-2-phenylindole staining and Annexin V-FITC/PI (propidium iodide) double-labeled staining in HepG2 cells. The results indicated a potential for N,N-dicyclohexylamine salt of hyperforin as a new antitumor drug.

Mitomycin C induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via a mitochondrial-mediated pathway.

PubMed

Yan, Chuqi; Kong, Dechao; Ge, Dong; Zhang, Yanming; Zhang, Xishan; Su, Changhui; Cao, Xiaojian

2015-01-01

Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway. © 2015 S. Karger AG, Basel.

Self-Assembly of Large-Scale Shape-Controlled DNA Nano-Structures

DTIC Science & Technology

2014-12-16

discharged carbon-coated TEM grids for 4 min and then stained for 1 min using a 2% aqueous uranyl formate solution containing 25 mM NaOH. Imaging was...temperature for 3 h in the dark. TEM imaging. For imaging, 2,5 pi annealed sample was adsorbed for 2 min onto glow- discharged , carbon-coated TEM grids...Imaging. For ’I’EM imaging, a 3.S //L sample (l—5 nM) was adsorbed onto glow discharged carbon-coated TEM grids for 4 min and then stained for 1 min or a

Long-term, repeated dose in vitro neurotoxicity of the glutamate receptor antagonist L-AP3, demonstrated in rat hippocampal slice cultures by using continuous propidium iodide incubation.

PubMed

Kristensen, Bjarne W; Blaabjerg, Morten; Noraberg, Jens; Zimmer, Jens

2007-05-01

Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2-3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100microM L-AP3, with PI (2microM) in the culture medium. Exposure to 100microM L-AP3 induced severe toxicity after 4-6 days, shown by massive PI uptake, LDH leakage, changes in MAP2 and GFAP immunostaining, and in Nissl and Timm staining. In contrast, 10microM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist NBQX, together with 100microM L-AP3, reduced neurodegeneration down to close to control values. It is concluded that continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies of inducible neuronal degeneration over time.

7 CFR 42.112 - Defects of containers: Tables IV, V, VI, and VII.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Rust (rust stain confined to the top or bottom double seam or rust that can be removed with a soft cloth is not scored a defect): (a) Rust stain (nonmilitary purchases) 206 (b) Rust stain (military purchases) 108 (c) Pitted rust 109 Wet cans (excluding refrigerated containers) 207 Dent: (a) Materially...

7 CFR 42.112 - Defects of containers: Tables IV, V, VI, and VII.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Rust (rust stain confined to the top or bottom double seam or rust that can be removed with a soft cloth is not scored a defect): (a) Rust stain (nonmilitary purchases) 206 (b) Rust stain (military purchases) 108 (c) Pitted rust 109 Wet cans (excluding refrigerated containers) 207 Dent: (a) Materially...

7 CFR 42.112 - Defects of containers: Tables IV, V, VI, and VII.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Rust (rust stain confined to the top or bottom double seam or rust that can be removed with a soft cloth is not scored a defect): (a) Rust stain (nonmilitary purchases) 206 (b) Rust stain (military purchases) 108 (c) Pitted rust 109 Wet cans (excluding refrigerated containers) 207 Dent: (a) Materially...

7 CFR 42.112 - Defects of containers: Tables IV, V, VI, and VII.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Rust (rust stain confined to the top or bottom double seam or rust that can be removed with a soft cloth is not scored a defect): (a) Rust stain (nonmilitary purchases) 206 (b) Rust stain (military purchases) 108 (c) Pitted rust 109 Wet cans (excluding refrigerated containers) 207 Dent: (a) Materially...

Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm.

PubMed

Cayli, Sevil; Sakkas, Denny; Vigue, Lynne; Demir, Ramazan; Huszar, Gabor

2004-05-01

The relationship between human sperm maturity and apoptosis is of interest because of the persistence of immature sperm in ejaculates in spite of various apoptotic processes during spermatogenesis. We assessed sperm maturity by HspA2 chaperone levels, and plasma membrane maturity by sperm binding to immobilized hyaluronic acid (HA). We also utilized objective morphometry. Sperm were stained with three antibody combinations: active caspase-3/creatine kinase (CK, a marker of cytoplasmic retention), caspase-3/the antiapoptotic Bcl-(XL), and CK/Bcl-(XL). In semen, 13% of sperm stained with CK, caspase-3 or Bcl-(XL), and 28% had stained with two markers. In the mature HA-bound sperm fraction, <4% were single- or double-stained. Regarding sperm regions, CK staining, whether alone or as double staining, occurred in the head and midpiece (15-20%), whereas caspase-3 and Bcl-(XL) were primarily (>80% of sperm) in the midpiece. Morphometrical attributes of clear, single- and double-stained sperm, in line with their more pronounced maturation arrest, showed an incremental increase in head size (due to cytoplasmic retention) and shorter tail length. We hypothesize that during faulty sperm development, three alternatives may occur: (i) elimination of aberrant germ cells by apoptosis; (ii) in surviving immature cells, caspase-3 is activated, and in response the antiapoptotic Bcl-(XL), and perhaps HspA2, provide protection; (iii) in a third type of immature sperm, in addition to the CK, caspase-3 and Bcl-(XL) expression, there are related manifestations of increased head size and shorter tail length. Thus, immature sperm may vary in the type of developmental arrest and in protection mechanisms for apoptosis. These variations are likely to explain the persistence of immature sperm in the ejaculate.

The influence of TLR4 agonist lipopolysaccharides on hepatocellular carcinoma cells and the feasibility of its application in treating liver cancer.

PubMed

Gu, Junsheng; Sun, Ranran; Shen, Shen; Yu, Zujiang

2015-01-01

This study was designed to explore the influence of Toll-like receptor 4 (TLR4) agonist lipopolysaccharides (LPS) on liver cancer cell and the feasibility to perform liver cancer adjuvant therapy. Human liver cancer cell lines HepG2, H7402, and PLC/PRF/5 were taken as models, and the expression of TLRs mRNA was detected by real time-polymerase chain reaction method semiquantitatively. WST-1 method was used to detect the influence of LPS on the proliferation ability of liver cancer cells; propidium iodide (PI) single staining and Annexin V/PI double staining were used to test the influence of LPS on the cell cycle and apoptosis, respectively, on human liver cancer cell line H7402. Fluorescent quantitative polymerase chain reaction and Western blot method were used to determine the change of expression of Cyclin D1. The results demonstrated that most TLRs were expressed in liver cancer cells; stimulating TLR4 by LPS could upregulate TLR4 mRNA and the protein level, activate NF-κB signaling pathway downstream of TLR4, and mediate the generation of inflammatory factors IL-6, IL-8, and TNF-α; LPS was found to be able to strengthen the proliferation ability of liver cancer cells, especially H7402 cells; the expression of Cyclin D1 rose and H7402 cells were promoted to transit from G1 stage to S stage under the stimulation of LPS, but cell apoptosis was not affected. It was also found that LPS was able to activate signal transducer and activator of transcription -3 (STAT3) signaling pathway in H7402 cells and meanwhile significantly increase the initiation activity of STAT3; proliferation promoting effect of LPS to liver cancer cells remarkably lowered once STAT3 was blocked or inhibited. Thus, TLR4 agonist LPS is proved to be able to induce liver cancer cells to express inflammation factors and mediate liver cancer cell proliferation and generation of multidrug resistance by activating the cyclooxygenase-2/prostaglandin signal axis as well as the STAT3 pathway.

Quercetin induced apoptosis of human oral cancer SAS cells through mitochondria and endoplasmic reticulum mediated signaling pathways.

PubMed

Ma, Yi-Shih; Yao, Chien-Ning; Liu, Hsin-Chung; Yu, Fu-Shun; Lin, Jen-Jyh; Lu, Kung-Wen; Liao, Ching-Lung; Chueh, Fu-Shin; Chung, Jing-Gung

2018-06-01

Oral cancer is a cause of cancer-associated mortality worldwide and the treatment of oral cancer includes radiation, surgery and chemotherapy. Quercetin is a component from natural plant products and it has been demonstrated that quercetin is able to induce cytotoxic effects through induction of cell apoptosis in a number of human cancer cell lines. However, there is no available information to demonstrate that quercetin is able to induce apoptosis in human oral cancer cells. In the present study, the effect of quercetin on the cell death via the induction of apoptosis in human oral cancer SAS cells was investigated using flow cytometry, Annexin V/propidium iodide (PI) double staining, western blotting and confocal laser microscopy examination, to test for cytotoxic effects at 6-48 h after treatment with quercetin. The rate of cell death increased with the duration of quercetin treatment based on the results of a cell viability assay, increased Annexin V/PI staining, increased reactive oxygen species and Ca 2+ production, decreased the levels of mitochondrial membrane potential (ΔΨ m ), increased proportion of apoptotic cells and altered levels of apoptosis-associated protein expression in SAS cells. The results from western blotting revealed that quercetin increased Fas, Fas-Ligand, fas-associated protein with death domain and caspase-8, all of which associated with cell surface death receptor. Furthermore, quercetin increased the levels of activating transcription factor (ATF)-6α, ATF-6β and gastrin-releasing peptide-78 which indicated an increase in endoplasm reticulum stress, increased levels of the pro-apoptotic protein BH3 interacting-domain death antagonist, and decreased levels of anti-apoptotic proteins B-cell lymphoma (Bcl) 2 and Bcl-extra large which may have led to the decreases of ΔΨ m . Additionally, confocal microscopy suggested that quercetin was able to increase the expression levels of cytochrome c , apoptosis-inducing factor and endonuclease G, which are associated with apoptotic pathways. Therefore, it is hypothesized that quercetin may potentially be used as a novel anti-cancer agent for the treatment of oral cancer in future.

Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

PubMed Central

Ahmad Mazian, Mu'adz; Salleh, Abu Bakar; Basri, Mahiran; Rahman, Raja Noor Zaliha Raja Abd.

2014-01-01

Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. PMID:25093119

Theoretical study of the A prime 5Sigma(+)g and C double prime 5Pi u states of N2 - Implications for the N2 afterglow

NASA Technical Reports Server (NTRS)

Partridge, Harry; Langhoff, Stephen R.; Bauschlicher, Charles W., Jr.; Schwenke, David W.

1988-01-01

Theoretical spectroscopic constants are reported for the A prime 5Sigma(+)g and C double prime 5Pi u states of N2 based on CASSCF/MRCI calculations using large ANO Gaussian basis sets. The calculated A prime Sigma(+)g potential differs qualitatively from previous calculations in that the inner well is significantly deeper (De = 3450/cm). This deeper well provides considerable support for the suggestion of Berkowitz et al. (1956) that A prime 5Sigma(+)g is the primary precursor state involved in the yellow Lewis-Rayleigh afterglow of N2.

Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme.

PubMed

Islam, Shah Md Asraful; Yeasmin, Shabina; Islam, Md Saiful; Islam, Md Shariful

2017-07-01

The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Exclusive Central $$\\pi^{+}\\pi^{-}$$ Production in Proton Antiproton Collisions at the CDF

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

Zurek, Maria

Exclusivemore » $$\\pi^{=}\\pi^{-}$$ production in proton-antiproton collisions at $$\\sqrt{s}$$ = 0.9 and 1.96 TeV in the Collider Detector at Fermilab has been measured. We select events with two particles with opposite charge in pseudorapidity region -1.3 < $$\\eta$$ < 1.3 with no other particles detected in -5.9 < $$\\eta$$ < 5.9. Particles are assumed to be pions. The $$\\pi^{+}\\pi^{-}$$system is required to have rapidity -1.0 < $y$ < 1.0. The data are expected to be dominated by the double pomeron exchange mechanism. Therefore, the quantum numbers of the central state are constrained. The data extend up to dipion mass M($$\\pi^{+}\\pi^{-}$$) = 5000 MeV/$c^2$. Resonance structures consistent with $$f_0$$ and $$f_2$$(1270) mesons are visible. The results are valuable for light hadron spectroscopy and for providing information about the nature of the pomeron in a region between non-perturbative and perturbative quantum chromodynamics« less

Computational Analysis of Mouse piRNA Sequence and Biogenesis

PubMed Central

Betel, Doron; Sheridan, Robert; Marks, Debora S; Sander, Chris

2007-01-01

The recent discovery of a new class of 30-nucleotide long RNAs in mammalian testes, called PIWI-interacting RNA (piRNA), with similarities to microRNAs and repeat-associated small interfering RNAs (rasiRNAs), has raised puzzling questions regarding their biogenesis and function. We report a comparative analysis of currently available piRNA sequence data from the pachytene stage of mouse spermatogenesis that sheds light on their sequence diversity and mechanism of biogenesis. We conclude that (i) there are at least four times as many piRNAs in mouse testes than currently known; (ii) piRNAs, which originate from long precursor transcripts, are generated by quasi-random enzymatic processing that is guided by a weak sequence signature at the piRNA 5′ends resulting in a large number of distinct sequences; and (iii) many of the piRNA clusters contain inverted repeats segments capable of forming double-strand RNA fold-back segments that may initiate piRNA processing analogous to transposon silencing. PMID:17997596

Directional properties in photodissociation: a probe for the symmetry and geometry of excited states of dimethylnitrosamine and tert-butyl nitrite

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

Lavi, R.; Schwartz-Lavi, I.B.; Rosenwaks, S.

1987-10-08

The rotational alignment, ..lambda..-doubling, and spin-orbit populations of the NO fragment following the photodissociation of (CH/sub 3/)/sub 2/NNO and (CH/sub 3/)/sub 3/CONO in different absorption bands are determined. The results for the S/sub 1/(n..pi..*) withdraws S/sub 0/ and S/sub 2/(..pi pi..*) withdraws S/sub 0/ transitions in both molecules indicate planarity of the fragmentation processes and an A'' symmetry of the S/sub 1/ state. For the S/sub 2/(..pi pi..*) withdraws S/sub 0/ transition in (CH/sub 3/)/sub 2/NNO the results imply an A' symmetry of the S/sub 2/ state. The values obtained for the alignment are indicative of the difference between themore » directions of the transition dipole moment for the S/sub 1/(n..pi..*) withdraws S/sub 0/ and the S/sub 2/(..pi pi..*) withdraws S/sub 0/ transitions.« less

Registration of two double rust resistant germplasms, HA-R12 and HA-R13 for confection sunflower

USDA-ARS?s Scientific Manuscript database

The confection sunflower (Helianthus annuus L.) germplasms HA-R12 (Reg. No. ______, PI 673104) and HA-R13 (Reg. No. ______, PI 673105) were developed by the USDA-ARS, Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station, and released in Jul...

Ectopic expression of a Catalpa bungei (Bignoniaceae) PISTILLATA homologue rescues the petal and stamen identities in Arabidopsis pi-1 mutant.

PubMed

Jing, Danlong; Xia, Yan; Chen, Faju; Wang, Zhi; Zhang, Shougong; Wang, Junhui

2015-02-01

PISTILLATA (PI) plays crucial roles in Arabidopsis flower development by specifying petal and stamen identities. To investigate the molecular mechanisms underlying organ development of woody angiosperm in Catalpa, we isolated and identified a PI homologue, referred to as CabuPI (C. bungei PISTILLATA), from two genetically cognate C. bungei (Bignoniaceae) bearing single and double flowers. Sequence and phylogenetic analyses revealed that the gene is closest related to the eudicot PI homologues. Moreover, a highly conserved PI-motif is found in the C-terminal regions of CabuPI. Semi-quantitative and quantitative real time PCR analyses showed that the expression of CabuPI was restricted to petals and stamens. However, CabuPI expression in the petals and stamens persisted throughout all floral development stages, but the expression levels were different. In 35S::CabuPI transgenic homozygous pi-1 mutant Arabidopsis, the second and the third whorl floral organs produced normal petals and a different number of stamens, respectively. Furthermore, ectopic expression of the CabuPI in transgenic wild-type or heterozygote pi-1 mutant Arabidopsis caused the first whorl sepal partially converted into a petal-like structure. These results clearly reveal the functional conservation of PI homologues between C. bungei and Arabidopsis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A double blind randomised controlled clinical trial comparing a novel anti-stain and calculus reducing dentifrice with a standard fluoride dentifrice.

PubMed

Jowett, Adrian K; Marlow, Ian; Rawlinson, Andrew

2013-04-01

This clinical trial tested the anti-stain efficacy at 3 and 6 months of a novel, sodium polyaspartate-containing, anti-stain dentifrice. In addition, the efficacy of the new dentifrice in controlling gingival inflammation and inhibition of calculus deposition was tested. Participants were recruited to this double blind randomised control clinical trial, and allocated to either test or control groups. The presence of stain and calculus were entry criteria. Measurements of stain, calculus and gingival inflammation were recorded using the Shaw and Murray Stain score, Volpe-Manhold Calculus score and the Modified Gingival Index respectively. Measurements were made at baseline, prior to the removal of stain and calculus, and after 3 and 6 months. Missing data were imputed by and the outcomes were analysed using univariate analysis. At three months, toothpaste containing sodium polyaspartate was better (difference of mean 1.13 with SEM 0.57) than control for the control of dental stain (p<0.05). Stain scores also showed a trend in favour of the test product (difference of mean 1.03 with SEM 0.78) at six months (p>0.05). There was no difference between toothpastes with respect to calculus deposition or gingival inflammation. Toothpaste containing sodium polyaspartate was more effective than a control toothpaste at preventing deposition of dental stain for 3 months after professional tooth cleaning but showed no significant effect at 6 months. Sodium polyaspartate toothpaste was more effective than a control toothpaste at preventing dental stain formation and maybe helpful in controlling staining between episodes of scaling and polishing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma.

PubMed

Maxwell, W M; Welch, G R; Johnson, L A

1996-01-01

Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.

OCT4B1 Regulates the Cellular Stress Response of Human Dental Pulp Cells with Inflammation

PubMed Central

Liu, Lu; Huang, Rong; Yang, Ruiqi

2017-01-01

Introduction. Infection and apoptosis are combined triggers for inflammation in dental tissues. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel spliced variant of OCT4 family, could respond to the cellular stress and possess antiapoptotic property. However, its specific role in dental pulpitis remains unknown. Methods. To investigate the effect of OCT4B1 on inflammation of dental pulp cells (DPCs), its expression in inflamed dental pulp tissues and DPCs was examined by in situ hybridization, real-time PCR, and FISH assay. OCT4B1 overexpressed DPCs model was established, confirmed by western blot and immunofluorescence staining, and then stimulated with Lipopolysaccharide (LPS). Apoptotic rate was determined by Hoechst/PI staining and FACS. Cell survival rate was calculated by CCK8 assay. Results. In situ hybridization, real-time PCR, and FISH assay revealed that OCT4B1 was extensively expressed in inflamed dental pulp tissues and DPCs with LPS stimulation. Western blot and immunofluorescence staining showed the expression of OCT4B1 and OCT4B increased after OCT4B1 transfection. Hoechst/PI staining and FACS demonstrated that less red/blue fluorescence was detected and apoptotic percentage decreased (3.45%) after transfection. CCK8 demonstrated that the survival rate of pCDH-OCT4B1-flag cells increased. Conclusions. OCT4B1 plays an essential role in inflammation and apoptosis of DPCs. OCT4B might operate synergistically with OCT4B1 to reduce apoptosis. PMID:28473980

Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation

PubMed Central

Rangiani, Afsaneh; Cao, Zheng-Guo; Liu, Ying; Voisey Rodgers, Anika; Jiang, Yong; Qin, Chun-Lin; Feng, Jian-Quan

2012-01-01

Deletion or mutation of dentin matrix protein 1 (DMP1) leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate (Pi) or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmp1-null (Dmp1−/−), Klotho-deficient (kl/kl), Dmp1/Klotho-double-deficient (Dmp1−/−/kl/kl) and wild-type (WT) mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography (μCT), scanning electron microscopy (SEM), histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmp1−/− (a low Pi level) or kl/kl (a high Pi level) mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice (a high Pi level) displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment (a high Pi level). PMID:23258378

Loss of ErbB2-PI3K/Akt signaling prevents zinc pyrithione-induced cardioprotection during ischemia/reperfusion.

PubMed

Thokala, Sandhya; Inapurapu, Santhipriya; Bodiga, Vijaya Lakshmi; Vemuri, Praveen Kumar; Bodiga, Sreedhar

2017-04-01

The purpose of this study was to determine if zinc homeostasis is affected during ischemia/reperfusion, if so, whether zinc pyrithione limits myocardial cell death and improves hemodynamics when administered as an adjunct to reperfusion and if ErbB receptor tyrosine kinases that are important for the long-term structural integrity of the heart are indispensable for reperfusion salvage. Isolated perfused rat hearts were subjected to 35min of global ischemia and reperfused for 120min to determine the relative intracellular zinc levels by TSQ staining. The hearts were reperfused in the presence of incremental concentrations of zinc pyrithione for the first 10min during reperfusion. Silencing or blockade of ErbB2 using a monoclonal antibody, ErbB2 kinase inhibition and PI3kinase inhibition was used to study their critical role in zinc pyrithione-induced cardioprotection. We found that there was a profound decrease in intracellular zinc after ischemia/reperfusion resulting in increased apoptosis, caspase-3 activation, and infarct size. A dose-dependent reduction of infarct size with zinc pyrithione in the range of 5-20μmol/l (optimal protection was seen at 10μmol/l with infarct size of 16±2% vs. I/R vehicle, 33±2%, p<0.01). Increased TUNEL staining and caspase-3 activity observed after ischemia/reperfusion were attenuated by zinc pyrithione administration during the reperfusion. Moreover, this protection was sensitive to silencing and blockade of ErbB2, inhibition of ErbB2 kinase activity or PI3-kinase activity. Western blot analysis revealed that zinc pyrithione resulted in decreased caspase-3 activation, rapid stabilization of ErbB2/ErbB1 heterodimers, and increased activation of PI3K/Akt signaling cascade. Zinc pyrithione attenuates lethal perfusion-induced injury in a manner that is reliant on ErbB2/PI3K/Akt activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Propidium Iodide Competes with Ca2+ to Label Pectin in Pollen Tubes and Arabidopsis Root Hairs1[W][OA

PubMed Central

Rounds, Caleb M.; Lubeck, Eric; Hepler, Peter K.; Winship, Lawrence J.

2011-01-01

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca2+ binding. We first show that Ca2+ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca2+ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca2+-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca2+ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca2+ in tip growth. PMID:21768649

Hydrogen postconditioning promotes survival of rat retinal ganglion cells against ischemia/reperfusion injury through the PI3K/Akt pathway.

PubMed

Wu, Jiangchun; Wang, Ruobing; Yang, Dianxu; Tang, Wenbin; Chen, Zeli; Sun, Qinglei; Liu, Lin; Zang, Rongyu

2018-01-22

Retinal ischemia/reperfusion injury (IRI) plays a crucial role in the pathophysiology of various ocular diseases. Our previous study have shown that postconditioning with inhaled hydrogen (H 2 ) (HPC) can protect retinal ganglion cells (RGCs) in a rat model of retinal IRI. Our further study aims to investigate potential mechanisms underlying HPC-induced protection. Retinal IRI was performed on the right eyes of rats and was followed by inhalation of 67% H 2 mixed with 33% oxygen immediately after ischemia for 1 h daily for one week. RGC density was counted using haematoxylin and eosin (HE) staining, retrograde labelling with cholera toxin beta (CTB) and TUNEL staining, respectively. Visual function was assessed using flash visual evoked potentials (FVEP) and pupillary light reflex (PLR). The phosphorylated Akt was analysed by RT-PCR and western blot. The results showed that administration of HPC significantly inhibited the apoptosis of RGCs and protected the visual function. Simultaneously, HPC treatment markedly increased the phosphorylations of Akt. Blockade of PI3K activity by inhibitors (LY294002) dramatically abolished its anti-apoptotic effect and lowered both visual function and Akt phosphorylation levels. Taken together, our results demonstrate that HPC appears to confer neuroprotection against retinal IRI via the PI3K/Akt pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

CINtec PLUS immunocytochemistry as a tool for the cytologic diagnosis of glandular lesions of the cervix uteri.

PubMed

Ravarino, Alberto; Nemolato, Sonia; Macciocu, Elena; Fraschini, Matteo; Senes, Giancarlo; Faa, Gavino; Negri, Giovanni

2012-11-01

Cytologic findings of glandular lesions of the cervix uteri are often difficult to evaluate. We studied the usefulness of CINtec PLUS p16/Ki-67 double stain (mtm laboratories, Heidelberg, Germany) for the diagnosis of glandular lesions. The study included 47 abnormal results on liquid-based cytologic tests with a subsequent histologic diagnosis of adenocarcinoma in situ or with early invasion, and 16 samples with negative results on follow-up. All samples were stained with CINtec PLUS p16/Ki-67 double stain. Of the neoplastic samples, 7 were excluded because of insufficient residual cellularity or loss of neoplastic cells. Of the samples that were adequate, 92.5% were stained with CINtec PLUS, whereas 7.5% were judged inconclusive. All inconclusive cases were at least 3 years old. Of the 16 negative samples, 15 (93.8%) stained negative and only 1 (6.2%) showed several positive clusters of cells. Our study shows that CINtec PLUS is a robust and useful tool for the diagnosis of glandular lesions of the cervix uteri.

Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

PubMed

Kaszuba-Zwoinska, J; Chorobik, P; Juszczak, K; Zaraska, W; Thor, P J

2012-10-01

Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early phase of apoptosis induced by both apoptosis inducing agents. The analysis of expression of the apoptosis related genes in MonoMac6 cultures treated with puromycin and exposed to PEMF performed in reverse transcription of polymerase chain reaction (PCR) assay has shown changes in mRNA of genes engaged in intrinsic apoptotic pathway and pathway with AIF abundance. The most influenced was expression of gene belonging to pro-apoptotic family of Bcl-2 and AIF agent. Examination of immunoblots developed with anti-AIF antibody showed that cytosol content of AIF protein was diminished after puromycin and PEMF treatment of MonoMac6 cells. The obtained results indicate that PEMF affects induction of apoptosis in MonoMac6 cells stimulated to death with inducing agents to a different extent. Main finding of the current results is that, PEMF stimulation of MonoMac6 cells simultaneously treated with puromycin caused changes in the Bcl-family genes expression as well as in caspase independent pathway of apoptosis inducing factor (AIF).

Core-Shell Double Gyroid Structure Formed by Linear ABC Terpolymer Thin Films.

PubMed

Antoine, Ségolène; Aissou, Karim; Mumtaz, Muhammad; Telitel, Siham; Pécastaings, Gilles; Wirotius, Anne-Laure; Brochon, Cyril; Cloutet, Eric; Fleury, Guillaume; Hadziioannou, Georges

2018-05-01

The synthesis and self-assembly in thin-film configuration of linear ABC triblock terpolymer chains consisting of polystyrene (PS), poly(2-vinylpyridine) (P2VP), and polyisoprene (PI) are described. For that purpose, a hydroxyl-terminated PS-b-P2VP (45 kg mol -1 ) building block and a carboxyl-terminated PI (9 kg mol -1 ) are first separately prepared by anionic polymerization, and then are coupled via a Steglich esterification reaction. This quantitative and metal-free catalyst synthesis route reveals to be very interesting since functionalization and purification steps are straightforward, and well-defined terpolymers are produced. A solvent vapor annealing (SVA) process is used to promote the self-assembly of frustrated PS-b-P2VP-b-PI chains into a thin-film core-shell double gyroid (Q 230 , space group: Ia3¯d) structure. As terraces are formed within PS-b-P2VP-b-PI thin films during the SVA process under a CHCl 3 vapor, different plane orientations of the Q 230 structure ((211), (110), (111), and (100)) are observed at the polymer-air interface depending on the film thickness. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphoinositide 3-kinase gamma regulates airway smooth muscle contraction by modulating calcium oscillations.

PubMed

Jiang, Haihong; Abel, Peter W; Toews, Myron L; Deng, Caishu; Casale, Thomas B; Xie, Yan; Tu, Yaping

2010-09-01

Phosphoinositide 3-kinase gamma (PI3Kgamma) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kgamma can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kgamma protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kgamma inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 muM. In contrast, inhibitors of PI3Kalpha, PI3Kbeta, or PI3Kdelta, at concentrations 40-fold higher than their reported IC(50) values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kgamma inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kgamma-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca(2+) transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca(2+) oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kgamma directly controls contractility of airways through regulation of Ca(2+) oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kgamma inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

PubMed Central

Jiang, Haihong; Abel, Peter W.; Toews, Myron L.; Deng, Caishu; Casale, Thomas B.; Xie, Yan

2010-01-01

Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kγ can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kγ protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kγ inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 μM. In contrast, inhibitors of PI3Kα, PI3Kβ, or PI3Kδ, at concentrations 40-fold higher than their reported IC50 values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kγ-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca2+ transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kγ directly controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kγ inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness. PMID:20501633

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

PubMed

Gallo, Alessandra; Boni, Raffaele; Tosti, Elisabetta

2018-01-01

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Anti-inflammatory and immunosuppressive effect of phloretin.

PubMed

Lu, Xiao-yu; Zeng, Yao-ying; Ye, Yan-xia; Zhou, Yu-ying; Mu, Jing-jing; Zhao, Xiao-hui

2009-05-01

This study investigated the effect of phloretin (Ph) on the proliferation, activation, and cell-cycle distribution of mouse T lymphocytes and NO production and phagocytosis of macrophages. Carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) staining plus flow cytometry assay was employed to obtain the proliferation-related index (PI) of lymphocytes. The expression levels of CD69 and CD25 on T lymphocytes stimulated with Con A were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Cell-cycle distribution of T lymphocytes was analyzed by propidium iodide staining. Griess kit was used to evaluate the NO production and fluorescent microbeads were used to analyze the phagocytosis ability of macrophages. Our results showed that phloretin (40, 60, and 80 micromol x L(-7)) significantly inhibited the proliferation of T lymphocytes and the PI reduced from 1.41 +/- 0.13 to 1.34 +/- 0.16, 1.19 +/- 0.12 and 1.07 +/- 0.06, respectively. Phloretin significantly inhibited the expression of CD69 and CD25 (P < 0.01). The cell cycle distribution analysis showed that phloretin could induce a cell cycle arrest at G0/G1 phase. NO production of LPS +IFN-gamma group of macrophages was (26.72 +/- 3.57) micromol x L(-1), and was significantly reduced by phloretin (P < 0.01). And phagocytosis rate of macrophages was significantly reduced by phloretin (P < 0.01). The results demonstrate that phloretin might be developed into a new immuosuppressive drug.

LED-activated pheophorbide a in ovarian cancer cells: Cytotoxicity and apoptosis induction

NASA Astrophysics Data System (ADS)

Liu, L.; Xu, C. S.; Xia, X. S.; Leung, A. W. N.

2011-02-01

Pheophorbide a (Pa) from Chinese herbal medicine Scutellaria Barbata and Silkworm excreta has been proved to be potential photosensitizer. The present study investigated the cytotoxicity of ovarian cancer cells induced by LED-activated Pa using light microscopy with the SRB staining. We further investigated the apoptosis of the cells 6 h after LED-activated Pa using of the flow cytometer with PI staining and nuclear staining. The results showed that LED-activated Pa remarkably caused cell death of ovarian cancer cells. The condensation of chromatin, nuclear fragmentations, and 12.3% of cells containing subdiploid levels of DNA were found in the ovarian cancer cells after the treatment of LED-activated Pa. These data demonstrated that LED-activated Pa could cause significant cytotoxicity and apoptosis of ovarian cancer cells.

Low-dose testosterone alleviates vascular damage caused by castration in male rats in puberty via modulation of the PI3K/AKT signaling pathway.

PubMed

Zhao, Jing; Liu, Ge-Li; Wei, Ying; Jiang, Li-Hong; Bao, Peng-Li; Yang, Qing-Yan

2016-09-01

The aim of the present study was to investigate the effect of testosterone on glucolipid metabolism and vascular injury in male rats, and examine the underlying molecular mechanisms. A total of 40 male Sprague-Dawley rats were divided into a control group (n=10), high-fat-diet + castration group (n=10), high‑fat‑diet + castration + low dose testosterone group (n=10), and high-fat-diet + castration + high dose testosterone group (n=10). Hematoxylin and eosin staining was performed to evaluate the morphology of the thoracic aortic tissues. Immunohistochemical staining was used to detect biomarkers of the phosphoinositide 3‑kinase (PI3K) signaling pathway. The mRNA and protein expression levels of PI3K, AKT, insulin receptor substrate‑1 (IRS‑1), glucose transporter type 4 (GLUT‑4), nuclear factor (NF)‑κB and tumor necrosis factor (TNF)‑α in the aortas were determined using quantitative polymerase chain reaction and Western blot analyses, respectively. Apoptosis in the aortic tissues was detected using a TUNEL assay. Castration induced apoptosis in the animals fed a high‑fat‑diet, whereas low dose testosterone replacement ameliorated the apoptosis in the aorta. However, the levels of apoptosis was more severe following high‑dose testosterone treatment. Low‑dose testosterone induced upregulation in the levels of IRS‑1, AKT, GLUT‑4 protein, NF‑κB, TNF‑α and PI3K, compared with those in the animals fed a high‑fat diet following castration. A high dose of testosterone resulted in a significant decrease in the levels of IRS‑1, AKT, GLUT‑4, NF‑κB, TNF‑α and PI3K. Compared with the rats in the high‑fat diet + castration group, a low dose of testosterone induced upregulation in the mRNA levels of IRS‑1, AKT and GLUT‑4, and downregulation of the mRNA levels of NF‑κB, TNF‑α and PI3K. A high dose of testosterone resulted in a significant decrease in the levels of IRS‑1, AKT and GLUT‑4, and marked increases in the mRNA levels of NF‑κB, TNF‑α and PI3K, compared with the low dose group. Castration induced marked disorders of glucolipid metabolism and vascular injuries in the pubescent male rats. Low‑dose testosterone treatment was found to ameliorate the vascular damage caused by castration via the PI3K/AKT signaling pathway.

Epigallocatechin-3-Gallate Reduces Neuronal Apoptosis in Rats after Middle Cerebral Artery Occlusion Injury via PI3K/AKT/eNOS Signaling Pathway

PubMed Central

Zhonghang, Xu; Tongtong, Liu; Wanshu, Guo

2018-01-01

Background/Aims Epigallocatechin-3-gallate (EGCG) has neuroprotective effects and the ability to resist amyloidosis. This study observed the protective effect of EGCG against neuronal injury in rat models of middle cerebral artery occlusion (MCAO) and investigated the mechanism of action of PI3K/AKT/eNOS signaling pathway. Methods Rat models of permanent MCAO were established using the suture method. Rat behavior was measured using neurological deficit score. Pathology and apoptosis were measured using HE staining and TUNEL. Oxidative stress and brain injury markers were examined using ELISA. Apoptosis-related proteins and PI3K/AKT/eNOS signaling pathway were determined using western blot assay and immunohistochemistry. Results EGCG decreased neurological function score, protected nerve cells, inhibited neuronal apoptosis, and inhibited oxidative stress injury and brain injury markers level after MCAO. EGCG reduced the apoptotic rate of neurons, increased the expression of Bcl-2, and decreased the expression of Caspase-3 and Bax. After LY294002 suppressed the PI3K pathway, the protective effect of EGCG decreased after administration of PI3K inhibitors. Conclusion EGCG has a protective effect on rat brain injury induced by MCAO, possibly by modulating the PI3K/AKT/eNOS signaling pathway. PMID:29770336

Target and double spin asymmetries for {rvec e} {rvec p} {yields} e{prime} p {pi}{sup 0}

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

Angela Biselli

2004-03-01

An extensive experimental program to measure the spin structure of the nucleons is carried out in Hall B with the CLAS detector at Jefferson Lab using a polarized electron beam incident on a polarized target. Spin degrees of freedom offer the possibility to test, in an independent way, existing models of resonance electroproduction. The present analysis selects the exclusive channel {rvec p}({rvec e}, e{prime}, p){pi}{sup 0} from data taken in 2000-2001, to extract single and double asymmetries in a Q{sup 2} range from 0.2 to 0.75 GeV{sup 2} and W range from 1.1 to 1.6 GeV/c{sup 2}. Results of themore » asymmetries will be presented as a function of the center of mass decay angles of the {pi}{sup 0} and compared with the unitary isobar model MAID, the dynamic model by Sato and Lee and the dynamic model DMT.« less

A Simple Visualization of Double Bond Properties: Chemical Reactivity and UV Fluorescence

ERIC Educational Resources Information Center

Grayson, Scott M.

2012-01-01

A simple, easily visualized thin-layer chromatography (TLC) staining experiment is presented that highlights the difference in reactivity between aromatic double bonds and nonaromatic double bonds. Although the stability of aromatic systems is a major theme in organic chemistry, the concept is rarely reinforced "visually" in the undergraduate…

Decreased expression of GST pi is correlated with a poor prognosis in human esophageal squamous carcinoma

PubMed Central

2010-01-01

Background Glutathione S-transferase pi (GST pi) is a subgroup of GST family, which provides cellular protection against free radical and carcinogenic compounds due to its detoxifying function. Expression patterns of GST pi have been studied in several carcinomas and its down-regulation was implicated to be involved in malignant transformation in patients with Barrett's esophagus. However, neither the exact role of GST pi in the pathogenesis nor its prognostic impact in squamous esophageal carcinoma is fully characterized. Methods Immunohistochemistry was used to investigate GST pi expression on 153 archival squamous esophageal carcinoma specimens with a GST pi monoclonal antibody. Statistic analyses were performed to explore its association with clinicopathological factors and clinical outcome. Results The GST pi expression was greatly reduced in tissues of esophageal carcinomas compared to adjacent normal tissues and residual benign tissues. Absent of GST pi protein expression in cytoplasm, nuclear and cytoplasm/nucleus was found in 51%, 64.7% and 48% of all the carcinoma cases, respectively. GST pi deficiency in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to poor differentiation (p < 0.001, p < 0.001 and p < 0.001, respectively). UICC stage and T stage were found significantly correlated to negative expression of GST pi in cytoplasm (p < 0.001 and p = 0.004, respectively) and cytoplasm/nucleus (p = 0.017 and p = 0.031, respectively). In univariate analysis, absent of GST pi protein expression in cytoplasm, nucleus and cytoplasm/nucleus was significantly associated with a shorter overall survival (p < 0.001, p < 0.001 and p < 0.001, respectively), whereas only GST pi cytoplasmic staining retained an independent prognostic significance (p < 0.001) in multivariate analysis. Conclusions Our results show that GST pi expression is down regulated in the squamous esophageal carcinoma, and that the lack of GST pi expression is associated with poor prognosis. Therefore, deficiency of GST pi protein expression may be an important mechanism involved in the carcinogenesis and progression of the squamous esophageal carcinoma, and the underlying mechanisms leading to decreased GST pi expression deserve further investigation. PMID:20602752

The impact of new polarization data from Bonn, Mainz and Jefferson Laboratory on $$\\gamma p \\rightarrow \\pi N$$ multipoles

DOE PAGES

Anisovich, A. V.; Beck, R.; Döring, M.; ...

2016-09-16

New data on pion-photoproduction off the proton have been included in the partial wave analyses Bonn-Gatchina and SAID and in the dynamical coupled-channel approach Julich-Bonn. All reproduce the recent new data well: the double polarization data for E, G, H, P and T inmore » $$\\gamma p \\to \\pi^0 p$$ from ELSA, the beam asymmetry $$\\Sigma$$ for $$\\gamma p \\to \\pi^0 p$$ and $$\\pi^+ n$$ from Jefferson Laboratory, and the precise new differential cross section and beam asymmetry data $$\\Sigma$$ for $$\\gamma p \\to \\pi^0 p$$ from MAMI. The new fit results for the multipoles are compared with predictions not taking into account the new data. Lastly, the mutual agreement is improved considerably but still far from being perfect.« less

The impact of new polarization data from Bonn, Mainz and Jefferson Laboratory on $$\\gamma p \\rightarrow \\pi N$$ multipoles

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

Anisovich, A. V.; Beck, R.; Döring, M.

New data on pion-photoproduction off the proton have been included in the partial wave analyses Bonn-Gatchina and SAID and in the dynamical coupled-channel approach Julich-Bonn. All reproduce the recent new data well: the double polarization data for E, G, H, P and T inmore » $$\\gamma p \\to \\pi^0 p$$ from ELSA, the beam asymmetry $$\\Sigma$$ for $$\\gamma p \\to \\pi^0 p$$ and $$\\pi^+ n$$ from Jefferson Laboratory, and the precise new differential cross section and beam asymmetry data $$\\Sigma$$ for $$\\gamma p \\to \\pi^0 p$$ from MAMI. The new fit results for the multipoles are compared with predictions not taking into account the new data. Lastly, the mutual agreement is improved considerably but still far from being perfect.« less

Central exclusive production of hadrons in CDF

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

Albrow, M. G.; Lontkovskyi, D.; Makarenko, I.

2012-01-01

At the Fermilab Tevatron withmore » $$\\sqrt{s}$$ = 900 and 1960 GeV, we have studied exclusive double pomeron exchange in the Collider Detector at Fermilab, CDF. With $$\\sim$$300,000 events we present the mass spectrum of two hadrons, $h^+h^-$, assumed to be pions, with $$|\\eta(\\pi)| <$$ 1.3 and two rapidity gaps $$\\Delta \\eta > 4.6$$. The mass spectrum shows resonance structures, including $$f_0(980),f_2(1270),$$ and$$ f_0(1370)$$. The cross section ratio 1960 GeV/900 GeV and the mean $$p_T(pair)$$ show mass-dependent structures, even above $M$ = 2 GeV where there are no established $$\\pi^+\\pi^-$$ resonances. The data extend above $M$ = 5 GeV. We place an upper limit on exclusive $$\\chi_{c0} \\rightarrow \\pi^+\\pi^-$$ and $K^+K^-$.« less

Tangeretin, a citrus polymethoxyflavonoid, induces apoptosis of human gastric cancer AGS cells through extrinsic and intrinsic signaling pathways.

PubMed

Dong, Yang; Cao, Aili; Shi, Jianrong; Yin, Peihao; Wang, Li; Ji, Guang; Xie, Jianqun; Wu, Dazheng

2014-04-01

Tangeretin, a natural polymethoxyflavone present in citrus peel oil, is known to have anticancer activities in breast cancer, colorectal carcinoma and lung carcinoma, yet, the underlying mechanisms of tangeretin in human gastric cancer AGS cells have not been investigated to date. In the present study, the apoptotic mechanisms of tangeretin in AGS cells were explored. It was observed that tangeretin increased the apoptotic rates of AGS cells following treatment with tangeretin for 48 h in a dose-dependent manner by Annexin V-FITC and PI double staining. In addition, characteristic apoptotic morphology such as nuclear shrinkage and apoptotic bodies was observed after Hoechst 33258 staining. Flow cytometric assay showed that treatment of AGS cells with tangeretin decreased the mitochondrial membrane potential (MMP) in a dose-dependent manner, which indicated that mitochondrial dysfunction was involved in the tangeretin-induced apoptosis. Caspase-3, -8 and -9 activities were increased by tangeretin in a dose-dependent manner. Western blotting showed that the protein levels of pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Bid, tBid, p53, p21/cip1, Fas and FasL were significantly upregulated by tangeretin. In addition, PFT-α (a p53 inhibitor) reduced the apoptotic rates and the expression of p53, p21, caspase-3 and caspase-9 induced by tangeretin, indicating that tangeretin-induced apoptosis was p53-dependent. In conclusion, these results suggest that tangeretin induces the apoptosis of AGS cells mainly through p53-dependent mitochondrial dysfunction and the Fas/FasL-mediated extrinsic pathway.

Efficacy of a Fixed Combination of Tetracycline, Chloramphenicol, and Colistimethate Sodium for Treatment of Candida albicans Keratitis.

PubMed

Blanco, Anna R; Nostro, Antonia; D'Angelo, Valeria; D'Arrigo, Manuela; Mazzone, Maria G; Marino, Andreana

2017-08-01

To evaluate the antifungal activity of a fixed antibiotic combination (AC) containing tetracycline (TET), chloramphenicol (CAF), and colistimethate sodium (CS). In vitro: Candida ATCC and clinical strains were used. The minimum inhibitory concentrations (MICs) of AC and of each antibiotic were determined. Fluconazole (FLC) was tested for comparison. Time-killing curves of selected strains were performed. Ex vivo keratitis: corneas were injected intrastromally with the selected strains. After the injection, corneas were divided into groups of treatments: AC, FLC, or saline. Then, the tissues were analyzed for colony-forming units per gram (CFU/g). Propidium iodide (PI) and MitoTracker (MTR) staining were used to investigate the mode of action. Values of MIC required to inhibit the growth of 90% of organisms for the antibiotics alone were higher than FLC. However, their activity was enhanced when used in combination against Candida yeasts. Time-killing curves showed that at 24 hours, AC reduced the load of both strains of approximately 1 Log10 CFU/g compared with the initial inoculum (P < 0.0001). This effect was also significant versus FLC. In ex vivo, AC was effective in decreasing the loads of both strains by 4 Log10 CFU/g with respect to the control. Moreover, it showed higher activity than FLC against Candida albicans ATCC 10231 (1 Log10 CFU/g, P < 0.01 versus control). PI staining demonstrated that CS changed the membrane's permeability, whereas MTR staining demonstrated that TET or CAF altered mitochondrial function. The cells treated with AC and stained showed both effects. In this study, AC showed antifungal efficacy versus Candida spp.; this activity can be due to the synergistic effects of antibiotics in it.

Increased Expression of TGF-β1 in Correlation with Liver Fibrosis during Echinococcus granulosus Infection in Mice.

PubMed

Liu, Yumei; Abudounnasier, Gulizhaer; Zhang, Taochun; Liu, Xuelei; Wang, Qian; Yan, Yi; Ding, Jianbing; Wen, Hao; Yimiti, Delixiati; Ma, Xiumin

2016-08-01

To investigate the potential role of transforming growth factor (TGF)-β1 in liver fibrosis during Echinococcus granulosus infection, 96 BALB/c mice were randomly divided into 2 groups, experimental group infected by intraperitoneal injection with a metacestode suspension and control group given sterile physiological saline. The liver and blood samples were collected at days 2, 8, 30, 90, 180, and 270 post infection (PI), and the expression of TGF-β1 mRNA and protein was determined by real-time quantitative RT-PCR and ELISA, respectively. We also evaluated the pathological changes in the liver during the infection using hematoxylin and eosin (H-E) and Masson staining of the liver sections. Pathological analysis of H-E stained infected liver sections revealed liver cell edema, bile duct proliferation, and structural damages of the liver as evidenced by not clearly visible lobular architecture of the infected liver, degeneration of liver cell vacuoles, and infiltration of lymphocytes at late stages of infection. The liver tissue sections from control mice remained normal. Masson staining showed worsening of liver fibrosis at the end stages of the infection. The levels of TGF-β1 did not show significant changes at the early stages of infection, but there were significant increases in the levels of TGF-β1 at the middle and late stages of infection (P<0.05). RT-PCR results showed that, when compared with the control group, TGF-β1 mRNA was low and comparable with that in control mice at the early stages of infection, and that it was significantly increased at day 30 PI and remained at high levels until day 270 PI (P<0.05). The results of this study suggested that increased expression of TGF-β1 during E. granulosus infection may play a significant role in liver fibrosis associated with E. granulosus infection.

Identification of novel alleles of the rice blast resistance gene Pi54

NASA Astrophysics Data System (ADS)

Vasudevan, Kumar; Gruissem, Wilhelm; Bhullar, Navreet K.

2015-10-01

Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.

Loss of PiT-1 Results in Abnormal Endocytosis in the Yolk Sac Visceral Endoderm

PubMed Central

Wallingford, Mary C.; Giachelli, Cecilia M.

2014-01-01

PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE. PMID:25138534

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells.

PubMed

Sarner, S; Kozma, R; Ahmed, S; Lim, L

2000-01-01

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.

Phosphatidylinositol 3-Kinase, Cdc42, and Rac1 Act Downstream of Ras in Integrin-Dependent Neurite Outgrowth in N1E-115 Neuroblastoma Cells

PubMed Central

Sarner, Shula; Kozma, Robert; Ahmed, Sohail; Lim, Louis

2000-01-01

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A RasH40C;G12V double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated RasG12V-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42G12V was Rac1 dependent. Cdc42G12V-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42G12V-induced outgrowth did not need Ras or PI 3-kinase activity. Active RhoG14V reduced outgrowth promoted by RasG12V. Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells. PMID:10594018

A Unique HMG-Box Domain of Mouse Maelstrom Binds Structured RNA but Not Double Stranded DNA

PubMed Central

Genzor, Pavol; Bortvin, Alex

2015-01-01

Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response. PMID:25807393

Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

PubMed Central

Dang, Yalong; Waxman, Susannah; Wang, Chao; Jensen, Adrianna; Loewen, Ralitsa T.; Bilonick, Richard A.

2017-01-01

Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture. PMID:28828244

Three-body Final State Interaction in η→3π

DOE PAGES

Guo, Peng; Danilkin, Igor V.; Schott, Diane; ...

2015-09-11

We present an unitary dispersive model for themore » $$\\eta \\to 3 \\pi$$ decay process based upon the Khuri-Treiman equations which are solved by means of the Pasquier inversion method. The description of the hadronic final-state interactions for the $$\\eta \\to 3\\pi$$ decay is essential to reproduce the available data and to understand the existing discrepancies between Dalitz plot parameters from experiment and chiral perturbation theory. Our approach incorporates substraction constants that are fixed by fitting the recent high-statistics WASA-at-COSY data for $$\\eta \\to \\pi^+ \\pi^- \\pi^0$$. Based on the parameters obtained we predict the slope parameter for the neutral channel to be $$\\alpha=-0.022\\pm 0.004$$. Through matching to next-to-leading order chiral perturbation theory we estimate the quark mass double ratio to be $$Q=21.4 \\pm 0.4$$.« less

Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica.

PubMed

Meamar, Mehrdad; Zribi, Nassira; Cambi, Marta; Tamburrino, Lara; Marchiani, Sara; Filimberti, Erminio; Fino, Maria Grazia; Biggeri, Annibale; Menezo, Yves; Forti, Gianni; Baldi, Elisabetta; Muratori, Monica

2012-08-01

To analyze the effect of cryopreservation on sperm DNA fragmentation (SDF) in two cytometric sperm populations, PI(brighter) and PI(dimmer), and to test the effects of Opuntia ficus-indica (OFI) extracts, which contain antioxidants and flavanoids, and of resveratrol on cryopreservation of human semen. In vitro prospective study. Institutional study. Twenty-one normozoospermic men undergoing semen analysis for couple infertility. Cryopreservation using the routine method in the presence of OFI extracts or resveratrol. Measurement of SDF by TUNEL/PI flow cytometric method to evaluate sperm motility (by automated motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI(br) and PI(dim). Cryopreservation induced an increase of SDF only in the PI(br) sperm population. The increase was negatively dependent on the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but statistically significantly reduced SDF in the PI(br) population without affecting the deleterious effect of cryopreservation on sperm motion parameters or viability. The increase of SDF in the PI(br) population, which is unrelated to semen quality, suggests that caution must be taken in using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunctional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Reducing Dental Plaque and Gingivitis With 0.6% Cortex Ilicis Rotundae Toothpaste: A Randomized, Double-Masked Clinical Trial.

PubMed

Liu, Hongchun; Yin, Wei

2016-03-01

Cortex Ilicis Rotundae has antioxidant and anti-inflammatory properties. Few studies have evaluated the effects of toothpastes containing Cortex Ilicis Rotundae. This study evaluates the antiplaque and antigingivitis effects of a test toothpaste containing 0.6% Cortex Ilicis Rotundae extract in a calcium carbonate base compared with a control toothpaste without any active ingredient. One hundred adults with a mean plaque index (PI) ≥ 1.5 and a mean gingival index (GI) ≥ 1.0 were enrolled in this randomized, double-masked, placebo-controlled clinical trial. They were assigned randomly to use a test toothpaste or a control toothpaste. At baseline, 6 weeks, and 12 weeks, they received examinations of oral hard and soft tissues, using Löe-Silness GI for gingivitis and the Turesky modification of the Quigley-Hein PI for PI. Adverse events were monitored. When the study was completed, the test group reported lower mean GI than the control group (1.13 ± 0.22 versus 1.30 ± 0.23; P = 0.001) and lower mean PI than the control group (2.53 ± 0.5 versus 2.93 ± 0.44; P < 0.001). Compared to the baseline, the test group had reductions in GI and PI of 14.39% and 17.86%, respectively (both P < 0.001); the control group had reductions in GI and PI of 3.7% and 3.93%, respectively (both P < 0.001). No adverse events were reported during the course of the study. The toothpaste containing 0.6% Cortex Ilicis Rotundae was effective in reducing dental plaque and gingivitis after 12 weeks of use compared with a negative control toothpaste.

Development of a constant surface pressure penetration langmuir balance based on axisymmetric drop shape analysis.

PubMed

Wege, H A; Holgado-Terriza, J A; Cabrerizo-Vílchez, M A

2002-05-15

A new constant pressure pendant-drop penetration surface balance has been developed combining a pendant-drop surface balance, a rapid-subphase-exchange technique, and a fuzzy logic control algorithm. Beside the determination of insoluble monolayer compression-expansion isotherms, it allows performance of noninvasive kinetic studies of the adsorption of surfactants added to the new subphase onto the free surface and of the adsorption/penetration/reaction of the former onto/into/with surface layers, respectively. The interfacial pressure pi is a fundamental parameter in these studies: by working at constant pi one controls the height of the energy barrier to adsorption/penetration and can select different regimes and steps of the adsorption/penetration process. In our device a solution drop is formed at the tip of a coaxial double capillary, connected to a double microinjector. Drop profiles are extracted from digital drop micrographs and fitted to the equation of capillarity, yielding pi, the drop volume V, and the interfacial area A. pi is varied changing V (and hence A) with the microinjector. Control is based on a case-adaptable modulated fuzzy-logic PID algorithm able to maintain constant pi (or A) under a wide range of experimental conditions. The drop subphase liquid can be exchanged quantitatively by the coaxial capillaries. The adsorption/penetration/reaction kinetics at constant pi are then studied monitoring A(t), i.e., determining the relative area change necessary at each instant to compensate the pressure variation due to the interaction of the surfactant in the subsurface with the surface layer. A fully Windows-integrated program manages the whole setup. Examples of experimental protein adsorption and monolayer penetration kinetics are presented.

Keratoacanthoma of the Lip

PubMed Central

Dillenburg, Caroline Siviero; Martins, Manoela Domingues; Meurer, Luise; Castilho, Rogerio Moraes; Squarize, Cristiane Helena

2015-01-01

Abstract The PI3K-PTEN-mTOR is one of the most important pathways involved in cancer development and progression; however, its role in keratoacanthoma (KA) is poorly understood. In this study, we investigated the activation of key proteins in the PI3K-mTOR pathway in lip KA. We analyzed the activation of the PI3K-PTEN-mTOR pathway using human tumor samples stained for well-established protein markers in this pathway, including pS6 and pAKT phosphoproteins. We assessed proliferation using Ki-67 and performed additional morphological and immunohistochemical analysis using anti-PTEN and anti-p16 antibodies. We found that the majority of KA labeled to pS6 and not pAKT. PTEN expression was inversely correlated with Ki-67 expression. In addition to PTEN expression, KA cells were positive for p16Ink4 senescence marker. PI3K-PTEN-mTOR pathway is activated in lip KA, leading to downstream activation of mTORC1, but not mTORC2. This pathway plays an important role in KA progression by promoting proliferation and activation of oncogenic-induced senescence. PMID:26402814

[Usefulness of systematic chromoendoscopy with a double dye staining technique for the detection of dysplasia in patients with premalignant gastric lesions].

PubMed

Yep-Gamarra, Víctor; Díaz-Vélez, Cristian; Araujo, Isis; Ginès, Àngels; Fernández-Esparrach, Gloria

2016-02-01

Premalignant gastric lesions have an increased risk to develop gastric cancer. To evaluate the usefulness of systematic endoscopy that includes chromoendoscopy with a double dye staining technique for the detection of dysplasia in patients with premalignant gastric lesions. This longitudinal, prospective study was performed in patients with gastric atrophy, intestinal metaplasia or dysplasia who were referred for endoscopy less than 6 months after the initial diagnosis. The second endoscopy was performed in three phases: phase 1, exhaustive and systematic review of the mucosa with photographic documentation and biopsies of suspicious areas; phase 2, chromoendoscopy with a double dye staining technique using acetic acid 1.2% and indigo carmine 0.5%; phase 3, topographic mapping and random biopsies. A total of 50 patients were included. Nine (18%) had atrophic gastritis, 38 (76%) had intestinal metaplasia, and 3 (6%) had low-grade dysplasia. Systematic endoscopy with chromoendoscopy using a double dye staining technique detected more patients with dysplasia (9 versus 3, p<.05), and a larger number of biopsies with the diagnosis of dysplasia were obtained. This occurred for visible (6 vs. 0, p<.05) and non-visible lesions (6 vs. 3, p=NS). In one patient, initial low-grade dysplasia was not detected again in the systematic endoscopy, giving a global endoscopic performance for the detection of lesions of 92%. Patients with premalignant gastric lesions have synchronous lesions with greater histological severity, which are detected when systematic endoscopy is conducted with indigo carmine dye added to acetic acid. Copyright © 2015 Elsevier España, S.L.U. and AEEH y AEG. All rights reserved.

Histochemical and cellular changes accompanying the appearance of lung fibrosis in an experimental mouse model for Hermansky Pudlak syndrome

PubMed Central

Lyerla, Timothy

2010-01-01

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1ep-Ap3b1pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lystbg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS. PMID:20603711

[Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte].

PubMed

Ji, Ying-Lei; Yan, Jun; Wang, Yan-Sha; Liu, Yi-Chang; Gu, Zhen-Yong

2014-02-01

To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

Oxymatrine extracted from Sophora flavescens inhibited cell growth and induced apoptosis in human osteosarcoma MG-63 cells in vitro.

PubMed

Wei, Jianghua; Zhu, Yin; Xu, Gang; Yang, Fan; Guan, Zhe; Wang, Mao; Fang, Yonghong

2014-11-01

Oxymatrine, one of the most active components of the ethanol extracts from Sophora flavescens, is known for its potent antitumor activity both in vitro and in vivo. However, the mechanism of its action in mediating the cell apoptosis remains elusive. In this study, we investigated the proliferation inhibitory and apoptotic activities of oxymatrine against human osteosarcoma MG-63 cells. The compound was found to markedly and dose-dependently inhibit the cell proliferation determined by 5-bromo-2-deoxyuridine incorporation. Oxymatrine also induced the cell apoptosis in a dose- and time-dependent manner as showed by the annexin V-FITC/PI double staining and TUNEL assay. Furthermore, a disruption of mitochondrial membrane potential and an up-regulation of cleaved caspases-3, and-9 and downregulation of Bax/Bcl-2 was evidenced in the oxymatrine-treated cells. These proteins have been known to play a pivotal role in the regulation of apoptosis. In conclusion, these observations indicate of the oxymatrine potential as an effective antitumor agent against osteosarcoma. Moreover, the compound appears to exert its anti-tumor action by stimulating the caspase-triggered signaling pathway.

Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation.

PubMed

Wang, Hui; Xu, Qian; Kong, You-Han; Chen, Yun; Duan, Jun-Ye; Wu, Wei-Hua; Chen, Yi-Fang

2014-04-01

The WRKY transcription factor family has more than 70 members in the Arabidopsis (Arabidopsis thaliana) genome, and some of them are involved in plant responses to biotic and abiotic stresses. This study evaluated the role of WRKY45 in regulating phosphate (Pi) uptake in Arabidopsis. WRKY45 was localized in the nucleus and mainly expressed in roots. During Pi starvation, WRKY45 expression was markedly induced, typically in roots. WRKY45 overexpression in Arabidopsis increased Pi content and uptake, while RNA interference suppression of WRKY45 decreased Pi content and uptake. Furthermore, the WRKY45-overexpressing lines were more sensitive to arsenate, the analog of Pi, compared with wild-type seedlings. These results indicate that WRKY45 positively regulates Arabidopsis Pi uptake. Quantitative real-time polymerase chain reaction and β-glucuronidase staining assays showed that PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression was enhanced in the WRKY45-overexpressing lines and slightly repressed in the WRKY45 RNA interference line. Chromatin immunoprecipitation and electrophoretic mobility shift assay results indicated that WRKY45 can bind to two W-boxes within the PHT1;1 promoter, confirming the role of WRKY45 in directly up-regulating PHT1;1 expression. The pht1;1 mutant showed decreased Pi content and uptake, and overexpression of PHT1;1 resulted in enhanced Pi content and uptake. Furthermore, the PHT1;1-overexpressing line was much more sensitive to arsenate than WRKY45-overexpressing and wild-type seedlings, indicating that PHT1;1 overexpression can enhance Arabidopsis Pi uptake. Moreover, the enhanced Pi uptake and the increased arsenate sensitivity of the WRKY45-overexpressing line was impaired by pht1;1 (35S:WRKY45-18::pht1;1), demonstrating an epistatic genetic regulation between WRKY45 and PHT1;1. Together, our results demonstrate that WRKY45 is involved in Arabidopsis response to Pi starvation by direct up-regulation of PHT1;1 expression.

Cross Sections for the Reactions e+e to K+ K- pi+pi-, K+ K- pi0pi0, and K+ K- K+ K- Measured Using Initial-State Radiation

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

Lees, J.P.; Poireau, V.; Prencipe, E.

2011-08-19

We study the processes e{sup +}e{sup -} {yields} K{sup +}K{sup -}{pi}{sup +}{pi}-{gamma}, K{sup +}K{sup -}{pi}{sup 0}{pi}{sup 0}{gamma}, and K{sup +}K{sup -}K{sup +}K{sup -}{gamma}, where the photon is radiated from the initial state. About 84000, 8000, and 4200 fully reconstructed events, respectively, are selected from 454 fb{sup -1} of BABAR data. The invariant mass of the hadronic final state defines the e{sup +}e{sup -} center-of-mass energy, so that the K{sup +}K{sup -}{pi}{sup +}{pi}{sup -}{gamma} data can be compared with direct measurements of the e{sup +}e{sup -} {yields} K{sup +}K{sup -}{pi}{sup +}{pi}{sup -} reaction. No direct measurements exist for the e{sup +}e{supmore » -} {yields} K{sup +}K{sup -}{pi}{sup 0}{pi}{sup 0} or e{sup +}e{sup -} {yields} K{sup +}K{sup -}K{sup +}K{sup -} reactions, and we present an update of our previous result with doubled statistics. Studying the structure of these events, we find contributions from a number of intermediate states, and extract their cross sections. In particular, we perform a more detailed study of the e{sup +}e{sup -} {yields} {phi}(1020){pi}{pi}{gamma} reaction, and confirm the presence of the Y (2175) resonance in the {phi}(1020)f{sub 0}(980) and K{sup +}K{sup -} f{sub 0}(980) modes. In the charmonium region, we observe the J/{psi} in all three final states and in several intermediate states, as well as the {phi}(2S) in some modes, and measure the corresponding branching fractions.« less

First measurement of the double spin asymmetry in (-->)e(-->)p-->e(prime)pi(+)n in the resonance region.

PubMed

De Vita, R; Anghinolfi, M; Burkert, V D; Dodge, G E; Minehart, R; Taiuti, M; Weller, H; Adams, G; Amaryan, M J; Anciant, E; Armstrong, D S; Asavapibhop, B; Asryan, G; Audit, G; Auger, T; Avakian, H; Bagdasaryan, H; Ball, J P; Barrow, S; Battaglieri, M; Beard, K; Bektasoglu, M; Bianchi, N; Biselli, A S; Boiarinov, S; Bonner, B E; Bosted, P; Bouchigny, S; Branford, D; Brooks, W K; Bueltmann, S; Calarco, J R; Capitani, G P; Carman, D S; Carnahan, B; Cazes, A; Ciciani, L; Cole, P L; Coleman, A; Connelly, J; Cords, D; Corvisiero, P; Crabb, D; Crannell, H; Cummings, J P; De Sanctis, E; Degtyarenko, P V; Demirchyan, R; Denizli, H; Dennis, L; Dharmawardane, K V; Dhuga, K S; Djalali, C; Doughty, D; Dragovitsch, P; Dugger, M; Dytman, S; Eckhause, M; Egiyan, H; Egiyan, K S; Elouadrhiri, L; Empl, A; Farhi, L; Fatemi, R; Feuerbach, R J; Ficenec, J; Forest, T A; Frolov, V; Funsten, H; Gaff, S J; Gai, M; Garçon, M; Gavalian, G; Gilad, S; Gilfoyle, G P; Giovanetti, K L; Girard, P; Golovatch, E; Griffioen, K; Guidal, M; Guillo, M; Gyurjyan, V; Hadjidakis, C; Hancock, D; Hardie, J; Heddle, D; Heimberg, P; Hersman, F W; Hicks, K; Hicks, R S; Holtrop, M; Hu, J; Hyde-Wright, C E; Ishkanov, B S; Ito, M M; Jenkins, D; Joo, K; Kelley, J H; Kellie, J D; Khandaker, M; Kim, K Y; Kim, K; Kim, W; Klein, A; Klein, F J; Klusman, M; Kossov, M; Kramer, L H; Kuang, Y; Kuhn, S E; Lachniet, J; Laget, J M; Lawrence, D; Li, Ji; Livingston, K; Longhi, A; Loukachine, K; Lucas, M; Major, W; Manak, J J; Marchand, C; McAleer, S; McCarthy, J; McNabb, J W C; Mecking, B A; Mestayer, M D; Meyer, C A; Mikhailov, K; Mirazita, M; Miskimen, R; Mokeev, V; Muccifora, V; Mueller, J; Mutchler, G S; Napolitano, J; Nelson, S O; Niculescu, G; Niculescu, I; Niczyporuk, B B; Niyazov, R A; Opper, A K; O'Rielly, G V; Osipenko, M; Park, K; Pasyuk, E; Peterson, G; Philips, S A; Pivnyuk, N; Pocanic, D; Pogorelko, O; Polli, E; Pozdniakov, S; Preedom, B M; Price, J W; Prok, Y; Protopopescu, D; Qin, L M; Raue, B A; Reolon, A R; Riccardi, G; Ricco, G; Ripani, M; Ritchie, B G; Rock, S; Ronchetti, F; Rossi, P; Rowntree, D; Rubin, P D; Sabatié, F; Sabourov, K; Salgado, C; Sapunenko, V; Sargsyan, M; Schumacher, R A; Serov, V S; Shafi, A; Sharabian, Y G; Shaw, J; Skabelin, A V; Smith, E S; Smith, T; Smith, L C; Sober, D I; Sorrell, L; Spraker, M; Stavinsky, A; Stepanyan, S; Stoler, P; Strakovsky, I I; Taylor, S; Tedeschi, D J; Thompson, R; Todor, L; Ungaro, M; Vineyard, M F; Vlassov, A V; Wang, K; Weinstein, L B; Weisberg, A; Weygand, D P; Whisnant, C S; Wolin, E; Yegneswaran, A; Yun, J; Zhang, B; Zhao, J; Zhou, Z

2002-02-25

The double spin asymmetry in the (-->)e(-->)p --> e(prime)pi(+)n reaction has been measured for the first time in the resonance region for four-momentum transfer Q2 = 0.35-1.5 GeV(2). Data were taken at Jefferson Lab with the CLAS detector using a 2.6 GeV polarized electron beam incident on a polarized solid NH3 target. Comparison with predictions of phenomenological models shows strong sensitivity to resonance contributions. Helicity-1/2 transitions are found to be dominant in the second and third resonance regions. The measured asymmetry is consistent with a faster rise with Q(2) of the helicity asymmetry A1 for the F(15)(1680) resonance than expected from the analysis of the unpolarized data.

Randomized Trial of Long-Acting Sustained-Release Naltrexone Implant vs Oral Naltrexone or Placebo for Preventing Relapse to Opioid Dependence

PubMed Central

Krupitsky, Evgeny; Zvartau, Edwin; Blokhina, Elena; Verbitskaya, Elena; Wahlgren, Valentina; Tsoy-Podosenin, Marina; Bushara, Natalia; Burakov, Andrey; Masalov, Dmitry; Romanova, Tatyana; Tyurina, Arina; Palatkin, Vladimir; Slavina, Tatyana; Pecoraro, Anna; Woody, George E.

2013-01-01

Context Sustained-release naltrexone implants may improve outcomes of nonagonist treatment of opioid addiction. Objective To compare outcomes of naltrexone implants, oral naltrexone hydrochloride, and nonmedication treatment. Design Six-month double-blind, double-dummy, randomized trial. Setting Addiction treatment programs in St Petersburg, Russia. Participants Three hundred six opioid-addicted patients recently undergoing detoxification. Interventions Biweekly counseling and 1 of the following 3 treatments for 24 weeks: (1) 1000-mg naltrexone implant and oral placebo (NI+OP group; 102 patients); (2) placebo implant and 50-mg oral naltrexone hydrochloride (PI+ON group; 102 patients); or (3) placebo implant and oral placebo (PI+OP group; 102 patients). Main Outcome Measure Percentage of patients retained in treatment without relapse. Results By month 6, 54 of 102 patients in the NI+OP group (52.9%) remained in treatment without relapse compared with 16 of 102 patients in the PI+ON group (15.7%) (survival analysis, log-rank test, P<.001) and 11 of 102 patients in the PI+OP group (10.8%) (P<.001). The PI+ON vs PI+OP comparison showed a nonsignificant trend favoring the PI+ON group (P=.07). Counting missing test results as positive, the proportion of urine screening tests yielding negative results for opiates was 63.6% (95% CI, 60%-66%) for the NI+OP group; 42.7% (40%-45%) for the PI+ON group; and 34.1% (32%-37%) for the PI+OP group (P<.001, Fisher exact test, compared with the NI+OP group). Twelve wound infections occurred among 244 implantations (4.9%) in the NI+OP group, 2 among 181 (1.1%) in the PI+ON group, and 1 among 148 (0.7%) in the PI+OP group (P=.02). All events were in the first 2 weeks after implantation and resolved with antibiotic therapy. Four local-site reactions (redness and swelling) occurred in the second month after implantation in the NI+OP group (P=.12), and all resolved with antiallergy medication treatment. Other nonlocal-site adverse effects were reported in 8 of 886 visits (0.9%) in the NI+OP group, 4 of 522 visits (0.8%) in the PI+ON group, and 3 of 394 visits (0.8%) in the PI+ON group; all resolved and none were serious. No evidence of increased deaths from overdose after naltrexone treatment ended was found. Conclusions The implant is more effective than oral naltrexone or placebo. More patients in the NI+OP than in the other groups develop wound infections or local irritation, but none are serious and all resolve with treatment. PMID:22945623

A novel histochemical method of simultaneous detection by a single- or double-immunofluorescence and Bielschowsky's silver staining in teased rat sciatic nerves.

PubMed

Segura-Anaya, Edith; Flores-Miranda, Rommel; Martínez-Gómez, Alejandro; Dent, Myrna A R

2018-07-01

The Golgi silver method has been widely used in neuroscience for the study of normal and pathological morphology of neurons. The method has been steadily improved and Bielschowsky's silver staining method (BSSM) is widely used in various pathological conditions, like Alzheimer's disease. In this work, teased sciatic nerves were silver impregnated using BSSM. We also developed simultaneous staining by silver impregnation and single- or double-immunofluorescence of the same section in teased nerve preparations. We immunostained against non-myelinating Schwann cells and different myelinating Schwann cell domains. BSSM teased nerves show a strong staining of axons (black) and a gold-brown staining of myelinating and non-myelinating Schwann cells. We were also able to stain by immunofluorescence these BSSM teased nerves with specific molecular markers against non-myelinating Schwann cells, also against non-compact myelin such as the Schmidt-Lanterman incisures or paranodal regions and compact myelin, but not axons. In peripheral nerves, several silver impregnation methods have been used to stain nerves in paraffin sections, but not in teased nerves to enable the assessment of isolated nerve fibers. In conclusion, BSSM gives accurate information of nerve morphology and combining the procedure with immunofluorescence it would be very useful to study the molecular nerve domain organization of the nerve fibers, and to study the molecular pathology of axon degeneration, or myelin disorders, or of any peripheral neuropathy, also to study demyelination diseases in the central nervous system. Copyright © 2018. Published by Elsevier B.V.

Characterization of the rhesus macaque (Macaca mulatta) scrub typhus model: Susceptibility to intradermal challenge with the human pathogen Orientia tsutsugamushi Karp

PubMed Central

Sunyakumthorn, Piyanate; Somponpun, Suwit J.; Im-erbsin, Rawiwan; Anantatat, Tippawan; Jenjaroen, Kemajittra; Dunachie, Susanna J.; Lombardini, Eric D.; Burke, Robin L.; Blacksell, Stuart D.; Jones, James W.; Mason, Carl J.; Richards, Allen L.; Day, Nicholas P. J.

2018-01-01

Background Scrub typhus is an important endemic disease in tropical Asia caused by Orientia tsutsugamushi for which no effective broadly protective vaccine is available. The successful evaluation of vaccine candidates requires well-characterized animal models and a better understanding of the immune response against O. tsutsugamushi. While many animal species have been used to study host immunity and vaccine responses in scrub typhus, only limited data exists in non-human primate (NHP) models. Methodology/Principle findings In this study we evaluated a NHP scrub typhus disease model based on intradermal inoculation of O. tsutsugamushi Karp strain in rhesus macaques (n = 7). After an intradermal inoculation with 106 murine LD50 of O. tsutsugamushi at the anterior thigh (n = 4) or mock inoculum (n = 3), a series of time course investigations involving hematological, biochemical, molecular and immunological assays were performed, until day 28, when tissues were collected for pathology and immunohistochemistry. In all NHPs with O. tsutsugamushi inoculation, but not with mock inoculation, the development of a classic eschar with central necrosis, regional lymphadenopathy, and elevation of body temperature was observed on days 7–21 post inoculation (pi); bacteremia was detected by qPCR on days 6–18 pi; and alteration of liver enzyme function and increase of white blood cells on day 14 pi. Immune assays demonstrated raised serum levels of soluble cell adhesion molecules, anti-O. tsutsugamushi-specific antibody responses (IgM and IgG) and pathogen-specific cell-mediated immune responses in inoculated macaques. The qPCR assays detected O. tsutsugamushi in eschar, spleen, draining and non-draining lymph nodes, and immuno-double staining demonstrated intracellular O. tsutsugamushi in antigen presenting cells of eschars and lymph nodes. Conclusions/Significance These data show the potential of using rhesus macaques as a scrub typhus model, for evaluation of correlates of protection in both natural and vaccine induced immunity, and support the evaluation of future vaccine candidates against scrub typhus. PMID:29522521

Efficacy test of a toothpaste in reducing extrinsic dental stain

NASA Astrophysics Data System (ADS)

Agustanti, A.; Ramadhani, S. A.; Adiatman, M.; Rahardjo, A.; Callea, M.; Yavuz, I.; Maharani, D. A.

2017-08-01

This clinical trial compared the external dental stain reduction achieved by tested toothpaste versus placebo in adult patients. In this double-blind, parallel, randomised clinical trial, 45 female volunteers with a mean age of 20 years old were included. All study subjects front teeth were topically applicated with Silver Diamine Fluoride (SDF) to create external dental stains. Subjects were randomized into test (n=22) and control (n=23) groups. Toothpastes were used for two days to analyse the effects of removing external stains on the labial surfaces of all anterior teeth. VITA Easyshade Advance 4.0 was used to measure dental extrinsic stains changes. The analysis showed statistically significant efficacy of the tested toothpaste in reducing external dental stain caused by SDF, comparing to the placebo toothpaste, after one and two days of usage. The tested toothpaste was effective in reducing dental stain.

Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

PubMed Central

Swathi, Marri; Mishra, Prashant K.; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Dutta-Gupta, Aparna; Padmasree, Kollipara

2016-01-01

Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus. PMID:27656149

Part I. Synthesis and Characterization of Donor-Pi-Acceptor Compounds with Pentadienyl-Bridged Indoline and Tetrahydroquinoline Donors and Aldehyde and Thiobarbituric Acid Acceptors Part II. Longitudinal Study Comparing Online versus Face-to-Face Course Delivery in Introductory Chemistry

ERIC Educational Resources Information Center

Greco, Patrick F.

2012-01-01

Part I. The design and development of organic second-order nonlinear optical (NLO) materials have attracted much interest due to their applications in optoelectronic devices and modern communications technology. Donor-pi-acceptor compounds, D-(CH=CH)[subscript n]-A, often exhibit hyperpolarizability that results in laser frequency doubling (second…

Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

PubMed Central

Neeper, I D; Patton, D L; Kuo, C C

1990-01-01

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma

PubMed Central

Liu, Wei-Lin; Gao, Ming; Tzen, Kai-Yuan; Tsai, Chiao-Ling; Hsu, Feng-Ming; Cheng, Ann-Lii; Cheng, Jason Chia-Hsien

2014-01-01

Tumor control of hepatocellular carcinoma by radiotherapy remains unsatisfactory. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in inhibiting cancer cell death. Elevated PI3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to determine whether the inhibition of PI3K/Akt activity by a PI3K inhibitor, BKM120, contributes to the increased sensitivity of liver cancer cells to irradiation. The hepatocellular carcinoma cell lines (Huh7 and BNL) were used to evaluate the in vitro synergism between BKM120 and irradiation. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy to assess the in vivo response. BKM120 increased cell killing by radiation, increased the expression of apoptotic markers, and suppressed the repair of radiation-induced DNA double-strand breaks. BKM120 pretreatment inhibited radiation-induced Akt phosphorylation and enhanced the tumor-suppressive effect and radiation-induced tumor cell apoptosis in ectopic xenografts. Inhibition of mTOR phosphorylation by rapamycin enhanced the radiosensitivity of BKM120-treated hepatocellular carcinoma cells. The synergism between BKM120 and irradiation likely inhibits the activation of Akt by radiation, leading to increased cell apoptosis and suppression of DNA-double-strand breaks repair in hepatocellular carcinoma cells. These data suggest that the BKM120/radiation combination may be a strategy worthy of clinical trials. PMID:25004403

Characterization of Novel Missense Variants of SERPINA1 Gene Causing Alpha-1 Antitrypsin Deficiency.

PubMed

Matamala, Nerea; Lara, Beatriz; Gomez-Mariano, Gema; Martínez, Selene; Retana, Diana; Fernandez, Taiomara; Silvestre, Ramona Angeles; Belmonte, Irene; Rodriguez-Frias, Francisco; Vilar, Marçal; Sáez, Raquel; Iturbe, Igor; Castillo, Silvia; Molina-Molina, María; Texido, Anna; Tirado-Conde, Gema; Lopez-Campos, Jose Luis; Posada, Manuel; Blanco, Ignacio; Janciauskiene, Sabina; Martinez-Delgado, Beatriz

2018-06-01

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway during Porcine Circovirus Type 2 Infection Facilitates Cell Survival and Viral Replication

PubMed Central

Wei, Li; Zhu, Shanshan; Wang, Jing

2012-01-01

Virus infection activates host cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates diverse cellular activities related to cell growth, survival, and apoptosis. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), a major causative agent of postweaning multisystemic wasting syndrome, which is an emerging and important swine disease, can transiently induce the PI3K/Akt pathway in cultured cells at an early step during PCV2 infection. Activation of the PI3K/Akt signal was also induced by UV-irradiated PCV2, indicating that virus replication was not required for this induction. Inhibition of PI3K activation leads to reduced virus yield, which is associated with decreased viral DNA replication and lower virus protein expression. However, inhibition of PI3K activation greatly enhanced apoptotic responses as evidenced by the cleavage of poly-ADP ribose polymerase and caspase-3 as well as DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining during the early stage of PCV2 infection. Furthermore, the pancaspase inhibitor zVAD.fmk alleviated the reduction in Akt phosphorylation levels by inhibiting PI3K activation, indicating that the signaling promotes cell survival and thereby favors viral replication. These results reveal that an antiapoptotic role for the PI3K/Akt pathway induced by PCV2 infection to suppress premature apoptosis for improved virus growth after infection, extending our understanding of the molecular mechanism of PCV2 infection. PMID:23035228

Valsartan regulates the interaction of angiotensin II type 1 receptor and endothelial nitric oxide synthase via Src/PI3K/Akt signalling.

PubMed

Su, Kuo-Hui; Tsai, Jin-Yi; Kou, Yu Ru; Chiang, An-Na; Hsiao, Sheng-Huang; Wu, Yuh-Lin; Hou, Hsin-Han; Pan, Ching-Chian; Shyue, Song-Kun; Lee, Tzong-Shyuan

2009-06-01

Valsartan, a selective angiotensin II type 1 receptor (AT1R) blocker, has beneficial effects in the cardiovascular system in part by its increase of nitric oxide (NO) bioavailability, yet the mechanisms are unclear. We investigated the molecular mechanisms underlying this effect in endothelial cells (ECs). NO production was examined by Griess reagent assay, DAF-2 DA fluorescence staining and cGMP ELISA kits. Protein interaction was determined by western blotting and immunoprecipitation. Treating bovine or human aortic ECs with valsartan increased NO production, as evidenced by elevated level of stable NO metabolites and intracellular cGMP. Valsartan increased the phosphorylation but not the protein level of endothelial NO synthase (eNOS). Inhibition of phosphoinositide-3 kinase (PI3K)/Akt and Src pathways by specific inhibitors suppressed valsartan-induced NO release. In addition, valsartan increased the tyrosine residue phosphorylation of AT1R, which was attenuated by inhibition of Src but not PI3K activities. Valsartan also suppressed the interaction of eNOS and AT1R, which was blocked by Src or PI3K inhibition. Valsartan-induced NO production in ECs is mediated through Src/PI3K/Akt-dependent phosphorylation of eNOS. Valsartan-induced AT1R phosphorylation depends on Src but not PI3K, whereas valsartan-induced suppression of AT1R-eNOS interaction depends on Src/PI3K/Akt signalling. These results indicate a novel vasoprotective mechanism of valsartan in upregulating NO production in ECs.

[Effect of Magnolol on Proliferation and Apoptosis of HL-60 Cells and Its Molecular Mechanism].

PubMed

Fang, Ke; Yuan, Xiao-Fen; Liao, Qiong; Zhang, Zhi-Yong; Song, Guan-Hua; Guo, Qiang; Ren, Xia; Jiang, Guo-Sheng

2016-04-01

To investigate the effect of magnolol on proliferation and apoptosis of HL-60 cells and its mechanism. MTT assay was used to measure the proliferation of HL-60 cells after treatment with different concentration of magnolol (5, 10, 20, 40, 80 and 160 µg/ml). The morphological changes of HL-60 cells were examined by light microscopy, and DAPI staining was performed to observe the nuclear morphology of HL-60 cells. The early cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI double-staining. RT-PCR was carried out to examine the mRNA expression of BAX and BCL-2. Western blot was performed to detect the protein expression of caspase family. The magnolol inhibited HL-60 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time- dependent manner (P < 0.05). HL-60 cells became small, even apoptotic bodies appeared after treatment with magnolol. In addition, nuclear condensation or fragmentation could be observed, which is the typical morphological features of apoptosis. When HL-60 cells were treated with 40 µg/ml of magnolol for 24 h, the ratio of early apoptotic cells reached to (11.7 ± 2.4) %, which was significant different from control (1.4 ± 1.1) % (P < 0.05). RT-PCR results showed that treatment of HL-60 cells with magnolol up-regulated the expression of BAX, whereas down-regulated the expression of BCL-2. Western blot results showed that the cleavages of caspase-3, -8 and -9 were significantly enhanced by magnolol. The magnolol can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through up-regulation of BAX, down-regulation of BCL-2 and the activation of caspases.

Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via mitochondria-dependent pathway.

PubMed

Lee, Jisun; Lee, Seul; Kim, Sun-Lim; Choi, Ji Won; Seo, Jeong Yeon; Choi, Doo Jin; Park, Yong Il

2014-12-05

Despite recent advances in prostate cancer diagnostics and therapeutics, the overall survival rate still remains low. This study was aimed to assess potential anti-cancer activity of maysin, a major flavonoid of corn silk (CS, Zea mays L.), in androgen-independent human prostate cancer cells (PC-3). Maysin was isolated from CS of Kwangpyeongok, a Korean hybrid corn, via methanol extraction and preparative C18 reverse phase column chromatography. Maysin cytotoxicity was determined by either monitoring cell viability in various cancer cell lines by MTT assay or morphological changes. Apoptotic cell death was assessed by annexin V-FITC/PI double staining, depolarization of mitochondrial membrane potential (MMP), expression levels of Bcl-2 and pro-caspase-3 and by terminal transferase mediated dUTP-fluorescein nick end labeling (TUNEL) staining. Underlying mechanism in maysin-induced apoptosis of PC-3 cells was explored by evaluating its effects on Akt and ERK pathway. Maysin dose-dependently reduced the PC-3 cell viability, with an 87% reduction at 200 μg/ml. Maysin treatment significantly induced apoptotic cell death, DNA fragmentation, depolarization of MMP, and reduction in Bcl-2 and pro-caspase-3 expression levels. Maysin also significantly attenuated phosphorylation of Akt and ERK. A combined treatment with maysin and other known anti-cancer agents, including 5-FU, etoposide, cisplatin, or camptothecin, synergistically enhanced PC-3 cell death. These results suggested for the first time that maysin inhibits the PC-3 cancer cell growth via stimulation of mitochondria-dependent apoptotic cell death and may have a strong therapeutic potential for the treatment of either chemo-resistant or androgen-independent human prostate cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Synthesis and cytotoxic activities of novel 4-methoxy-substituted and 5-methyl-substituted (3′S,4′S)-(−)-cis-khellactone derivatives that induce apoptosis via the intrinsic pathway

PubMed Central

Chen, Jingrun; Liu, Junjie; Cui, Dongxiao; Yan, Chaoqun; Meng, Liqiang; Sun, Liqian; Ban, Shurong; Ge, Rui; Liang, Taigang; Li, Qingshan

2017-01-01

This study deals with the design and synthesis of a series of novel 4-methoxy-substituted and 5-methyl-substituted (3′S,4′S)-(−)-cis-khellactones. The newly synthesized compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C-NMR, mass spectrometry, and elemental analysis. All the derivatives were subjected to in vitro cytotoxicity screening against HEPG-2 (human liver carcinoma), SGC-7901 (human gastric carcinoma), and LS174T (human colon carcinoma), by using the MTT assay. The results revealed that several of the 4-methoxy-substituted compounds exhibited potent cytotoxicity. Among these, compound 12e showed the highest activity against cancer cells which 50% inhibitory concentration (IC50) values were in the range of 6.1–9.2 μM with low toxicity on normal human hepatocyte. Preliminary investigation of possible mechanisms of action of compound 12e against HEPG-2 cells indicated possible induction of apoptosis, as determined by morphological observations and Annexin V/propidium iodide (PI) double staining, in addition to apparent dissipation of mitochondrial membrane potential (MMP), as measured by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining in combination with the activation of caspase-9 and caspase-3 by Western blot analysis. Overall, the data suggest that compound 12e may be a promising potential anti-cancer agent that could act primarily by inducing apoptosis through the mitochondria-mediated intrinsic pathway in human hepatoma cells. PMID:28694689

Determination of cell cycle phases in live B16 melanoma cells using IRMS.

PubMed

Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

2013-07-21

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

The Effects of Cadmium at Low Environmental Concentrations on THP-1 Macrophage Apoptosis

PubMed Central

Olszowski, Tomasz; Baranowska-Bosiacka, Irena; Gutowska, Izabela; Piotrowska, Katarzyna; Mierzejewska, Katarzyna; Korbecki, Jan; Kurzawski, Mateusz; Tarnowski, Maciej; Chlubek, Dariusz

2015-01-01

Cadmium at environmental concentrations is a risk factor for many diseases, including cardiovascular and neurodegenerative diseases, in which macrophages play an important role. The aim of this study was to evaluate the effects of cadmium at low environmental (nanomolar) concentrations on apoptotic processes in THP-1(acute monocytic leukemia cells line)-derived macrophages, with special focus on mitochondrial events involved. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM and 2 µM CdCl2. Cell viability was measured using flow cytometry. Flow cytometric measurement (annexin V/FITC (annexin V/fluorescein isothiocyanate) and PI (propidium iodide) double staining) was used to quantify the extent of apoptosis. Fluorescence and confocal microscopy were used for imaging of apoptosis process. Changes in mitochondrial membrane potential were monitored using cytofluorimetry after cell staining with JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyane iodide) probe. Mitochondrial ROS (reactive oxygen species) levels were measured cytofluorimetrically after incubation of cells with mitochondrial superoxide indicator (MitoSOX) red fluorescent marker. The mRNA expression of Bcl-2 and Bax was analysed with qRT-PCR. Our study demonstrates that cadmium, even at low environmental concentrations, exerts mitochondrial toxicity in THP-1 macrophages. Forty-eight-hour exposure to very low concentrations reduces cell viability and results in cell death by apoptosis and necrosis. The decrease in mitochondrial membrane potential, increased ROS production, increased Bax and decreased Bcl-2 mRNA expression are mitochondrial events involved in cadmium-induced apoptosis. PMID:26370970

Induction of apoptosis in human ovarian cancer cells by new anticancer compounds, epothilone A and B.

PubMed

Rogalska, Aneta; Marczak, Agnieszka; Gajek, Arkadiusz; Szwed, Marzena; Śliwińska, Agnieszka; Drzewoski, Józef; Jóźwiak, Zofia

2013-02-01

Epothilones are a new group of compounds with action mechanisms similar to taxanes. The aim of this study was to compare the effects of epothilone A (Epo A) and epothilone B (Epo B) on ovarian cancer cell line SKOV-3 with those of paclitaxel (PTX), a classic taxane. We evaluate glycoprotein P (P-gp) activity in the ovarian cancer cell line. Apoptotic and necrotic cell levels were measured by double staining with Hoechst 33258 and propidium iodide (PI) as well as Annexin V staining. The production of reactive oxygen species (ROS) and changes in mitochondrial membrane potential (ΔΨm) in cells exposed to Epo A, Epo B and PTX were studied using specific fluorescence probes, DCFH(2)-DA (2',7'-dichlorodihydrofluorescein diacetate) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine). The cytotoxic activity of the drugs was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) test. All probes were analyzed in both the presence and absence of the antioxidant N-acetylcysteine (NAC). The results obtained demonstrated that the antiproliferative capacity of Epo A and Epo B in SKOV-3 cell line (measured as IC(50) after 72 h continuous treatment) was six and five times greater than that of PTX's respectively. Epothilones induced timecourse-dependent apoptosis and necrosis. Apoptotic and necrotic events were partially blocked by NAC, indicating ROS played a substantial role in epothilone-induced apoptosis. Cell death was also associated with a decrease in mitochondrial membrane potential, which was more pronounced after treatment with epothilones as compared to paclitaxel. Copyright © 2012 Elsevier Ltd. All rights reserved.

Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

PubMed

Yang, Shucai; Ma, Jing; Xiao, Jianbing; Lv, Xiaohong; Li, Xinlei; Yang, Huike; Liu, Ying; Feng, Sijia; Zhang, Yafang

2012-08-01

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Copyright © 2012 Wiley Periodicals, Inc.

Soft X-Ray Absorption Spectroscopy of High-Abrasion-Furnace Carbon Black

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

Muramatsu, Yasuji; Harada, Ryusuke; Gullikson, Eric M.

2007-02-02

The soft x-ray absorption spectra of high-abrasion-furnace carbon black were measured to obtain local-structure/chemical-states information of the primary particles and/or crystallites. The soft x-ray absorption spectral features of carbon black represent broader {pi}* and {sigma}* peak structures compared to highly oriented pyrolytic graphite (HOPG). The subtracted spectra between the carbon black and HOPG, (carbon black) - (HOPG), show double-peak structures on both sides of the {pi}* peak. The lower-energy peak, denoted as the 'pre-peak', in the subtracted spectra and the {pi}*/{sigma}* peak intensity ratio in the absorption spectra clearly depend on the specific surface area by nitrogen adsorption (NSA). Therefore,more » it is concluded that the pre-peak intensity and the {pi}*/{sigma}* ratio reflect the local graphitic structure of carbon black.« less

Measurement of the Branching Fractions and CP Asymmetries of B{sup -} --> D{sup 0}{sub (CP)}K{sup -} Decays with the BABAR Detector

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

Aubert, B

The authors have reconstructed B{sup -} --> D{sup 0}K{sup -} decays with D{sup 0} mesons decaying to non-CP (K{sup -}{pi}{sup +}), CD-even (K{sup -}K{sup +}, {pi}{sup -}{pi}{sup +}) and CP-odd (K{sup 0}{sub s}{pi}{sup 0}) eigenstates. They have measured the CP asymmetries A{sub CP{sup +}} = 0.40 {+-} 0.15(stat) {+-} 0.08(syst), A{sup CP{sup -}} = 0.21 {+-} 0.17(stat) {+-} 0.07(syst), and the double ratio of branching fractions R{sub +} = 0.87 {+-} 0.14(stat) {+-} 0.06(syst), R{sub -} = 0.80 {+-} 0.14(stat) {+-} 0.08(syst). These results improve the previous existing measurements from BABAR. All results presented in this document are preliminary.

CIP-13F, a novel aminopeptidase N (APN/CD13) inhibitor, inhibits Lewis lung carcinoma growth and metastasis in mice.

PubMed

Pei, Ke-Ling; Yuan, Yi; Qin, San-Hai; Wang, Yan; Zhou, Ling; Zhang, Hou-Li; Qu, Xian-Jun; Cui, Shu-Xiang

2012-04-01

Aminopeptidase N (APN/CD13) is highly expressed on the surface of cancer cells and is thought to be involved in cancer growth and metastasis. The research of APN/CD13 inhibitors is considered as a strategy of cancer treatment. We aimed to evaluate the efficacy of CIP-13F, a novel APN/CD13 inhibitor, using a Lewis lung carcinoma (LLC) implantation mouse model. C57BL/6 mice were subcutaneously inoculated with LLC cells in anterior flank. Then, 0, 50 and 100 mg/kg of CIP-13F were injected via vena caudalis. Bestatin was used as the positive control. Administration of CIP-13F or bestatin was performed daily for 3 consecutive weeks. Mice were killed, and the tumors in anterior flank and metastasis nodules in lungs were examined. The assays of immunohistochemical staining, immunofluorescent flow cytometry and western blotting were performed to estimate the expression of APN/CD13 in LLC cells. We carried out the experiments of Annexin-V/PI staining, DNA fragmentation analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to determine apoptotic cells in LLC tissues. Using immunohistochemical staining with CD34, the antiangiogenesis of CIP-13F was evaluated in LLC tissue sections. CIP-13F treatment resulted in a significant delay of LLC growth in anterior flank. Examination of lungs showed that the number of metastatic nodules of LLC was also markedly decreased. The inhibitory effect of CIP-13F on LLC growth was further evidenced by the induction of LLC apoptosis, showing the increases in Annexin-V/PI staining cells, DNA fragmentation and TUNEL staining cells. Molecular analyses of LLC tissues in CIP-13F-treated mice suggested that the decrease in APN/CD13 expression by CIP-13F might account for its actions of mechanism. Further, the inhibition of angiogenesis in LLC tissues was determined, showing the decreases in microvessel density (MVD) and angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-α). Our results showed that CIP-13F effectively inhibited LLC growth and pulmonary metastasis in mice and suggested that CIP-13F is a potential drug for the treatment for cancers with positive APN/CD13 expression.

Targeting Phosphatidylinositol 4-Kinase IIIα for Radiosensitization: A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent

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

Kwon, Jeanny; Kim, Dan Hyo; Park, Ji Min

Purpose: To investigate which isotype of phosphatidylinositol 4-kinase (PI4K) may affect radiosensitivity and examine whether anti–hepatitis C viral (HCV) agents, some of which have been shown to inhibit PI4K IIIα activity, could be repositioned as a radiosensitizer in human cancer cells. Methods and Materials: U251, BT474, and HepG2 cell lines and normal human astrocyte were used. Ribonucleic acid interference, clonogenic assays, Western blotting, immunofluorescence, annexin V assay, lysotracker staining, and β-galactosidase assay were performed. Results: Of the 4 PI4K isotypes, specific inhibition of IIIα increased radiosensitivity. For pharmacologic inhibition of PI4K IIIα, we screened 9 anti-HCV agents by half-maximal inhibitorymore » concentration assay. Simeprevir was selected, and its inhibition of PI4K IIIα activity was confirmed. Combination of simeprevir treatment and radiation significantly attenuated expression of phospho-phospho-PKC and phospho-Akt and increased radiation-induced cell death in tested cell lines. Pretreatment with simeprevir prolonged γH2AX foci formation and down-regulation of phospho-DNA-PKcs, indicating impairment of nonhomologous end-joining repair. Cells pretreated with simeprevir exhibited mixed modes of cell death, including apoptosis and autophagy. Conclusion: These data demonstrate that targeting PI4K IIIα using an anti-HCV agent is a viable approach to enhance the therapeutic efficacy of radiation therapy in various human cancers, such as glioma, breast, and hepatocellular carcinoma.« less

Enhancing the Area of a Raman Atom Interferometer Using a Versatile Double-Diffraction Technique

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

Leveque, T.; Gauguet, A.; Michaud, F.

2009-08-21

In this Letter, we demonstrate a new scheme for Raman transitions which realize a symmetric momentum-space splitting of 4(Planck constant/2pi)k, deflecting the atomic wave packets into the same internal state. Combining the advantages of Raman and Bragg diffraction, we achieve a three pulse state labeled an interferometer, intrinsically insensitive to the main systematics and applicable to all kinds of atomic sources. This splitting scheme can be extended to 4N(Planck constant/2pi)k momentum transfer by a multipulse sequence and is implemented on a 8(Planck constant/2pi)k interferometer. We demonstrate the area enhancement by measuring inertial forces.

Altered gravity causes the changes in the proteins NoA100 in plant cell nucleoli

NASA Astrophysics Data System (ADS)

Sobol, Margarita A.; Gonzalez-Camacho, Fernando; Kordyum, Elizabeth L.; Medina, Francisco Javier

2005-08-01

A nucleolar protein homologous to the mammalian nucleolin and to the onion nucleolin-like protein NopA100 was detected in nuclear soluble protein fraction from Lepidium sativum root meristematic cells, using the specific silver staining method and the cross-reaction with the anti-NopA100 antibody. In 2D Western blots of soluble nuclear fraction, NopA100 was revealed as a smear extending through a certain range of pI. In extracts obtained from seedlings grown under clinorotation, the extension of the pI range was shorter than in the stationary control indicating a lower phosphorylation of the protein. This suggests that altered gravity causes a decrease in the rate of nucleolar activity.

Bisphosphonates in the Prevention of Post-Traumatic Osteoarthritis

DTIC Science & Technology

2014-07-01

OARSI-describe PTOA scoring system was brought into the lab and 4 individuals have been trained on it. Currently, the safranin-O stained samples are...of chondrocyte proliferation (Ki-67), apoptosis (activated caspase-3), hypertrophy (collagen type X), anabolism, catabolism, and inflammation within...surgery training and technique import into P-PI laboratory complete. o Baseline, DMM-only, and age-matched cohort groups completed for D0 through

Berberine displays antitumor activity in esophageal cancer cells in vitro.

PubMed

Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

2017-04-14

To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G 2 /M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

Berberine displays antitumor activity in esophageal cancer cells in vitro

PubMed Central

Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

2017-01-01

AIM To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients. PMID:28465635

Receptor interacting protein 3-induced RGC-5 cell necroptosis following oxygen glucose deprivation.

PubMed

Ding, Wei; Shang, Lei; Huang, Ju-Fang; Li, Na; Chen, Dan; Xue, Li-Xiang; Xiong, Kun

2015-08-04

Necroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of various diseases. Receptor-interacting protein 3 (RIP3), a member of the RIP family of proteins, has been reported as an important necroptotic pathway mediator in regulating a variety of human diseases, such as myocardial ischemia, inflammatory bowel disease, and ischemic brain injury. Our previous study showed that RIP3 was expressed in rat retinal ganglion cells (RGCs), where it was significantly upregulated during the early stage of acute high intraocular pressure. Furthermore, RIP3 expression was co-localized with propidium iodide (PI)-positive staining (necrotic cells). These results suggested that RIP3 up-regulation might be involved in the necrosis of injured RGCs. In this study, we aimed to reveal the possible involvement of RIP3 in oxygen glucose deprivation (OGD)-induced retinal ganglion cell-5 (RGC-5) necroptosis. RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8 h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. RIP3 expression was detected by western blot and flow cytometry was used to detect the effect of RIP3 on RGC-5 necroptosis following OGD in rip3 knockdown cells. Malondialdehyde (MDA) lipid peroxidation assay was performed to determine the degree of oxidative stress. PI staining showed that necrosis was present in the early stage of OGD-induced RGC-5 cell death. The presence of RGC-5 necroptosis after OGD was detected by flow cytometry using necrostatin-1, a necroptosis inhibitor. Western blot demonstrated that RIP3 up-regulation may be involved in RGC-5 necroptosis. Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown. Furthermore, MDA levels in the normal RGC-5 cells were much higher than in the rip3-knockdown cells after OGD. Our findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.

Detection of microinvasion in vulvar and cervical intraepithelial neoplasia using double immunostaining for cytokeratin and basement membrane components.

PubMed

Rush, Demaretta; Hyjek, Elizabeth; Baergen, Rebecca N; Ellenson, Lora H; Pirog, Edyta C

2005-06-01

Identification of early invasion in vulvar intraepithelial neoplasia 3 (VIN 3) and cervical intraepithelial neoplasia 3 (CIN 3) may be difficult with the use of routine hematoxylin-eosin staining. Presence of obscuring inflammation and tangential tissue sectioning are the most common diagnostic pitfalls. To examine the utility of double immunostaining for cytokeratin-collagen IV or cytokeratin-laminin in the detection of early invasion in VIN 3 and CIN 3. The study group consisted of 10 cases of "VIN 3, suspicious for invasion" and 10 cases of "CIN 3, suspicious for invasion." The negative control group consisted of VIN 3 (n = 15) and CIN 3 (n = 10). The positive control group consisted of cases of invasive vulvar carcinoma (n = 11) and invasive cervical carcinoma (n = 25). All cases were double immunostained for cytokeratin and collagen IV and, in a separate reaction, for cytokeratin and laminin. The continuity of the basement membrane and the presence of stromal invasion were assessed in the stained sections. The staining for collagen IV and laminin yielded identical results. A well-defined, continuous basement membrane was visualized in all cases of VIN 3 and CIN 3. A discontinuous or absent basement membrane was observed around the malignant cells on the invasive tumor front in all cases of vulvar and cervical carcinoma. In 2 of 10 cases of VIN 3, suspicious for invasion and in 4 of 10 cases of CIN 3, suspicious for invasion definitive foci of microinvasion were identified with the use of double immunostaining. A well-defined, continuous basement membrane was present in the remaining cases "suspicious for invasion." Double immunostaining for cytokeratin- collagen IV or cytokeratin-laminin is useful for evaluation of early invasion in equivocal cases of VIN 3 and CIN 3.

P110β Inhibition Reduces Histone H3K4 Di-Methylation in Prostate Cancer.

PubMed

Pang, Jun; Yang, Yue-Wu; Huang, Yiling; Yang, Jun; Zhang, Hao; Chen, Ruibao; Dong, Liang; Huang, Yan; Wang, Dongying; Liu, Jihong; Li, Benyi

2017-02-01

Epigenetic alteration plays a major role in the development and progression of human cancers, including prostate cancer. Histones are the key factors in modulating gene accessibility to transcription factors and post-translational modification of the histone N-terminal tail including methylation is associated with either transcriptional activation (H3K4me2) or repression (H3K9me3). Furthermore, phosphoinositide 3-kinase (PI3 K) signaling and the androgen receptor (AR) are the key determinants in prostate cancer development and progression. We recently showed that prostate-targeted nano-micelles loaded with PI3 K/p110beta specific inhibitor TGX221 blocked prostate cancer growth in vitro and in vivo. Our objective of this study was to determine the role of PI3 K signaling in histone methylation in prostate cancer, with emphasis on histone H3K4 methylation. PI3 K non-specific inhibitor LY294002 and p110beta-specific inhibitor TGX221 were used to block PI3 K/p110beta signaling. The global levels of H3K4 and H3K9 methylation in prostate cancer cells and tissue specimens were evaluated by Western blot assay and immunohistochemical staining. A synthetic androgen R1881 was used to stimulate AR activity in prostate cancer cells. A castration-resistant prostate cancer (CRPC) specific human tissue microarray (TMA) was used to assess the global levels of H3K4me2 methylation by immunostaining approach. Our data revealed that H3K4me2 levels were significantly elevated after androgen stimulation. With RNA silencing and pharmacology approaches, we further defined that inhibition of PI3 K/p110beta activity through gene-specific knocking down and small chemical inhibitor TGX221 abolished androgen-stimulated H3K4me2 methylation. Consistently, prostate cancer-targeted delivery of TGX221 in vivo dramatically reduced the global levels of H3K4me2 as assessed by immunohistochemical staining on tissue section of mouse xenografts from CRPC cell lines 22RV1 and C4-2. Finally, immunostaining data revealed a strong H3K4me2 immunosignal in CRPC tissues compared to primary tumors and benign prostate tissues. Taken together, our results suggest that PI3 K/p110beta-dependent signaling is involved in androgen-stimulated H3K4me2 methylation in prostate cancer, which might be used as a novel biomarker for disease prognosis and targeted therapy. Prostate 77:299-308, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Transition probabilities in OH A 2 sigma + - X 2 pi i: Bands with v prime = 0 and 1, v double prime = 0 to 4

NASA Technical Reports Server (NTRS)

Copeland, Richard A.; Jeffries, Jay B.; Crosley, David R.

1986-01-01

Experimental results for relative vibrational band transition probabilities for v prime = 0 and 1, and v double prime = 0 to 4 in the A-X electronic system of OH are presented. The measurements, part of a larger set involving v prime = 0 to 4 and v double prime = 0 to 6, were made using spectrally dispersed laser-induced fluorescence (LIF) in the burnt gases of a flame. These Einstein coefficients will be useful in dynamics experiments for quantitative LIF determinations of OH radical concentrations in high v double prime.

High resolution absorption cross sections for the A2Pi-X2Pi system of ClO

NASA Technical Reports Server (NTRS)

Wine, P. H.; Ravishankara, A. R.; Philen, D. L.; Davis, D. D.; Watson, R. T.

1977-01-01

High-resolution ultraviolet absorption cross-sections for the ClO molecule are obtained, with the aim of facilitating studies of ozone depletion resulting from the injection of chlorofluorocarbons into the atmosphere. The spectroscopic analysis, which involves a frequency-doubled tunable dye laser with a bandwidth of 0.015 A, is described. Studies of the rotational lines of the ClO A 2Pi 3/2-X2Pi 3/2 9-10 band were conducted. Peak cross-sections for the P and R lines of the 9-0 band are found to be 10.0, 9.6, 8.6, 10.6, 10.3, and 9.2 times ten to the negative seventeenth power cm squared, with estimated accuracy of plus or minus 25%. Problems in distinguishing between Cl-35 and Cl-37 absorption are also considered.

The double capsules in macro-textured breast implants.

PubMed

Giot, Jean-Philippe; Paek, Laurence S; Nizard, Nathanael; El-Diwany, Mostafa; Gaboury, Louis A; Nelea, Monica; Bou-Merhi, Joseph S; Harris, Patrick G; Danino, Michel A

2015-10-01

Breast implants are amongst the most widely used types of permanent implants in modern medicine and have both aesthetic and reconstructive applications with excellent biocompatibility. The double capsule is a complication associated with textured prostheses that leads to implant displacement; however, its etiology has yet to be elucidated. In this study, 10 double capsules were sampled from breast expander implants for in-depth analysis; histologically, the inner capsular layer demonstrated highly organized collagen in sheets with delamination of fibers. At the prosthesis interface (PI) where the implant shell contacts the inner capsular layer, scanning electron microscopy (SEM) revealed a thin layer which mirrored the three-dimensional characteristics of the implant texture; the external surface of the inner capsular layer facing the intercapsular space (ICS) was flat. SEM examination of the inner capsule layer revealed both a large bacterial presence as well as biofilm deposition at the PI; a significantly lower quantity of bacteria and biofilm were found at the ICS interface. These findings suggest that the double capsule phenomenon's etiopathogenesis is of mechanical origin. Delamination of the periprosthetic capsule leads to the creation of the ICS; the maintained separation of the 2 layers subsequently alters the biostability of the macro-textured breast implant. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simple and rapid staining for detection of Entamoeba cysts and other protozoans with fluorochromes.

PubMed

Kawamoto, F; Mizuno, S; Fujioka, H; Kumada, N; Sugiyama, E; Takeuchi, T; Kobayashi, S; Iseki, M; Yamada, M; Matsumoto, Y

1987-02-01

Three fluorochromes were applied to stain various parasitic protozoans. By double staining with 4',6-diamidino-2-phenylindole and propidium iodide, differentiation of the nuclei from the cytoplasm can easily be achieved within several seconds. The chromatoid bodies in Entamoeba cysts were stained bright red. Plasmodium yoelii at all stages except late trophozoites and young gametocytes was easily identified. In the oocysts of Cryptosporidium sp., the nuclei and cytoplasm of the sporozoites fluoresced bluish white and red, respectively, whereas the residual body appeared blue or green. The third fluorochrome, Calcofluor white M2R, was suitable for detecting the cysts of Entamoeba spp. and Chilomastix mesnili.

β-elemene reverses the drug resistance of lung cancer A549/DDP cells via the mitochondrial apoptosis pathway.

PubMed

Yao, Cheng-Cai; Tu, Yuan-Rong; Jiang, Jie; Ye, Sheng-Fang; Du, Hao-Xin; Zhang, Yi

2014-05-01

β-elemene (β-ELE) is a new anticancer drug extracted from Curcuma zedoaria Roscoe and has been widely used to treat malignant tumors. Recent studies have demonstrated that β-ELE reverses the drug resistance of tumor cells. To explore the possible mechanisms of action of β-ELE, we investigated its effects on cisplatin-resistant human lung adenocarcinoma A549/DDP cells. The effects of β-ELE on the growth of A549/DDP cells in vitro were determined by MTT assay. Apoptosis was assessed by fluorescence microscopy with Hoechst 33258 staining and flow cytometry with Annexin V-FITC/PI double staining. Mitochondrial membrane potential was assessed using JC-1 fluorescence probe and laser confocal scanning microscopy, and intracellular reactive oxygen species levels were measured by 2',7'-dichlorofluorescein-diacetate staining and flow cytometry. Cytosolic glutathione content was determined using GSH kits. The expression of cytochrome c, caspase-3, procaspase-3 and the Bcl-2 family proteins was assessed by western blotting. The results demonstrated that β-ELE inhibited the proliferation of A549/DDP cells in a time- and dose-dependent manner. Furthermore, β-ELE enhanced the sensitivity of A549/DDP cells to cisplatin and reversed the drug resistance of A549/DDP cells. Consistent with a role in activating apoptosis, β-ELE decreased mitochondrial membrane potential, increased intracellular reactive oxygen species concentration and decreased the cytoplasmic glutathione levels in a time- and dose-dependent manner. The combination of β-ELE and cisplatin enhanced the protein expression of cytochrome c, caspase-3 and Bad, and reduced protein levels of Bcl-2 and procaspase-3 in the A549/DDP lung cancer cells. These results define a pathway of procaspase‑3-β-ELE function that involves decreased mitochondrial membrane potential, leading to apoptosis triggered by the release of cytochrome c into the cytoplasm and the modulation of apoptosis-related genes. The reversal of drug resistance of the A549/DDP cell line by β-ELE may be derived from its effect in inducing apoptosis.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

PubMed

Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

2015-09-01

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Impact on photon/phi 3 meson measurement from CLEO-c binned neutral D meson decaying to neutral kaon(short,long) positive pion negative pion Dalitz analysis

NASA Astrophysics Data System (ADS)

He, Qing

The Dalitz plot analysis of the three-body decay D˜0 → K0S pi+pi-- (D0 or D0) from the B+/- → DK+/- process provides today the best measurement of the CKM angle gamma/φ3. However, the unbinned D˜0 → K0S Dalitz analysis is sensitive to the choice of the model used to describe the three-body decay, which introduces 7° ˜ 10° uncertainty on gamma determination. For LHCb and future Super-B factories, this uncertainty will become a major limitation. A model independent approach has been proposed and the quantum correlated D0/ D0 data produced at the resonance psi(3770) can provide important information on the strong phase difference between D 0 → K0S pi+pi-- and D 0 → K0S pi+pi-- processes. Using the 818 pb-1 data collected by the CLEO-c detector at the Cornell Electron Storage Ring, we measure the strong phase parameters, c i, si, using fully reconstructed D0 D0 pairs with K0S pi+pi-- vs. flavor states, CP eigenstates and double K0S pi+pi-- samples. We also include K0L pi+pi-- data to further improve the determination of ci and si in a global fit. The ci and si results for the 8 phase bin case are shown in the following table. With the ci, si measurements from CLEO, the uncertainty of gamma determination can be lowered down to about 2° if one assumes plenty of B+/- → D˜K +/- decays.* *Please refer to dissertation for diagrams.

Genetic and mechanistic diversity of piRNA 3' end formation

PubMed Central

Handler, Dominik; Mohn, Fabio; Ameres, Stefan L.; Brennecke, Julius

2016-01-01

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and thereby play important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and siRNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. piRNAs—the 22-30 nt long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated Dicer-independently from single-stranded precursors2,3. piRNA 5' ends are defined either by Zucchini, a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3' ends is poorly understood. Here, we find that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler/Mut-710–13. The relative activity of these two pathways dictates the extent to which piRNAs are fueled into cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Strikingly, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway where piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data establish a coherent blueprint for piRNA biogenesis, and set the stage for the mechanistic dissection of the processes that govern piRNA 3' end formation. PMID:27851737

Protease inhibitor-associated bone mineral density loss is related to hypothyroidism and related bone turnover acceleration.

PubMed

Kinai, Ei; Gatanaga, Hiroyuki; Mizushima, Daisuke; Nishijima, Takeshi; Aoki, Takahiro; Genka, Ikumi; Teruya, Katsuji; Tsukada, Kunihisa; Kikuchi, Yoshimi; Oka, Shinichi

2017-05-01

Clinical and experiments evidence indicate that protease inhibitors (PI) can cause bone mineral density (BMD) loss. However, the mechanism of such loss remains obscure. This single-center, cross-sectional study included 184 HIV-infected patients treated with PI who underwent dual-energy X-ray absorptiometry scan. Serum phosphorus, percentage of tubular reabsorption of phosphate (%TRP), thyroid and parathyroid function (iPTH), vitamin D, osteocalcin (OC), urinary deoxypyridinoline (DPD), and urinary cross-linked N-telopeptide of type I collagen (u-NTx) were measured. The rate of hypothyroidism in PI-users [32/117 (27%)] was double that in non-PI users [8/67 (12%), p = 0.016] and was significantly associated with PI use in multivariate analysis [odds ratio (OR) 11.37, 95% confidence interval (CI) 1.358-95.17, p = 0.025]. Spine BMD was significantly lower in hypothyroid patients than euthyroid, for both total population (-1.37 vs. -1.00, p = 0.041) and PI users (-1.56 vs. -1.13, p = 0.029). Multivariate regression analysis identified inverse correlation between hypothyroidism and spine BMD [estimate -0.437, 95% CI -0.858 to -0.024, p = 0.042]. OC, DPD and u-NTx were significantly higher in PI users than in non-PI users (p = 0.01, 0.05, and 0.01, respectively). PI use is associated with hypothyroidism as well as bone turnover acceleration, which worsens PI-associated BMD loss. In PI-treated patients, thyroid function tests are warranted to prevent further progression of PI-associated BMD loss. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean1[W][OA

PubMed Central

Qin, Lu; Zhao, Jing; Tian, Jiang; Chen, Liyu; Sun, Zhaoan; Guo, Yongxiang; Lu, Xing; Gu, Mian; Xu, Guohua; Liao, Hong

2012-01-01

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance. PMID:22740613

Glutathione S-transferases in neonatal liver disease.

PubMed Central

Mathew, J.; Cattan, A. R.; Hall, A. G.; Hines, J. E.; Nelson, R.; Eastham, E.; Burt, A. D.

1992-01-01

AIMS: To investigate the distribution of alpha and pi class glutathione S-transferases (GST) in normal fetal, neonatal, and adult liver; and to examine changes in GST expression in neonatal liver disease. METHODS: alpha and pi class GST were immunolocalised in sections of formalin fixed liver tissue obtained from human fetuses (n = 21), neonates (n = 8), young children (n = 9) and adults (n = 10), and from neonates with extrahepatic biliary atresia (n = 15) and neonatal hepatitis (n = 12). Monospecific rabbit polyclonal antibodies were used with a peroxidase-antiperoxidase method. RESULTS: Expression of pi GST was localised predominantly within biliary epithelial cells of developing and mature bile ducts of all sizes from 16 weeks' gestation until term and in neonatal and adult liver. Coexpression of pi and alpha GST was seen in hepatocytes of developing fetal liver between 16 and 34 weeks' gestation. Although pi GST was seen in occasional hepatocytes up to six months of life, this isoenzyme was not expressed by hepatocytes in adult liver. By contrast, alpha GST continued to be expressed by hepatocytes in adult liver; this isoenzyme was also seen in some epithelial cells of large bile ducts in adult liver. No change was observed in the distribution of alpha GST in either neonatal hepatitis or extrahepatic biliary atresia. However, aberrant expression of pi GST was identified in hepatocytes of all but one case of extrahepatic biliary atresia but in only two cases of neonatal hepatitis. CONCLUSIONS: The phenotypic alterations noted in extrahepatic biliary atresia may result from the effect of cholate stasis. Evaluation of the pattern of pi and alpha GST distribution by immunohistochemical staining may provide valuable information in distinguishing between these two forms of neonatal liver disease. Images PMID:1401176

Long-term in vivo left ventricular assist device study for 284 days with Gyro PI pump.

PubMed

Ohtsuka, G; Nakata, K; Yoshikawa, M; Takano, T; Glueck, J; Sankai, Y; Takami, Y; Mueller, J; Sueoka, A; Letsou, G; Schima, H; Schmallegger, H; Wolner, E; Koyanagi, H; Fujisawa, A; Baldwin, J C; Nosé, Y

1999-06-01

A totally implantable centrifugal artificial heart has been developed. The plastic prototype, the Gyro PI 601, passed 2 day hemodynamic tests as a functional total artificial heart (TAH), 2 week screening tests for anti-thrombogenecity, and a 1 month system feasibility study. Based upon these results, a metallic prototype, the Gyro PI 700 series, was subjected to long-term in vivo left ventricular assist device (LVAD) studies of over 1 month. The Gyro PI 700 series has the same inner dimension and same characteristics of the Gyro PI 601 such as an eccentric inlet port, a double pivot bearing system, and a magnet coupling system. The PI metallic pump is also driven with the Vienna DC brushless motor actuator like the PI 601. The pump-actuator package was implanted in 3 calves in the preperitoneal space, bypassing from the left ventricular (LV) apex to the descending aorta. Case 1 achieved a 284 day survival. Case 2 was euthanized early at 72 postoperative days as a result of the functional obstruction of the inlet port due to the excessive growth of the calf. There was no blood clot inside the pumps of either case. Case 3 is on-going (22 days on July 24, 1998). During these periods, all cases showed no physiological abnormalities. In conclusion, the PI 700 series pump has excellent results as a long-term implantable LVAD.

Electrical properties of double layer dielectric structures for space technology

NASA Astrophysics Data System (ADS)

Lian, Anqing

1993-04-01

Polymeric films such as polyimide (PI) and polyethylene terephthalate (PET) are used in space technology as thermal blankets. Thin SiO2 and SiN coatings plasma deposited onto PI and PET surfaces were proposed to protect the blanket materials against the space environment. The electrical properties of this kind of dual layer dielectric structure were investigated to understand the mechanisms for suppressing charge accumulation and flashover. Bulk and surface electrical conductivities of thin single-layer PI and PET samples and of the dual layer SiO2 and SiN combinations with PI and PET were measured in a range of applied electrical fields. The capacitance voltage (CV) technique was used for analyzing charge transport and distribution in the structures. The electric current in the bulk of the SiO2/PI and SiN/PI samples was found to depend on the polarity of the electric field. Other samples did not exhibit any such polarity effect. The polarity dependence is attributed to charge trapping at the PI/plasma deposit interface. The CV characteristics of the Al-PI-SiO2-Si structure confirm that charges which can modify the local electric field can be trapped near the interface. A model is proposed to interpret the properties of the currents in dual layer structures. This model can semi-quantitatively explain all the observed results.

Experimental Study of the NaK 3(1)Pi State.

PubMed

Laub; Mazsa; Webb; La Civita J; Prodan; Jabbour; Namiotka; Huennekens

1999-02-01

We report the results of an optical-optical double resonance experiment to determine the NaK 3(1)Pi state potential energy curve. In the first step, a narrow band cw dye laser (PUMP) is tuned to line center of a particular 2(A)1Sigma+(v', J') <-- 1(X)1Sigma+(v", J") transition, and its frequency is then fixed. A second narrowband tunable cw Ti:Sapphirelaser (PROBE) is then scanned, while 3(1)Pi --> 1(X)1Sigma+ violet fluorescence is monitored. The Doppler-free signals accurately map the 3(1)Pi(v, J) ro-vibrational energy levels. These energy levels are then fit to a Dunham expansion to provide a set of molecular constants. The Dunham constants, in turn, are used to construct an RKR potential curve. Resolved 3(1)Pi(v, J) --> 1(X)1Sigma+(v", J") fluorescence scans are also recorded with both PUMP and PROBE laser frequencies fixed. Comparison between observed and calculated Franck-Condon factors is used to determine the absolute vibrational numbering of the 3(1)Pi state levels and to determine the variation of the 3(1)Pi --> 1(X)1Sigma+ transitiondipole moment with internuclear separation. The recent theoretical calculation of the NaK 3(1)Pi state potential reported by Magnier and Millié (1996, Phys. Rev. A 54, 204) is in excellent agreement with the present experimental RKR curve. Copyright 1999 Academic Press.

Efficacy and safety of topical diclofenac/menthol gel for ankle sprain: A randomized, double-blind, placebo- and active-controlled trial

PubMed Central

Collaku, Agron; Reed, Kenneth

2017-01-01

Purpose This study was performed to evaluate topical 1% diclofenac/3% menthol gel in treating ankle sprain. Design In this randomized, double-blind, placebo-controlled trial, adolescents and adults with acute ankle sprain (N = 385) applied 4 g of gel containing 1% diclofenac/3% menthol (n = 117), 1% diclofenac (n = 112), 3% menthol (n = 77), or placebo (n = 75) four times daily. The primary outcome was the area under the curve of pain intensity (PI) on movement [0 (no pain) to 10 (extreme pain)] from 24 to 72 hours post-application (AUC1–3 days). Secondary outcomes included pain relief (PR); PI; time to onset of PR, meaningful PR, cooling, and complete recovery; PI difference; sum of PI difference; total PR; reduction in ankle swelling; and the patient’s global assessment of response to treatment. Results There were no statistically significant differences in AUC1–3 between 1% diclofenac/3% menthol and placebo, diclofenac, or menthol gels and no meaningful advantages of 1% diclofenac/3% menthol for any secondary outcome. There was a higher incidence of skin and application-site events with 1% diclofenac/3% menthol than with placebo or 1% diclofenac. Conclusion No significant improvement was observed with topical 1% diclofenac/3% menthol gel compared with placebo, 1% diclofenac, or 3% menthol gel in treating pain from ankle sprain. ClinicalTrials.Gov Identifier: NCT02100670 PMID:28345425

Retrospective, Demographic, and Clinical Investigation of the Causes of Postoperative Infection in Patients With Lumbar Spinal Stenosis Who Underwent Posterior Stabilization.

PubMed

Yaldiz, Can; Yaldiz, Mahizer; Ceylan, Nehir; Kacira, Ozlem Kitiki; Ceylan, Davut; Kacira, Tibet; Kizilcay, Gokhan; Tanriverdi, Taner

2015-07-01

Owing to the increasing population of elderly patients, a large number of patients with degenerative spondylosis are currently being surgically treated. Although basic measures for decreasing postoperative surgical infections (PSIs) are considered, it still remains among the leading causes of morbidity and mortality. The aim of this retrospective analysis is to present possible causes leading to PSI in patients who underwent surgery for lumbar degenerative spondylosis and highlight how it can be avoided to decrease morbidity and mortality. The study included 540 patients who underwent posterior stabilization due to degenerative lumbar stenosis between January 2013 and January 2014. The data before and after surgery was retrieved from the hospital charts. Patients with degenerative lumbar stenosis who were operated upon in this study had >2 levels of laminectomy and facetectomy. For this reason, posterior stabilization was performed for all the patients included in this study. Determining the causes of postoperative infection (PI) following spinal surgeries performed with instrumentation is a struggle. Seventeen different parameters that may be related to PI were evaluated in this study. The presence of systemic diseases, unknown glove perforations, and perioperative blood transfusions were among the parameters that increased the prevalence of PI. Alternatively, prolene sutures, double-layered gloves, and the use of rifampicin Sv (RIS) decreased the incidence of PI. Although the presence of systemic diseases, unnoticed glove perforations, and perioperative blood transfusions increased PIs, prolene suture material, double-layered gloves, and the use of RIS decreased PIs.

A chemical genetic strategy identify the PHOSTIN, a synthetic molecule that triggers phosphate starvation responses in Arabidopsis thaliana.

PubMed

Bonnot, Clémence; Pinson, Benoît; Clément, Mathilde; Bernillon, Stéphane; Chiarenza, Serge; Kanno, Satomi; Kobayashi, Natsuko; Delannoy, Etienne; Nakanishi, Tomoko M; Nussaume, Laurent; Desnos, Thierry

2016-01-01

Plants display numerous strategies to cope with phosphate (Pi)-deficiency. Despite multiple genetic studies, the molecular mechanisms of low-Pi-signalling remain unknown. To validate the interest of chemical genetics to investigate this pathway we discovered and analysed the effects of PHOSTIN (PSN), a drug mimicking Pi-starvation in Arabidopsis. We assessed the effects of PSN and structural analogues on the induction of Pi-deficiency responses in mutants and wild-type and followed their accumulation in plants organs by high pressure liquid chromotography (HPLC) or mass-spectrophotometry. We show that PSN is cleaved in the growth medium, releasing its active motif (PSN11), which accumulates in plants roots. Despite the overaccumulation of Pi in the roots of treated plants, PSN11 elicits both local and systemic Pi-starvation effects. Nevertheless, albeit that the transcriptional activation of low-Pi genes by PSN11 is lost in the phr1;phl1 double mutant, neither PHO1 nor PHO2 are required for PSN11 effects. The range of local and systemic responses to Pi-starvation elicited, and their dependence on the PHR1/PHL1 function suggests that PSN11 affects an important and early step of Pi-starvation signalling. Its independence from PHO1 and PHO2 suggest the existence of unknown pathway(s), showing the usefulness of PSN and chemical genetics to bring new elements to this field. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Cellular phosphoinositides and the maturation of bluetongue virus, a non-enveloped capsid virus

PubMed Central

2013-01-01

Background Bluetongue virus (BTV), a member of Orbivirus genus in the Reoviridae family is a double capsid virus enclosing a genome of 10 double-stranded RNA segments. A non-structural protein of BTV, NS3, which is associated with cellular membranes and interacts with outer capsid proteins, has been shown to be involved in virus morphogenesis in infected cells. In addition, studies have also shown that during the later stages of virus infection NS3 behaves similarly to HIV protein Gag, an enveloped viral protein. Since Gag protein is known to interact with membrane lipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] and one of the known binding partners of NS3, cellular protein p11 also interacts with annexin a PI(4,5)P2 interacting protein, this study was designed to understand the role of this negatively charged membrane lipid in BTV assembly and maturation. Methods Over expression of cellular enzymes that either depleted cells of PI(4,5)P2 or altered the distribution of PI(4,5)P2, were used to analyze the effect of the lipid on BTV maturation at different times post-infection. The production of mature virus particles was monitored by plaque assay. Microscopic techniques such as confocal microscopy and electron microscopy (EM) were also undertaken to study localization of virus proteins and virus particles in cells, respectively. Results Initially, confocal microscopic analysis demonstrated that PI(4,5)P2 not only co-localized with NS3, but it also co-localized with VP5, one of the outer capsid proteins of BTV. Subsequently, experiments involving depletion of cellular PI(4,5)P2 or its relocation demonstrated an inhibitory effect on normal BTV maturation and it also led to a redistribution of BTV proteins within the cell. The data was supported further by EM visualization showing that modulation of PI(4,5)P2 in cells indeed resulted in less particle production. Conclusion This study to our knowledge, is the first report demonstrating involvement of PI(4,5)P2 in a non-enveloped virus assembly and release. As BTV does not have lipid envelope, this finding is unique for this group of viruses and it suggests that the maturation of capsid and enveloped viruses may be more closely related than previously thought. PMID:23497128

Three-body final state interaction in η → 3π updated

DOE PAGES

Guo, P.; Danilkin, I. V.; Fernandez-Ramirez, C.; ...

2017-06-07

In view of the recent high-statistic KLOE-2 data for themore » $$\\eta \\to \\pi^+ \\pi^- \\pi^0$$ decay, a new determination of the quark mass double ratio has been done. Our approach relies on a unitary dispersive model that takes into account rescattering effects between three pions. The latter is essential to reproduce the Dalitz plot distribution. A simultaneous description of the KLOE-2 and WASA-at-COSY data is achieved in terms of just two real parameters. From a global fit, we determine $$Q=21.6 \\pm 0.4$$. Here, the predicted slope parameter for the neutral channel $$\\alpha=-0.025\\pm 0.004$$ is in a reasonable agreement with the PDG average value.« less

The protective effects of chitooligosaccharides against glucose deprivation-induced cell apoptosis in cultured cortical neurons through activation of PI3K/Akt and MEK/ERK1/2 pathways.

PubMed

Xu, Yanyan; Zhang, Qi; Yu, Shu; Yang, Yumin; Ding, Fei

2011-02-23

Chitooligosaccharides (COSs), the biodegradation product of chitosan, possess a wide range of biological activities. In this study, we investigated the influences of COSs on primary cultured cortical neurons exposed to glucose deprivation (GD). The cell viability assessment by MTT assay, in couple with cell apoptosis analysis by Hoechst 33342 and TUNEL staining, indicated that GD-induced cell apoptosis in cultured cortical neurons was attenuated by COSs co-treatment in a dose-dependent manner. Light micrography following tetramethylrhodamine methyl ester staining revealed that COSs protected cultured cortical neurons from GD insult through the stabilization of mitochondrial membrane potentials. COSs co-treatment also led to the increase in Bcl-2/Bax protein ratio and the inhibition of caspase-3 activation in cultured cortical neurons exposed to GD insult. We further found that COSs were able to transiently cause the activation of Akt and ERK1/2 proteins, and anti-apoptotic effects of COSs could be blocked by chemical inhibition of PI3K and MEK. Taken together, the results suggest that COSs hold a promise to serve as a potential neuroprotective agent for treating cerebral ischemic stroke and neurodegenerative diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Cytotoxic analysis and chemical characterization of fractions of the hydroalcoholic extract of the Euterpe oleracea Mart. seed in the MCF-7 cell line.

PubMed

Freitas, Dayanne da S; Morgado-Díaz, José A; Gehren, Adriana S; Vidal, Flávia C B; Fernandes, Raquel Maria T; Romão, Wanderson; Tose, Lilian V; Frazão, Fabiola N S; Costa, Maria Célia P; Silva, Dulcelena F; Nascimento, Maria do Desterro S B

2017-06-01

To analyse the antineoplastic activity of fractions derived from the hydroalcoholic extract of Euterpe oleracea Mart. seed in the MCF-7 cell line and to identify the compounds responsible for the antineoplastic action. Cells were treated with 10, 20, 40 and 60 μg/ml with the hexane, chloroform and ethyl acetate fraction (EAF) of the hydroalcoholic extract of açaí seed, for 24 and 48 h. After treatment, cell viability was measured using MTT assay and cell death was assessed using the Annexin-Pi assay. The most cytotoxic fraction under study was analysed by mass spectrometry using an electrospray ionization source and a cyclotron analyser coupled to a Fourier transform. Data were analysed statistically by analysis of variance (ANOVA) or by Student's t-test, where appropriate. All fractions caused significant reduction in the cell viability, but the EAF was the most cytotoxic (P < 0.001). It was observed the absence of significant annexin staining but increase Pi staining (P < 0.001). The EAF is composed of epicatechin, proanthocyanidin A 2 and trimeric and tetrameric procyanidins. In this study, we demonstrated that EAF was the most effective fraction in reducing cell viability and causing necroptosis in the MCF-7 cell. © 2017 Royal Pharmaceutical Society.

Optogalvanic spectroscopy of the C"5Pi ui-A' 5Sigma+g electronic system of N2.

PubMed

Pirali, O; Tokaryk, D W

2006-11-28

We have recorded spectra involving the 3-1, 4-2, 2-0, and 2-2 bands of the C" 5Pi(ui)-A' (5)Sigma+(g) electronic system of N(2) using optogalvanic detection in a discharge through a supersonic jet expansion of argon mixed with a trace of nitrogen gas. The spectra have an effective rotational temperature of about 45 K. They involve all five spin-orbit components of the C" 5Pi(ui) state, which has allowed for precise determination of the spin-orbit coupling in this state. Analysis of the C" 5Pi(ui) state Lambda-doubling shows that it is caused primarily by a first-order spin-spin effect rather than by interaction with Sigma(u) (+/-) states. Our results allow us to assign lines in the 4-2 and 2-0 bands observed in a fluorescence depletion experiment conducted over ten years ago [Ch. Ottinger and A. F. Vilesov, J. Chem. Phys. 103, 9929 (1995)], and to comment on the suggestion that perturbations to the C (3)Pi(u) v=1 level of N(2) arise from interactions with the C" 5Pi(ui) state.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

PubMed

Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

2015-06-01

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PubMed

Chen, Shuai; Zhu, Haifeng; Wang, Gangliang; Xie, Ziang; Wang, Jiying; Chen, Jian

2018-06-16

To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

Training of resistance to proactive interference and working memory in older adults: a randomized double-blind study.

PubMed

Loosli, Sandra V; Falquez, Rosalux; Unterrainer, Josef M; Weiller, Cornelius; Rahm, Benjamin; Kaller, Christoph P

2016-03-01

Working memory (WM) performance is often decreased in older adults. Despite the growing popularity of WM trainings, underlying mechanisms are still poorly understood. Resistance to proactive interference (PI) constitutes a candidate process that contributes to WM performance and might influence training or transfer effects. Here, we investigated whether PI resistance can be enhanced in older adults using a WM training with specifically increased PI-demands. Further, we investigated whether potential effects of such a training were stable and entailed any transfer on non-trained tasks. Healthy old adults (N = 25, 68.8 ± 5.5 years) trained with a recent-probes and an n-back task daily for two weeks. Two different training regimens (high vs. low PI-amount in the tasks) were applied as between-participants manipulation, to which participants were randomly assigned. Near transfer tasks included interference tasks; far transfer tasks assessed fluid intelligence (gF) or speed. Immediate transfer was assessed directly after training; a follow-up measurement was conducted after two months. Both groups similarly improved in PI resistance in both training tasks. Thus, PI susceptibility was generally reduced in the two training groups and there was no difference between WM training with high versus low PI demands. Further, there was no differential near or far transfer on non-trained tasks, neither immediately after the training nor in the follow-up. PI-demands in WM training tasks do not seem critical for enhancing WM performance or PI resistance in older adults. Instead, improved resistance to PI appears to be an unspecific side-effect of a WM training.

Phosphate-Dependent Root System Architecture Responses to Salt Stress1[OPEN

PubMed Central

Sommerfeld, Hector Montero; ter Horst, Anneliek; Haring, Michel A.

2016-01-01

Nutrient availability and salinity of the soil affect the growth and development of plant roots. Here, we describe how inorganic phosphate (Pi) availability affects the root system architecture (RSA) of Arabidopsis (Arabidopsis thaliana) and how Pi levels modulate responses of the root to salt stress. Pi starvation reduced main root length and increased the number of lateral roots of Arabidopsis Columbia-0 seedlings. In combination with salt, low Pi dampened the inhibiting effect of mild salt stress (75 mm) on all measured RSA components. At higher salt concentrations, the Pi deprivation response prevailed over the salt stress only for lateral root elongation. The Pi deprivation response of lateral roots appeared to be oppositely affected by abscisic acid signaling compared with the salt stress response. Natural variation in the response to the combination treatment of salt and Pi starvation within 330 Arabidopsis accessions could be grouped into four response patterns. When exposed to double stress, in general, lateral roots prioritized responses to salt, while the effect on main root traits was additive. Interestingly, these patterns were not identical for all accessions studied, and multiple strategies to integrate the signals from Pi deprivation and salinity were identified. By genome-wide association mapping, 12 genomic loci were identified as putative factors integrating responses to salt stress and Pi starvation. From our experiments, we conclude that Pi starvation interferes with salt responses mainly at the level of lateral roots and that large natural variation exists in the available genetic repertoire of accessions to handle the combination of stresses. PMID:27208277

Crocin prevents retinal ischaemia/reperfusion injury-induced apoptosis in retinal ganglion cells through the PI3K/AKT signalling pathway.

PubMed

Qi, Yun; Chen, Li; Zhang, Lei; Liu, Wen-Bo; Chen, Xiao-Yan; Yang, Xin-Guang

2013-02-01

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) and has been reported to be useful in the treatment of neuronal damage. In the present study, we investigated the neuroprotective effect of crocin on retinal ganglion cells (RGCs) after retinal ischaemia/reperfusion (IR) injury, and our results show that crocin acts through the PI3K/AKT signalling pathway. Retinal IR injury was induced by raising the intraocular pressure of Sprague-Dawley rats to 110 mmHg for 60 min. The neuroprotective effect of crocin was determined by quantifying the surviving RGCs and apoptotic RGCs following IR injury by means of retrograde labelling and TUNEL staining, respectively. The phosphorylated AKT protein level was determined by western blot and immunohistochemical analysis. To determine the extent to which the PI3K/AKT pathway contributes to the neuroprotective effect of crocin, experiments were also performed using the PI3K inhibitor LY294002. Compared with the IR + vehicle group, crocin (50 mg/kg) treatment enhanced RGC survival by approximately 36% and decreased RGC apoptosis by 44% after retinal IR injury. Western blot and immunohistochemical analysis demonstrated that the PI3K/AKT pathway was activated by crocin in the ganglion cell layer after retinal IR injury. Intravitreal injection of LY294002 blocked the neuroprotective effect of crocin on IR-induced RGC death. In conclusion, crocin prevents retinal IR-induced apoptosis of RGCs by activating the PI3K/AKT signalling pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bioequivalence of a Liquid Formulation of Alpha1-Proteinase Inhibitor Compared with Prolastin®-C (Lyophilized Alpha1-PI) in Alpha1-Antitrypsin Deficiency.

PubMed

Barker, Alan F; Campos, Michael A; Brantly, Mark L; Stocks, James M; Sandhaus, Robert A; Lee, Douglas; Steinmann, Kimberly; Lin, Jiang; Sorrells, Susan

2017-12-01

This study evaluated the bioequivalence, safety, and immunogenicity of a new liquid formulation of human plasma-derived alpha 1 -proteinase inhibitor, Liquid Alpha 1 -PI, compared with the Lyophilized Alpha 1 -PI formulation (Prolastin®-C), for augmentation therapy in patients with alpha 1 -antitrypsin deficiency (AATD). In this double-blind, randomized, 20-week crossover study, 32 subjects with AATD were randomized to receive 8 weekly infusions of 60 mg/kg of Liquid Alpha 1 -PI or Lyophilized Alpha 1 -PI. Serial blood samples were drawn for 7 days after the last dose followed by 8 weeks of the alternative treatment. The primary endpoint was bioequivalence at steady state, as measured by area under the concentration versus time curve from 0 to 7 days (AUC 0-7 days ) postdose using an antigenic content assay. Bioequivalence was defined as 90% confidence interval (CI) for the ratio of the geometric least squares (LS) mean of AUC 0-7 days for both products within the limits of 0.80 and 1.25. Safety and immunogenicity were assessed. Mean alpha 1 -PI concentration versus time curves for both formulations were superimposable. Mean AUC 0-7 days was 20 320 versus 19 838 mg × h/dl for Liquid Alpha 1 -PI and Lyophilized Alpha 1 -PI, respectively. The LS mean ratio of AUC 0-7 days (90% CI) for Liquid Alpha 1 -PI versus Lyophilized Alpha 1 -PI was 1.05 (1.03-1.08), indicating bioequivalence. Liquid Alpha 1 -PI was well tolerated and adverse events were consistent with Lyophilized Alpha 1 -PI. Immunogenicity to either product was not detected. In conclusion, Liquid Alpha 1 -PI is bioequivalent to Lyophilized Alpha 1 -PI, with a similar safety profile. The liquid formulation would eliminate the need for reconstitution and shorten preparation time for patients receiving augmentation therapy for AATD.

Selective Toxicity of Apigenin on Cancerous Hepatocytes by Directly Targeting their Mitochondria.

PubMed

Seydi, Enayatollah; Rasekh, Hamid R; Salimi, Ahmad; Mohsenifar, Zhaleh; Pourahmad, Jalal

2016-01-01

hepatocellular carcinoma (HCC) is the third cause of mortality due to cancer throughout the world. The main goal of the current research was to evaluate the selective toxicity of apigenin (APG) on hepatocytes and mitochondria obtained from the liver of HCC rats). In this research, HCC induced by a single dose of diethylnitrosamine (DEN); 200 mg/kg, i.p, and 2-acetylaminofluorene (2-AAF) (0.02%, through dietary) for 14 days. For confirmation of HCC, histopathological evaluations and determination of serum concentrations of liver toxicity enzymes and specific liver cancer marker; alpha-fetoprotein (AFP) were performed. Then, cancerous and non- cancerous hepatocytes were isolated by using the collagen perfusion method. Eventually, mitochondria isolated from HCC and normal hepatocytes were tested for every eventual toxic effects of APG. After confirmation of HCC, the results of this research showed that APG (10, 20 and 40 μM) increased mitochondrial parameters such as, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) level, mitochondrial swelling and cytochrome c expulsion only in cancerous hepatocytes. Apoptotic effect of APG on HCC cells was confirmed by caspase-3 activation and Annexin V-FITC and PI double staining analysis. These results propose the eligibility of the flavonoid APG as a complementary therapeutic agent for patients with hepatocellular carcinoma.

[Silymarin Protects Umbilical Cord-Derived Mesenchymal Stem Cells against Apoptosis Induced by Serum-Deprivation].

PubMed

Wei, Xiao-Juan; Zhang, Hong-Chao; Guo, Zi-Kuan; Zheng, Hai-Bin; Yang, Lei-Lei; Liu, Chao-Zhong

2015-10-01

To investigate the protection of silymarin against the human mesenchymal stem cell (MSC) apoptosis induced by serum deprivation and its underlying mechanism. Human umbilical cord MSCs were cultured in the absence of serum, and the silymain of different concentration (1-10 µg/ml) was added into the medium. MTT test was performed to observe the cell proliferation status. After being cultured for 72 hours, the cells were collected, and flow cytometry with Annexin-V-PI double-staining was used to detect the apoptotic cells from the control and silymarin-treated groups. Furthermore, the intracellular contents of BAX and BCL-2 were detected by Western blot for exploring the potential mechanism. The silymarin promoted the proliferation of human UC-MSCs in a dose-dependent manner, reaching its maximal at a dose of 5 µg/ml. Moreover, silymarin could inhibit the serum deprivation-induced apoptosis of MSCs and, the inhibitory rate reached up to 30% when it was added at a concentration of 5 µg/ml. The content of intracellular BAX was obviously elevated after serum-deprivation treatment, and this increase could be blunted by the addition of silymarin. Meanwhile, the content of BCL-2 was not obviously changed. The silymarin can stimulate MSC growth and inhibit the apoptosis of MSCs probably by the mitochondria pathway.

The Neuroprotective Effects of SIRT1 on NMDA-Induced Excitotoxicity

PubMed Central

Si, Peipei; Qin, Huaping; Yin, Litian

2017-01-01

Silent information regulator 1 (SIRT1), an NAD+-dependent deacetylase, is involved in the regulation of gene transcription, energy metabolism, and cellular aging and has become an important therapeutic target across a range of diseases. Recent research has demonstrated that SIRT1 possesses neuroprotective effects; however, it is unknown whether it protects neurons from NMDA-mediated neurotoxicity. In the present study, by activation of SIRT1 using resveratrol (RSV) in cultured cortical neurons or by overexpression of SIRT1 in SH-SY5Y cell, we aimed to evaluate the roles of SIRT1 in NMDA-induced excitotoxicity. Our results showed that RSV or overexpression of SIRT1 elicited inhibitory effects on NMDA-induced excitotoxicity including a decrease in cell viability, an increase in lactate dehydrogenase (LDH) release, and a decrease in the number of living cells as measured by CCK-8 assay, LDH test, and Calcein-AM and PI double staining. RSV or overexpression of SIRT1 significantly improved SIRT1 deacetylase activity in the excitotoxicity model. Further study suggests that overexpression of SIRT1 partly suppressed an NMDA-induced increase in p53 acetylation. These results indicate that SIRT1 activation by either RSV or overexpression of SIRT1 can exert neuroprotective effects partly by inhibiting p53 acetylation in NMDA-induced neurotoxicity. PMID:29081884

A hypothesis of target cell formation in sickle cell disease.

PubMed

Wong, P

2016-08-01

A fraction of erythrocytes appear as target cells in stained blood smears in sickle cell disease, due to a inheritance of the hemoglobin variant Hb S, polymerizing upon deoxygenation. These cells appear in a three dimension as thin cups. A process of their formation in this disease is proposed based on a band 3-based mechanism of the erythrocyte shape control, able to explain the erythrocyte echinocytosis by glucose depletion. It indicates that their formation is due to a stomatocytogenic slow outward transport of the dibasic form of endogenous Pi with an H(+) by band 3, promoted by the decrease of the Donnan ratio, which decreases cell pH and volume, attributed by a decrease of cell KCl concentration by the higher efflux of K(+)Cl(-) cotransport and Ca(2+) activation of the Gardos channel. Its implications are briefly discussed with respect to target cells per se, target cell formation in other hemoglobinopathies, acquired and inherited disorders of the lipid metabolism and dehydrated hereditary stomatocytosis as well as a stomatocyte presence in a double heterozygote of Hb S and Hb C and of an involvement of the process of target cell formation in acanthocytosis in acquired and inherited disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

The metastasis suppressor SOX11 is an independent prognostic factor for improved survival in gastric cancer

PubMed Central

QU, YING; ZHOU, CHENFEI; ZHANG, JIANIAN; CAI, QU; LI, JIANFANG; DU, TAO; ZHU, ZHENGGANG; CUI, XIAOJIANG; LIU, BINGYA

2014-01-01

SOX11 is involved in gastrulation and in malignant diseases. The aim of this study was to investigate the role of SOX11 in gastric cancer and its expression pattern and clinical significance. SOX11 overexpression cell model was used to examine in vitro and in vivo the role of SOX11 in cell growth and metastasis. Cell cycle analysis and Annexin V/PI double staining were used to investigate the effect of SOX11 on cell cycle progression and apoptosis. The expression of SOX11 in human gastric cancer was examined by immunohistochemistry. The correlation of SOX11 expression with clinicopathological characteristics and survival of patients was analyzed by Pearson’s χ2 and Kaplan-Meier analyses, respectively. Cox’s proportional hazard model was employed in multivariate analysis. SOX11 overexpression did not inhibit cell growth but strongly suppressed cell migration/invasion in vitro and in vivo. We found a significant correlation between high SOX11 protein levels and Lauren’s classification (intestinal type), differentiation status (high and medium), and early TNM stage. SOX11 is an independent prognostic factor for improved survival in gastric cancer patients. SOX11 was a potential tumor-suppressor and an independent positive prognostic factor in gastric cancer patients with less advanced clinicopathological features. PMID:24604109

Exosomal DNMT1 mediates cisplatin resistance in ovarian cancer.

PubMed

Cao, Ya-Lei; Zhuang, Ting; Xing, Bao-Heng; Li, Na; Li, Qin

2017-08-01

Ovarian cancer is the most common malignancy in women. Owing to late syndromic presentation and lack of efficient early detection, most cases are diagnosed at advanced stages. Surgery and platinum-based chemotherapy are still the standard care currently. However, resistance invoked often compromises the clinical value of the latter. Expression of DNA methyltransferase 1 (DNMT1) was analysed by gene array. Protein was determined by immunoblotting. Exosome was isolated with commercial kit. Cell proliferation was measured by CCK8 method. Annexin V-PI double staining was performed for apoptosis evaluation. Xenograft model was established and administrated with exosome. Tumour growth and overall survival were monitored. We demonstrated the upregulation of DNMT1 in both tumour and derived cell line. DNMT1 transcripts were highly enriched in exosomes from conditioned medium of ovarian cells. Co-incubation with exosomes stimulated endogenous expression and rendered host cell the resistance to cytotoxicity of cisplatin. In vivo administration of DNMT1-containing exosomes exacerbated xenograft progression and reduced overall survival significantly. Moreover, treatment with exosome inhibitor GW4869 almost completely restored sensitivity in resistant cells. Our data elucidated an unappreciated mechanism of exosomal DNMT1 in cisplatin resistance in ovarian cancer, also indicating the potential of the combination of exosome inhibitor with cisplatin in resistant patients. Copyright © 2017 John Wiley & Sons, Ltd.

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

Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id; Department of Mathematic Faculty of MIPA Universitas Gadjah Mada; Kusumo, F. A.

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present amore » mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.« less

A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

NASA Astrophysics Data System (ADS)

Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

2016-04-01

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

Fourier-transform MW spectroscopy of the SH({sup 2}{Pi}{sub i})-Ar and SD-Ar radical complexes

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

Sumiyoshi, Yoshihiro; Endo, Yasuki; Ohshima, Yasuhiro

1996-12-31

The authors have studied the SH({sup 2}{Pi}{sub i})-Ar and SD-Ar radical complexes with FTMW spectroscopy. The complexes were produced in a supersonic free jet by a pulsed discharge of H{sub 2}S or D{sub 2}S, which was diluted to 0.35% in Ar with a stagnation pressure of 2 atm. R-branch transitions in the lower spin-orbit component ({Omega}=3/2) for the linear {sup 2}{Pi}{sub i} radicals were observed for J{double_prime} = 3/2 to J{double_prime} = 15/2 in the 8-26 GHz region. The transitions were split into two parity components owing to the parity doubling. Each parity component was split further due to themore » magnetic hyperfine interaction associated with the H/D nucleus. Rotational constants for SH-Ar and SD-Ar were determined to be 1569.656(2) and 1567.707(2)MHz respectively. The value for SH-Ar agrees well with that of a previous LIF study. From the SH/SD data, it was confirmed that the argon atom is located at the hydrogen side of the SH radical. With an assumption that the S-H bond length is equal to that in the monomer, the H-Ar distance is calculated to be 2.900 {Angstrom}, which is about 0.1 {Angstrom} longer than that in OH-Ar. The effective D{sub J} constants of SH-Ar and SD-Ar were found to have negative values of -58.4(7) and -50.7(6), kHz respectively.« less

Non-thermal Plasma Induces Apoptosis in Melanoma Cells via Production of Intracellular Reactive Oxygen Species

PubMed Central

Sensenig, Rachel; Kalghatgi, Sameer; Cerchar, Ekaterina; Fridman, Gregory; Shereshevsky, Alexey; Torabi, Behzad; Arjunan, Krishna Priya; Podolsky, Erica; Fridman, Alexander; Friedman, Gary; Azizkhan-Clifford, Jane; Brooks, Ari D.

2012-01-01

Non-thermal atmospheric pressure dielectric barrier discharge (DBD) plasma may provide a novel approach to treat malignancies via induction of apoptosis. The purpose of this study was to evaluate the potential of DBD plasma to induce apoptosis in melanoma cells. Melanoma cells were exposed to plasma at doses that did not induce necrosis, and cell viability and apoptotic activity were evaluated by Trypan blue exclusion test, Annexin-V/PI staining, caspase-3 cleavage, and TUNEL® analysis. Trypan blue staining revealed that non-thermal plasma treatment significantly decreased the viability of cells in a dose-dependent manner 3 and 24 h after plasma treatment. Annexin-V/PI staining revealed a significant increase in apoptosis in plasma-treated cells at 24, 48, and 72 h post-treatment (p<0.001). Caspase-3 cleavage was observed 48 h post-plasma treatment at a dose of 15 J/cm2. TUNEL® analysis of plasma-treated cells demonstrated an increase in apoptosis at 48 and 72 h post-treatment (p<0.001) at a dose of 15 J/cm2. Pre-treatment with N-acetyl-L-cysteine (NAC), an intracellular reactive oxygen species (ROS) scavenger, significantly decreased apoptosis in plasma-treated cells at 5 and 15 J/cm2. Plasma treatment induces apoptosis in melanoma cells through a pathway that appears to be dependent on production of intracellular ROS. DBD plasma production of intracellular ROS leads to dose-dependent DNA damage in melanoma cells, detected by γ-H2AX, which was completely abrogated by pre-treating cells with ROS scavenger, NAC. Plasma-induced DNA damage in turn may lead to the observed plasma-induced apoptosis. Since plasma is non-thermal, it may be used to selectively treat malignancies. PMID:21046465

[Development of an Enterococcus faecalis periapical biofilm model for in vitro morphological study].

PubMed

Cao, Ridan; Hou, Benxiang

2014-08-01

This study aims to develop and observe a model system of the periapical biofilm structure of Enterococcus faecalis (E. faecalis). A total of 24 intact human single-rooted premolars extracted for orthodontic reasons were collected and randomly divided into eight groups (n = 3). The specimens were subjected to ultraviolet disinfection, inoculated with E. faecalis (ATCC 29212) suspension adjusted to 1 x 10(8) CFU x mL(-1), and incubated at 37 degrees C for 1, 2, and 7 d. Specimen groups were prepared for scanning electron microscope to examine the biofilm formation. The specimens in the confocal laser scanning microscope (CLSM) groups were stained with propidium iodide (PI) and ConA-fluorescein isothiocyanate (ConA-FITC) to examine the biofilm formation. The images were randomized, and biofilm coverage (%) was assessed using Photoshop CS5. The biofilm coverage (%) on the cementum increased with increasing incubation period. The biofilm coverage of the 7 d group was significantly higher than those of the 1 and 2 d groups (P < 0.05). The values of the latter two groups were not significantly different (P > 0.05). Dense aggregations composed of E. faecalis and the amorphous matrix were observed on the root cementum surfaces of the specimens in the 7 d group. The bacteria were stained red by PI, and the matrix was stained green by ConA-FITC under CLSM observation. The biofilm coverage (%) on the samples in the 7 d group was 17.23% +/- 1.52%, showing multi-level space structure and water channels. E. faecalis forms bacterial biofilms on the root cementum surface in 7 d. The biofilms were composed of E. faecalis and the amorphous matrix.

Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds.

PubMed

Lema, Carolina; Varela-Ramirez, Armando; Aguilera, Renato J

As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z' factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC 50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity.

Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds

PubMed Central

LEMA, Carolina; VARELA-RAMIREZ, Armando; AGUILERA, Renato J.

2016-01-01

As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z′ factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity. PMID:27042697

Hepatocellular apoptosis associated with cytotoxic T/natural killer-cell infiltration in chronic active EBV infection.

PubMed

Nomura, Yuko; Kimura, Hiroshi; Karube, Kennosuke; Yoshida, Shiro; Sugita, Yasuo; Niino, Daisuke; Shimizu, Kei; Kimura, Yoshizo; Aoki, Ryosuke; Kiyasu, Junichi; Takeuchi, Masanori; Hashikawa, Keiko; Hirose, Shinichi; Ohshima, Koichi

2009-07-01

The aim of the present study was to identify the mechanism of hepatocellular apoptosis induced by EBV-infected cytotoxic T/natural killer (NK) cells in chronic active EBV infection (CAEBV). Eight patients with CAEBV were studied, and infected T-cell expansion and NK-cell expansion were detected in four patients each. Biopsy or necropsy was performed on lymph node, liver, or spleen, and each specimen was subjected to immunohistochemical double staining of CD3 plus caspase-3 with the addition of cytotoxic markers of T-cell restricted intracellular antigen-1 (TIA-1), perforin, and granzyme B, as well as EBV in situ hybridization (EBV-ISH). In the liver, some of the infiltrating CD3-positive lymphocytes stained positively for EBV-ISH and cytotoxic markers. Double staining of CD3 plus caspase-3 indicated caspase-3 positive hepatocytes with apoptotic features, accompanied by extensive infiltration of CD3-positive cells, which were directly attached to the apoptotic caspase-3 positive hepatocytes. In contrast, far fewer cells stained positive for caspase-3 in lymph node and spleen than in liver. The present findings suggest that in patients with CAEBV, cytotoxic T/NK cells may directly induce hepatocytes to undergo apoptosis more frequently than they do cells in other organs of the reticulo-endothelial system.

TLR2 ligands induce cardioprotection against ischaemia/reperfusion injury through a PI3K/Akt-dependent mechanism

PubMed Central

Ha, Tuanzhu; Hu, Yulong; Liu, Li; Lu, Chen; McMullen, Julie R.; Kelley, Jim; Kao, Race L.; Williams, David L.; Gao, Xiang; Li, Chuanfu

2010-01-01

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN- or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism. PMID:20421349

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

PubMed Central

Yuen, Hiu-Fung; Chan, Ka-Kui; Grills, Claire; Murray, James T.; Platt-Higgins, Angela; Eldin, Osama Sharaf; O’Byrne, Ken; Janne, Pasi; Fennell, Dean A.; Johnston, Patrick G.; Rudland, Philip S.; El-Tanani, Mohamed

2011-01-01

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown compared to normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK and PI3K/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry (PI and Annexin V staining) and MTT assay in cancer cells grown under different conditions after knockdown of Ran.. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. KRas mutated, c-Met amplified and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of KRas or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. PMID:22090358

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus

PubMed Central

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-01-01

Abstract DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure. PMID:29800455

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

PubMed

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-05-01

DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

Possible involvement of long chain fatty acids in the spores of Ganoderma lucidum (Reishi Houshi) to its anti-tumor activity.

PubMed

Fukuzawa, Masataka; Yamaguchi, Rie; Hide, Izumi; Chen, Zhiqing; Hirai, Yuko; Sugimoto, Akiko; Yasuhara, Tadashi; Nakata, Yoshihiro

2008-10-01

During our isolation of biologically active substances from the spores of Ganoderma lucidum (Reishi Houshi, G. lucidum) guided by the inhibitory activity on HL-60 cell proliferation, NMR spectroscopic and mass spectrometric data indicate the substance contains a mixture of several long chain fatty acids. Hence, in this study, we have examined the inhibitory effects of an ethanolic extract of the spores of G. lucidum as the spore extract, on the proliferation of various human cancer cell lines by comparison with several authentic long chain fatty acids. Of the fatty acids we examined nonadecanoic acid (C19:0) showed the highest inhibitory activity for HL-60 cell proliferation with IC(50) values of 68+/-7 microM followed by heptadecanoic acid (C17:0, 120+/-23 microM), octa- (C18:0, 127+/-4 microM) and hexadecanoic acids (C16:0, 132+/-25 microM), respectively. The corresponding unsaturated fatty acids containing one double bond such as cis-10-nonadecenoic acid (C19:1), cis-9-octadecenoic acid (C18:1), cis-10-heptadecenoic acid (C17:1) and cis-9-hexadecenoic acid (C16:1) were less effective. The ethanolic extract of spores of G. lucidum were shown by annexin-V FITC/PI double staining to induce apoptosis of HL-60 cells in a similar way to cis-10-nonadecenoic acid (C19:1). These unsaturated fatty acids probably inhibit tumor necrosis factor production induced by lipopolysaccharide in a mouse macrophage preparation. Our results suggest the spores of G. lucidum contain 19-carbon fatty acids as one of the components for characteristics of its physiological effects.

EMP-1 promotes tumorigenesis of NSCLC through PI3K/AKT pathway.

PubMed

Lai, Senyan; Wang, Guihua; Cao, Xiaonian; Li, Zhaoming; Hu, Junbo; Wang, Jing

2012-12-01

This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and immunohistochemically detected to evaluate the correlation of EMP-1 expression to the clinical features of NSCLC. Recombinant adenovirus was constructed to over-express EMP-1 and then infect PC9 cells. Cell proliferation was measured by Ki67 staining. Western blotting was performed to examine the effect of EMP-1 on the PI3K/AKT signaling. Moreover, tumor xenografts were established by subcutaneous injection of PC9 cell suspension (about 5×10(7)/mL in 100 μL of PBS) into the right hind limbs of athymic nude mice. The results showed EMP-1 was significantly up-regulated in NSCLC patients as compared with those with benign lung diseases. Over-expression of EMP-1 promoted proliferation of PC9 cells, which coincided with the activation of the PI3K/AKT pathway. EMP-1 promoted the growth of xenografts of PC9 cells in athymic nude mice. It was concluded that EMP-1 expression may contribute to the development and progress of NSCLC by activating PI3K/AKT pathway.

Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

PubMed

Novak, K D; Peterson, M D; Reedy, M C; Titus, M A

1995-12-01

The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems.

PubMed

Duedu, Kwabena O; French, Christopher E

2017-04-01

Monitoring bacterial growth is an important technique required for many applications such as testing bacteria against compounds (e.g. drugs), evaluating bacterial composition in the environment (e.g. sewage and wastewater or food suspensions) and testing engineered bacteria for various functions (e.g. cellulose degradation). T?=1,^FigItem(1) ^ReloadFigure=Yesraditionally, rapid estimation of bacterial growth is performed using spectrophotometric measurement at 600nm (OD600) but this estimation does not differentiate live and dead cells or other debris. Colony counting enumerates live cells but the process is laborious and not suitable for large numbers of samples. Enumeration of live bacteria by flow cytometry is a more suitable rapid method with the use of dual staining with SYBR I Green nucleic acid gel stain and Propidium Iodide (SYBR-I/PI). Flow cytometry equipment and maintenance costs however are relatively high and this technique is unavailable in many laboratories that may require a rapid method for evaluating bacteria growth. We therefore sought to adapt and evaluate the SYBR-I/PI technique of enumerating live bacterial cells for a cheaper platform, a fluorimeter. The fluorimetry adapted SYBR-I/PI enumeration of bacteria in turbid growth media had direct correlations with OD600 (p>0.001). To enable comparison of fluorescence results across labs and instruments, a fluorescence intensity standard unit, the equivalent fluorescent DNA (EFD) was proposed, evaluated and found useful. The technique was further evaluated for its usefulness in enumerating bacteria in turbid media containing insoluble particles. Reproducible results were obtained which OD600 could not give. An alternative method based on the assessment of total protein using the Pierce Coomassie Plus (Bradford) Assay was also evaluated and compared. In all, the SYBR-I/PI method was found to be the quickest and most reliable. The protocol is potentially useful for high-throughput applications such as monitoring of growth of live bacterial cells in 96-well microplates and in assessing in vivo activity of cellulose degrading enzyme systems. Copyright © 2017 Elsevier B.V. All rights reserved.

The inhibitory mechanism of Cordyceps sinensis on cigarette smoke extract-induced senescence in human bronchial epithelial cells.

PubMed

Liu, Ailing; Wu, Jinxiang; Li, Aijun; Bi, Wenxiang; Liu, Tian; Cao, Liuzhao; Liu, Yahui; Dong, Liang

2016-01-01

Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or stress. The bronchial epithelial cell is often injured by inhaled toxic substances, such as cigarette smoke. In the present study, we investigated whether exposure to cigarette smoke extract (CSE) induces senescence of bronchial epithelial cells; and Cordyceps sinensis mechanism of inhibition of CSE-induced cellular senescence. Human bronchial epithelial cells (16HBE cells) cultured in vitro were treated with CSE and/or C. sinensis. p16, p21, and senescence-associated-galactosidase activity were used to detect cellular senescence with immunofluorescence, quantitative polymerase chain reaction, and Western blotting. Reactive oxygen species (ROS), PI3K/AKT/mTOR and their phosphorylated proteins were examined to testify the activation of signaling pathway by ROS fluorescent staining and Western blotting. Then, inhibitors of ROS and PI3K were used to further confirm the function of this pathway. Cellular senescence was upregulated by CSE treatment, and C. sinensis can decrease CSE-induced cellular senescence. Activation of ROS/PI3K/AKT/mTOR signaling pathway was enhanced by CSE treatment, and decreased when C. sinensis was added. Blocking ROS/PI3K/AKT/mTOR signaling pathway can attenuate CSE-induced cellular senescence. CSE can induce cellular senescence in human bronchial epithelial cells, and ROS/PI3K/AKT/mTOR signaling pathway may play an important role in this process. C. sinensis can inhibit the CSE-induced senescence.

Molecular alterations of EGFR and PIK3CA in uterine serous carcinoma.

PubMed

Hayes, Monica Prasad; Douglas, Wayne; Ellenson, Lora Hedrick

2009-06-01

Uterine serous carcinoma (USC) is an aggressive endometrial cancer associated with poor prognosis despite comprehensive surgical staging and adjuvant chemotherapy and radiation therapy. Biologic targets have yet to be fully explored in this disease and research on such targets could lead to clinical trials utilizing a new class of therapeutics. This study sought to evaluate primary USC tumors for molecular alterations in epidermal growth factor receptor (EGFR) and the recently characterized oncogene PIK3CA, which encodes the catalytic p110-alpha subunit of phosphatidylinositol 3-kinase (PI3K) and thus activates the AKT-mTOR oncogenic pathway. Paraffin-embedded archival tissue of 45 primary USC tumors was utilized in this study. Immunohistochemical analysis of EGFR was performed and cases given a score of 0 to 12 calculated as the product of staining intensity (0 to 3+) and the percentage of positively stained cells (0-4), with 1=1-25%, 2=26-50%, 3=51-75%, and 4=76-100%. For mutational analysis, neoplastic tissue was microdissected and DNA was extracted with phenol-chloroform. Exons 18 through 21 of EGFR and exons 9 and 20 of PIK3CA, the most commonly mutated exons of these genes, were amplified and directly sequenced. When EGFR was evaluated, moderate or strong EGFR membranous staining was observed in 25/45 (56%) USC cases. Thus, a mutational analysis was performed on 35 cases, including all cases with moderate and strong EGFR staining. No mutations were identified in EGFR. In contrast, PIK3CA mutations were confirmed in 5/34 (15%) of USC cases. Four cases were mutated in exon 20 and one case was mutated in exon 9. Since optimal treatment of uterine serous carcinoma remains unknown, novel therapeutic approaches need to be actively pursued. In the current study of primary USC tumors, oncogenic mutations of the PIK3CA gene were seen in 15% of USC cases. This represents the first report of this gene mutation in USC. In addition, EGFR stained positively in the majority of cases, suggesting a possible target protein. These findings warrant further investigation and suggest a potential role for therapeutic agents targeting the PI3K-AKT-mTOR pathway, such as rapamycin, as well as possible targets of EGFR in the treatment of uterine serous carcinoma.

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.

Identification of Psilocybe cubensis spore allergens by immunoprinting.

PubMed

Helbling, A; Horner, W E; Lehrer, S B

1993-01-01

Previous studies established that Psilocybe cubensis contains potent allergens, and that a significant percentage of atopic subjects were sensitized to P. cubensis spores. The objective of this study was to identify P. cubensis spore allergens using isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoprinting. Coomassie blue staining of IEF gels detected approximately 20 bands between pI 3.6 and 9.3. Immunoprints obtained with 15 P. cubensis skin test- and RAST-positive sera revealed 13 IgE-binding bands; the most reactive were at pI 5.0 (80%), 5.6 (87%), 8.7 (80%) and 9.3 (100%). SDS-PAGE resolved 27 proteins ranging from about 13 to 112 kD. SDS-PAGE immunoprints conducted with 11 skin test- and RAST-positive sera demonstrated 18 IgE-binding bands; most sera reacted to 16 (82%), 35 (100%) and 76 kD (91%) allergens. Both electrophoretic procedures demonstrated a single allergen (at pI 9.3 and 35 kD) that reacted with all sera tested. This study corroborates the allergenic significance of P. cubensis spores and identifies the allergens of greatest importance.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937

Dissociative and double photoionization cross sections of NO from threshold to 120 A

NASA Technical Reports Server (NTRS)

Samson, J. A. R.; Masuoka, T.; Pareek, P. N.

1985-01-01

The partial photoionization cross sections for producing the NO(+) parent ion and the O(+), N(+), and NO(2+) fragmentations from neutral NO are presented from 120 to 614 A. The results indicate predissociation of the 3 pi (21.72 eV) and B-prime 1Sigma(+) (22.73 eV) electronic states of NO(+). The photoionization threshold for double ionization was found to be 39.4 + or - 0.12 eV.

Emergence of late-onset placental dysfunction: relationship to the change in uterine artery blood flow resistance between the first and third trimesters.

PubMed

Llurba, Elisa; Turan, Ozhan; Kasdaglis, Tania; Harman, Chris R; Baschat, Ahmet A

2013-06-01

To test if emergence of third-trimester (T3) placental dysfunction is related to the impedance change in uterine artery blood flow resistance between the first trimester (T1) and T3. Mean T1 and T3 uterine artery (mUtA) pulsatility index (PI) was measured in 1098 singletons. Each patient's individual mUtA-PI change was calculated ([(T3 PI - T1 PI/interval in days)] × 100; ΔmUtA-PI). This parameter and T1 and T3 mUtA-PI z-scores were related to placenta-related disease (PRD) and to constitutionally small neonates (CS). Forty-seven (5%) women had PRD and 83 (8.7%) delivered a CS neonate. T1 and T3 mUtA-PI z-scores were higher with PRD (0.418 versus -0.097 and 1.06 versus -0.13, p < 0.001 for all). Change in mUtA-PI (ΔmUtA PI) was similar for patients with PRD. However, the prevalence of PRD doubled with rising ΔmUtA-PI (11.1% versus 5.2%, p = 0.041). T3 uterine artery Doppler performs significantly better in detecting patients at risk for late-onset PRD than T1 or the gestational age change in uterine artery Doppler resistance This suggests that a proportion of late emerging PRD is not amenable to early screening by uterine artery Doppler. Further research is essential to identify the optimal screening strategy for late-onset placental dysfunction. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Retrospective, Demographic, and Clinical Investigation of the Causes of Postoperative Infection in Patients With Lumbar Spinal Stenosis Who Underwent Posterior Stabilization

PubMed Central

Yaldiz, Can; Yaldiz, Mahizer; Ceylan, Nehir; Kacira, Ozlem Kitiki; Ceylan, Davut; Kacira, Tibet; Kizilcay, Gokhan; Tanriverdi, Taner

2015-01-01

Abstract Owing to the increasing population of elderly patients, a large number of patients with degenerative spondylosis are currently being surgically treated. Although basic measures for decreasing postoperative surgical infections (PSIs) are considered, it still remains among the leading causes of morbidity and mortality. The aim of this retrospective analysis is to present possible causes leading to PSI in patients who underwent surgery for lumbar degenerative spondylosis and highlight how it can be avoided to decrease morbidity and mortality. The study included 540 patients who underwent posterior stabilization due to degenerative lumbar stenosis between January 2013 and January 2014. The data before and after surgery was retrieved from the hospital charts. Patients with degenerative lumbar stenosis who were operated upon in this study had >2 levels of laminectomy and facetectomy. For this reason, posterior stabilization was performed for all the patients included in this study. Determining the causes of postoperative infection (PI) following spinal surgeries performed with instrumentation is a struggle. Seventeen different parameters that may be related to PI were evaluated in this study. The presence of systemic diseases, unknown glove perforations, and perioperative blood transfusions were among the parameters that increased the prevalence of PI. Alternatively, prolene sutures, double-layered gloves, and the use of rifampicin Sv (RIS) decreased the incidence of PI. Although the presence of systemic diseases, unnoticed glove perforations, and perioperative blood transfusions increased PIs, prolene suture material, double-layered gloves, and the use of RIS decreased PIs. PMID:26200620

Assessing the response to opioids in cancer patients: a methodological proposal and the results.

PubMed

Corli, O; Roberto, A; Greco, M T; Montanari, M

2015-07-01

The efficacy of treatment with opioids in cancer pain is variable. To evaluate this variability, we (1) applied two parameters, changes in pain intensity (PI) and opioid daily doses (DDs), to distinguish different responses to opioids. The need to switch to another opioid was recorded. We then (2) evaluated the distribution of the responses depending on these parameters, alone and taken together, in cancer patients with pain. The cutoffs between positive and negative responses related to PI and DD were defined on the basis of the literature. For PI, responders were patients who obtained simultaneously a decrease of 30% or more and a final score ≤4 points (numerical rating scale 0 to 10). For DD changes, we applied the opioid escalation index percentage, a positive response corresponding to a dose increase ≤5%. These criteria were applied to 201 cancer patients treated with WHO step III "strong" opioids for 21 days. The results were mainly analyzed case by case. Of the patients, 63.7% obtained a positive analgesic response and 80.1% a dose-related positive response. Combining the parameters, the response was double positive in 55.2% of cases, double negative in 11.4%, a good analgesic response with a large dose escalation in 8.5%, and no pain relief with a stable dose in 24.9%. Switches were made 21 times, 15 because of the lack of analgesia. Different degrees of response to opioids were observed, PI and DD changes both contributing. Only over half the patients had a full positive response.

Genes and Structural Proteins of the Phage Syn5 of the Marine Cyanobacteria Synechococcus

DTIC Science & Technology

2005-09-01

typhimurium phage P22, a podoviridae, was shown to possess extensive genomic similarity to coliphage lambda, a siphoviridae (Botstein and Herskowitz...are found among cyanobacteria in the surface waters during the winter. Temperature influences the number of infectious particles produced during lytic...grids and stained with 1% uranyl acetate for 15 minutes, washed three times in double-distilled water , stained in 1% lead citrate for 4 minutes, and

Long-term in vivo left ventricular assist device study with a titanium centrifugal pump.

PubMed

Ohtsuka, G; Nakata, K; Yoshikawa, M; Mueller, J; Takano, T; Yamane, S; Gronau, N; Glueck, J; Takami, Y; Sueoka, A; Letsou, G; Schima, H; Schmallegger, H; Wolner, E; Koyanagi, H; Fujisawa, A; Baldwin, J C; Nosé, Y

1998-01-01

A totally implantable centrifugal artificial heart has been developed. The plastic prototype, Gyro PI 601, passed 2 day hemodynamic tests as a functional total artificial heart, 2 week screening tests for antithrombogenicity, and 1 month system feasibility. Based on these results, a metallic prototype, Gyro PI 702, was subjected to in vivo left ventricular assist device (LVAD) studies. The pump system employed the Gyro PI 702, which has the same inner dimensions and the same characteristics as the Gyro PI 601, including an eccentric inlet port, a double pivot bearing system, and a magnet coupling system. The PI 702 is driven with the Vienna DC brushless motor actuator. For the in vivo LVAD study, the pump actuator package was implanted in the preperitoneal space in two calves, from the left ventricular apex to the descending aorta. Case 1 achieved greater than 9 month survival without any complications, at an average flow rate of 6.6 L/min with 10.2 W input power. Case 2 was killed early due to the excessive growth of the calf, which caused functional obstruction of the inlet port. There was no blood clot inside the pump. During these periods, neither case exhibited any physiologic abnormalities. The PI 702 pump gives excellent results as a long-term implantable LVAD.

Double-masked, placebo-controlled safety and efficacy trial of diquafosol tetrasodium (INS365) ophthalmic solution for the treatment of dry eye.

PubMed

Tauber, J; Davitt, W F; Bokosky, J E; Nichols, K K; Yerxa, B R; Schaberg, A E; LaVange, L M; Mills-Wilson, M C; Kellerman, D J

2004-11-01

To investigate the safety and efficacy of diquafosol tetrasodium, a P2Y2 receptor agonist that stimulates fluid and mucin secretion on the ocular surface, as a novel topical treatment of dry eye disease. Subjects with dry eye (n=527) were evaluated in a randomized, double-masked, parallel-group trial comparing 24 weeks of treatment with 2 concentrations of diquafosol (1% and 2%) versus placebo instilled 4 times daily. Corneal staining, conjunctival staining, Schirmer tests, and subjective symptoms of dry eye were evaluated. Use of artificial tears was permitted as necessary. Subjects treated with 2% diquafosol had significantly lower corneal staining scores compared with placebo at the 6-week, primary efficacy time point (P<0.001), and superiority continued throughout the 24-week study. Reductions in corneal staining were observed as early as after 2 weeks of treatment, were maintained throughout the 24-week study, and were observed to worsen slightly (toward baseline) when diquafosol treatment was discontinued (week 25). Results for conjunctival staining were consistent with those observed for corneal staining. Schirmer scores at week 6 were significantly higher with diquafosol treatment than with placebo (P

NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

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

Cowan, Catherine M.; Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095; Zhang, Xinli

2012-06-08

Highlights: Black-Right-Pointing-Pointer NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. Black-Right-Pointing-Pointer NELL-1 significantly increases intracellular inorganic phosphate levels. Black-Right-Pointing-Pointer NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 ormore » rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.« less

Gene transfer of inducible nitric oxide synthase complementary DNA regresses the fibrotic plaque in an animal model of Peyronie's disease.

PubMed

Davila, Hugo H; Magee, Thomas R; Vernet, Dolores; Rajfer, Jacob; Gonzalez-Cadavid, Nestor F

2004-11-01

The goal of the present study was to investigate the antifibrotic role of inducible nitric oxide synthase (iNOS) in Peyronie's disease (PD) by determining whether a plasmid expressing iNOS (piNOS) injected into a PD-like plaque can induce regression of the plaque. A PD-like plaque was induced with fibrin in the penile tunica albuginea of mice and then injected with a luciferase-expressing plasmid (pLuc), either alone or with piNOS, following luciferase expression in vivo by bioluminescence imaging. Rats were treated with either piNOS, an empty control plasmid (pC), or saline. Other groups were treated with pC or piNOS, in the absence of fibrin. Tissue sections were stained for collagen, transforming growth factor (TGF) beta1, and plasminogen-activator inhibitor (PAI-1) as profibrotic factors; copper-zinc superoxide dismutase (CuZn SOD) as scavenger of reactive oxygen species (ROS); and nitrotyrosine to detect nitric oxide reaction with ROS. Quantitative image analysis was applied. Both iNOS and xanthine oxido-reductase (XOR; oxidative stress) were estimated by Western blot analysis. Luciferase reporter expression was restricted to the penis, peaked at 3 days after injection, but continued for at least 3 wk. In rats receiving piNOS, iNOS expression also peaked at 3 days, but expression decreased at the end of treatment, when a considerable reduction of plaque size occurred. Protein nitrotyrosine, XOR, and CuZn SOD increased, and TGFbeta1 and PAI-1 decreased. The piNOS gene transfer regressed the PD plaque and expression of profibrotic factors, supporting the view that endogenous iNOS induction in PD is defense mechanism by the tissue against fibrosis.

The role of necroptosis in status epilepticus-induced brain injury in juvenile rats.

PubMed

Cai, Qianyun; Gan, Jing; Luo, Rong; Qu, Yi; Li, Shiping; Wan, Chaomin; Mu, Dezhi

2017-10-01

To study the role of necroptosis in status epilepticus (SE)-induced injury in the developing brain and the possible associations of necroptosis with epileptogenesis and cognitive dysfunction. The lithium-pilocarpine epilepsy model was reproduced in male rats at postnatal day 25. Propidium iodide (PI) staining was used to detect cell death after SE. Transmission electron microscopy (TEM) was performed to observe morphological changes in injured neurons. Western blot and immunofluorescence (IF) staining were used to investigate the expression of receptor interacting protein kinase-3 (RIP3), mixed lineage kinase domain-like (MLKL), and p-MLKL after SE. EEG was monitored during the chronic epileptic period. The Morris water maze test was performed to evaluate spatial learning and memory in juvenile rats after SE. Massive PI-positive (PI + ) neurocytes were observed mainly in the amygdala and piriform cortex 24h to 7days after SE, with the most prominent changes observed after 72h. Injured neurons observed via TEM exhibited necroptotic morphological features, including loss of ribosomes, autophagosome formations, deformed nuclei with condensed and marginated chromatin, and disruptive cell membranes. The expression of RIP3 and p-MLKL increased after 24h, peaked at 72h, and decreased 7days after SE. In addition, IF staining revealed that MLKL was expressed in cell plasma membranes present in the amygdala and piriform cortex. This finding was concomitant with the fact that MLKL is involved in executing necroptosis by binding and disrupting the plasma membrane. During the chronic epileptic period, spontaneous recurrent seizures were observed behaviorally and interictal spikes and sharp waves were recorded by EEG in the SE group. The Morris water maze test revealed that in the place navigation test, the escape latency of the SE group was longer than that of the control group (p<0.05). In the spatial probe test, the number of times the rats in the SE group passed through the original platform site was lesser than that of the rats in the control group (p<0.05). SE-induced brain injury leads to neuronal necroptosis in juvenile rats. MLKL may play a significant role in the execution of SE-induced necroptosis. Further studies are required to determine whether inhibiting necroptosis can prevent chronic epileptogenesis and improve cognitive ability for juvenile rats. Copyright © 2017 Elsevier Inc. All rights reserved.

A comparative clinical study evaluating stain removal efficacy of a new sensitivity whitening dentifrice compared to commercially available whitening dentifrices.

PubMed

Hughes, Nathan; Maggio, Brenda; Sufi, Farzana; Mason, Stephen; Kleber, Carl J

2009-01-01

To assess the extrinsic stain removal efficacy of a new sensitivity dentifrice containing sodium tripolyphosphate (STP) to marketed whitening toothpastes after six weeks of product use. This was a single-center, double-blind, stratified, six-week clinical study comparing the reduction in stain area and intensity of Sensodyne Extra Whitening to Crest Maximum Strength Sensitivity Protection Whitening plus Scope and Colgate Tartar Control Plus Whitening Mint dentifrice, as measured by MacPherson's Modification of the Lobene Stain Index (MMLSI) in a forced stain model. Two-hundred and ninety-five subjects completed the study. Tooth stain MMSLI scores showed significant differences between Sensodyne and Crest dentifrices in favor of Sensodyne for all surface sites (p = 0.014), and individually for facial (p = 0.023), lingual (p = 0.027), and interproximal (p = 0.014) surfaces. No significant statistical differences between Sensodyne and Colgate dentifrices were observed for any of the surfaces. Results from this stain removal clinical study demonstrate significant extrinsic stain removal efficacy for all dentifrices relative to baseline. Significant differences between the two marketed sensitivity whitening dentifrices were demonstrated in favor of the new Sensodyne Sensitivity Whitening dentifrice.

Immunomodulatory activity of interleukin-27 in human chronic periapical diseases.

PubMed

Li, Juan; Wang, Rong; Huang, Shi-Guang

2017-01-01

This study aims to observe expression of IL-27 on different cells in periapical tissues of different types of human chronic periapical diseases. Periapical tissue specimens of 60 donors, including healthy control (n=20), periapical granuloma group (n=20) and radicular cysts group (n=20), were fixed in 10% buffered formalin, stained with hematoxylin and eosin for histopathology. Then specimens were stained with double- immuno-fluorescence assay for identification of IL-27-tryptase (mast cells, MCs), IL-27-CD14 (mononuclear phagocyte cells, MPs) and IL-27-CD31 (endothelial cells, ECs) double-positive cells in periapical tissues. The results indicated that compared with healthy control, the densities (cells/mm 2 ) of IL-27-tryptase, IL-27-CD14 and IL-27-CD31 double-positive cells were significantly increased in human chronic periapical diseases (periapical granuloma group and radicular cysts group) ( P <0.001). The density of IL-27-tryptase double positive cells in radicular cysts group was significantly higher than those in periapical granuloma group ( P <0.001). Densities of IL-27-CD14 and IL-27-CD31 double-positive cells in periapical granuloma group had no significant difference with those in radicular cysts group ( P =0.170 and 0.138, respectively). IL-27-CD14 double positive cells density achieved to peak among three cell groups in radicular cysts groups. In conclusion, IL-27 expressed in MCs, MPs and ECs of human chronic periapical diseases with different degrees. IL-27-tryptase double-positive cells may participate in pathogenic mechanism of chronic periapical diseases, especially for formation of fibrous in periapical cysts. IL-27-CD14 and IL-27-CD31 double-positive cells may participate in immunologic response to resist periapical infection, and they may play an dual role in pathogenesis and localization of periapical diseases.

Protective Effect of 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione, Isolated from Averrhoa Carambola L., Against Palmitic Acid-Induced Inflammation and Apoptosis in Min6 Cells by Inhibiting the TLR4-MyD88-NF-κB Signaling Pathway.

PubMed

Xie, Qiuqiao; Zhang, Shijun; Chen, Chunxia; Li, Juman; Wei, Xiaojie; Xu, Xiaohui; Xuan, Feifei; Chen, Ning; Pham, Thithaihoa; Qin, Ni; He, Junhui; Ye, Fangxing; Huang, Wansu; Huang, Renbin; Wen, Qingwei

2016-01-01

Studies have demonstrated that 2-dodecyl-6-methoxycyclohexa-2, 5-diene-1, 4-dione (DMDD), isolated from the roots of Averrhoa carambola L., has significant therapeutic potential for the treatment of diabetes. However, the protective effect of DMDD against pancreatic beta cell dysfunction has never been reported. We investigated whether DMDD protected against palmitic acid-induced dysfunction in pancreatic β-cell line Min6 cells by attenuating the inflammatory response and apoptosis and to shed light on its possible mechanism. Cell viability was assessed by CCK-8. Glucose-stimulated insulin secretion levels and inflammatory cytokines levels were examined by ELISA. Apoptosis was assessed by Annexin V-FITC/PI Flow cytometry assay, Hoechst 33342/PI double-staining assay, and Transmission electron microscopy assay. Relative quantitative real-time PCR and western blot were used to determine the expressions of genes and proteins. Cell viability and glucose-stimulated insulin secretion levels were increased in DMDD-pretreated Min6 cells. DMDD inhibited inflammatory cytokines IL-6, TNF-α and MCP-1 generations in palmitic acid (PA)-induced Min6 cells. Moreover, DMDD protected against PA-induced Min6 cells apoptosis and the expression of Cleaved-Caspase-3, -8 and -9 were down-regulated and the Bcl-2/Bax ratio was increased in DMDD-pretreated Min6 cells. In addition, the expression of TLR4, MyD88 and NF-κB were down-regulated in DMDD-pretreated Min6 cells and TAK-242-pretreated group cells. DMDD protected Min6 cells against PA-induced dysfunction by attenuating the inflammatory response and apoptosis, and its mechanism of this protection was associated with inhibiting the TLR4-MyD88-NF-κB signaling pathway. © 2016 The Author(s) Published by S. Karger AG, Basel.

NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.

PubMed

Wree, Alexander; Eguchi, Akiko; McGeough, Matthew D; Pena, Carla A; Johnson, Casey D; Canbay, Ali; Hoffman, Hal M; Feldstein, Ariel E

2014-03-01

Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry-based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death. Our data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage. © 2014 by the American Association for the Study of Liver Diseases.

Cooperation between both Wnt/β-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion

PubMed Central

Perry, John M.; He, Xi C.; Sugimura, Ryohichi; Grindley, Justin C.; Haug, Jeffrey S.; Ding, Sheng; Li, Linheng

2011-01-01

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and β-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of β-catenin alone results in apoptosis of HSCs. However, together, the Wnt/β-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, β-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/β-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity. PMID:21890648

Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging

PubMed Central

Ohnishi, Mutsuko; Razzaque, M. Shawkat

2010-01-01

Identifying factors that accelerate the aging process can provide important therapeutic targets for slowing down this process. Misregulation of phosphate homeostasis has been noted in various skeletal, cardiac, and renal diseases, but the exact role of phosphate toxicity in mammalian aging is not clearly defined. Phosphate is widely distributed in the body and is involved in cell signaling, energy metabolism, nucleic acid synthesis, and the maintenance of acid-base balance by urinary buffering. In this study, we used an in vivo genetic approach to determine the role of phosphate toxicity in mammalian aging. Klotho-knockout mice (klotho−/−) have a short life span and show numerous physical, biochemical, and morphological features consistent with premature aging, including kyphosis, uncoordinated movement, hypogonadism, infertility, severe skeletal muscle wasting, emphysema, and osteopenia, as well as generalized atrophy of the skin, intestine, thymus, and spleen. Molecular and biochemical analyses suggest that increased renal activity of sodium-phosphate cotransporters (NaPi2a) leads to severe hyperphosphatemia in klotho−/− mice. Genetically reducing serum phosphate levels in klotho−/− mice by generating a NaPi2a and klotho double-knockout (NaPi2a−/−/klotho−/−) strain resulted in amelioration of premature aging-like features. The NaPi2a−/−/klotho−/− double-knockout mice regained reproductive ability, recovered their body weight, reduced their organ atrophy, and suppressed ectopic calcifications, with the resulting effect being prolonged survival. More important, when hyperphosphatemia was induced in NaPi2a−/−/klotho−/− mice by feeding with a high-phosphate diet, premature aging-like features reappeared, clearly suggesting that phosphate toxicity is the main cause of premature aging in klotho−/− mice. The results of our dietary and genetic manipulation studies provide in vivo evidence for phosphate toxicity accelerating the aging process and suggest a novel role for phosphate in mammalian aging.—Ohnishi, M., Razzaque, M. S. Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging. PMID:20418498

Efficacy and safety of emtricitabine/tenofovir alafenamide (FTC/TAF) vs. emtricitabine/tenofovir disoproxil fumarate (FTC/TDF) as a backbone for treatment of HIV-1 infection in virologically suppressed adults: subgroup analysis by third agent of a randomized, double-blind, active-controlled phase 3 trial^.

PubMed

Post, Frank A; Yazdanpanah, Yazdan; Schembri, Gabriel; Lazzarin, Adriano; Reynes, Jacques; Maggiolo, Franco; Yan, Mingjin; Abram, Michael E; Tran-Muchowski, Cecilia; Cheng, Andrew; Rhee, Martin S

2017-05-01

FTC/TAF was shown to be noninferior to FTC/TDF with advantages in markers of renal and bone safety. To evaluate the efficacy and safety of switching to FTC/TAF from FTC/TDF by third agent (boosted protease inhibitor [PI] vs. unboosted third agent). We conducted a 48-week subgroup analysis based on third agent from a randomized, double blind study in virologically suppressed adults on a FTC/TDF-containing regimen who switched to FTC/TAF vs. continued FTC/TDF while remaining on the same third agent. We randomized (1:1) 663 participants to either switch to FTC/TAF (N = 333) or continue FTC/TDF (N = 330), each with baseline third agent stratifying by class of third agent in the prior treatment regimen (boosted PI 46%, unboosted third agent 54%). At week 48, significant differences in renal biomarkers and bone mineral density were observed favoring FTC/TAF over FTC/TDF (p < 0.05 for all), with similar improvements in the FTC/TAF arm in those who received boosted PI vs. unboosted third agents. At week 48, virologic success rates were similar between treatment groups for those who received a boosted PI (FTC/TAF 92%, FTC/TDF 93%) and for those who received an unboosted third agent (97% vs. 93%). In virologically suppressed patients switching to FTC/TAF from FTC/TDF, high rates of virologic suppression were maintained, while renal and bone safety parameters improved, regardless of whether participants were receiving a boosted PI or an unboosted third agent. FTC/TAF offers safety advantages over FTC/TDF and can be an important option as an NRTI backbone given with a variety of third agents.

An Application of Response Surface Methodology to a Macroeconomic Model.

DTIC Science & Technology

1985-12-01

L21,ILI,ISl ,KI,SBI,PYl ,MI,P11,Q1 double precision TE,TW,TC,TN,TR,G,W2,R2,T,H,NP,NL,N8,NE,FR, :: & PF,LB A double precision CLI, SCLI ,PCL1IDL1 ,WlLl...TCLI-TNLI- SCLI )*PILI/PRLI) & .0.012*FR)+0.5*R1 PR=0.5*(-131. 17+2.32.PI)+0.5*PR *The monetary sector is omitted (See Chapter IV) C L1=0.5*(0.14*(M-TW

Dissociative and double photoionization cross sections of NO from threshold to 120 A

NASA Technical Reports Server (NTRS)

Samson, J. A. R.; Masuoka, T.; Pareek, P. N.

1985-01-01

The partial photoionization cross sections for producing the NO(+) parent ion and the O(+), N(+), and NO2(+) fragment ions from neutral NO are presented from 120 to 614 A. The results indicate predissociation of the c(sup3) pi (21.72 eV) and B prime (sup 1) sigma (+) (22.73 eV) electronic states of NO(+). The photoionization threshold for double ionization was found to be 39.4 + or 0.12 eV.

Absolute measurement of hadronic branching fractions of the Ds+ meson.

PubMed

Alexander, J P; Berkelman, K; Cassel, D G; Duboscq, J E; Ehrlich, R; Fields, L; Gibbons, L; Gray, R; Gray, S W; Hartill, D L; Heltsley, B K; Hertz, D; Jones, C D; Kandaswamy, J; Kreinick, D L; Kuznetsov, V E; Mahlke-Krüger, H; Mohapatra, D; Onyisi, P U E; Patterson, J R; Peterson, D; Riley, D; Ryd, A; Sadoff, A J; Shi, X; Stroiney, S; Sun, W M; Wilksen, T; Athar, S B; Patel, R; Yelton, J; Rubin, P; Eisenstein, B I; Karliner, I; Mehrabyan, S; Lowrey, N; Selen, M; White, E J; Wiss, J; Mitchell, R E; Shepherd, M R; Besson, D; Pedlar, T K; Cronin-Hennessy, D; Gao, K Y; Hietala, J; Kubota, Y; Klein, T; Lang, B W; Poling, R; Scott, A W; Zweber, P; Dobbs, S; Metreveli, Z; Seth, K K; Tomaradze, A; Libby, J; Powell, A; Wilkinson, G; Ecklund, K M; Love, W; Savinov, V; Lopez, A; Mendez, H; Ramirez, J; Ge, J Y; Miller, D H; Sanghi, B; Shipsey, I P J; Xin, B; Adams, G S; Anderson, M; Cummings, J P; Danko, I; Hu, D; Moziak, B; Napolitano, J; He, Q; Insler, J; Muramatsu, H; Park, C S; Thorndike, E H; Yang, F; Artuso, M; Blusk, S; Khalil, S; Li, J; Mountain, R; Nisar, S; Randrianarivony, K; Sultana, N; Skwarnicki, T; Stone, S; Wang, J C; Zhang, L M; Bonvicini, G; Cinabro, D; Dubrovin, M; Lincoln, A; Rademacker, J; Asner, D M; Edwards, K W; Naik, P; Briere, R A; Ferguson, T; Tatishvili, G; Vogel, H; Watkins, M E; Rosner, J L

2008-04-25

The branching fractions of D(s)(+/-) meson decays serve to normalize many measurements of processes involving charm quarks. Using 298 pb(-1) of e(+)e(-) collisions recorded at a center of mass energy of 4.17 GeV, we determine absolute branching fractions for eight D(s)(+/-) decays with a double tag technique. In particular we determine the branching fraction B(D(s)(+)-->K(-)K(+}pi(+))=(5.50+/-0.23+/-0.16)%, where the uncertainties are statistical and systematic, respectively. We also provide partial branching fractions for kinematic subsets of the K(-)K(+)pi(+) decay mode.

A Study of the X(sup 2) Sigma(sup +) and A(sup 2) Pi States of MgAr(sup +) and MgKr(sup +)

NASA Technical Reports Server (NTRS)

Bauschlicher, Charles W., Jr.; Partridge, Harry; Langhoff, Stephen R. (Technical Monitor)

1995-01-01

The ground (sup 2)Sigma(sup +) and lowest excited (sup 2)Pi states of MgAr(sup +) and MgKr(sup +) are studied using the singles and doubles configuration interaction (SDCI) approach, in conjunction with large basis sets. The effect of Mg core correlation and core polarization are accounted for using the core-polarization potential (CPP) approach. Franck-Condon factors, oscillator strengths, radiative lifetimes, dissociation energies, bond lengths, and excitation energies are reported. The computed results are in good agreement with the available experimental data.

Localized CT-Guided Irradiation Inhibits Neurogenesis in Specific Regions of the Adult Mouse Brain

PubMed Central

Ford, E. C.; Achanta, P.; Purger, D.; Armour, M.; Reyes, J.; Fong, J.; Kleinberg, L.; Redmond, K.; Wong, J.; Jang, M. H.; Jun, H.; Song, H-J.; Quinones-Hinojosa, A.

2011-01-01

Radiation is used in the study of neurogenesis in the adult mouse both as a model for patients undergoing radiation therapy for CNS malignancies and as a tool to interrupt neurogenesis. We describe the use of a dedicated CT-guided precision device to irradiate specific sub-regions of the adult mouse brain. Improved CT visualization was accomplished with intrathecal injection of iodinated contrast agent, which enhances the lateral ventricles. T2-weighted MRI images were also used for target localization. Visualization of delivered beams (10 Gy) in tissue was accomplished with immunohistochemical staining for the protein γ-H2AX, a marker of DNA double-strand breaks. γ-H2AX stains showed that the lateral ventricle wall could be targeted with an accuracy of 0.19 mm (n = 10). In the hippocampus, γ-H2AX staining showed that the dentate gyrus can be irradiated unilaterally with a localized arc treatment. This resulted in a significant decrease of proliferative neural progenitor cells as measured by Ki-67 staining (P < 0.001) while leaving the contralateral side intact. Two months after localized irradiation, neurogenesis was significantly inhibited in the irradiated region as seen with EdU/NeuN double labeling (P < 0.001). Localized radiation in the rodent brain is a promising new tool for the study of neurogenesis. PMID:21449714

Analysis of carrier transport and carrier trapping in organic diodes with polyimide-6,13-Bis(triisopropylsilylethynyl)pentacene double-layer by charge modulation spectroscopy and optical second harmonic generation measurement

NASA Astrophysics Data System (ADS)

Lim, Eunju; Taguchi, Dai; Iwamoto, Mitsumasa

2014-08-01

We studied the carrier transport and carrier trapping in indium tin oxide/polyimide (PI)/6,13-Bis(triisopropylsilylethynyl)pentacene (TIPS-pentacene)/Au diodes by using charge modulation spectroscopy (CMS) and time-resolved electric field induced optical second harmonic generation (TR-EFISHG) measurements. TR-EFISHG directly probes the spatial carrier behaviors in the diodes, and CMS is useful in explaining the carrier motion with respect to energy. The results clearly indicate that the injected carriers move across TIPS-pentacene thorough the molecular energy states of TIPS-pentacene and accumulate at the PI/TIPS-pentacene interface. However, some carriers are trapped in the PI layers. These findings take into account the capacitance-voltage and current-voltage characteristics of the diodes.

Qualitative Aspects of UV-Vis Spectrophotometry of Beta-Carotene and Lycopene.

ERIC Educational Resources Information Center

Tan, Barrie; Soderstrom, David N.

1989-01-01

Explores the structural behavior of polyenic pi systems such as isomerization and conjugation. Uses the simultaneous spectrophotometric analysis of a beta-carotene and lycopene mixture. Presents an empirical method to determine the number of double bonds in the polyenic carotenoid. (MVL)

Application of image processing to calculate the number of fish seeds using raspberry-pi

NASA Astrophysics Data System (ADS)

Rahmadiansah, A.; Kusumawardhani, A.; Duanto, F. N.; Qoonita, F.

2018-03-01

Many fish cultivator in Indonesia who suffered losses due to the sale and purchase of fish seeds did not match the agreed amount. The loss is due to the calculation of fish seed still using manual method. To overcome these problems, then in this study designed fish counting system automatically and real-time fish using the image processing based on Raspberry Pi. Used image processing because it can calculate moving objects and eliminate noise. Image processing method used to calculate moving object is virtual loop detector or virtual detector method and the approach used is “double difference image”. The “double difference” approach uses information from the previous frame and the next frame to estimate the shape and position of the object. Using these methods and approaches, the results obtained were quite good with an average error of 1.0% for 300 individuals in a test with a virtual detector width of 96 pixels and a slope of 1 degree test plane.

Role of Phosphatidylinositol-3 Kinase Pathway in NMDA Preconditioning: Different Mechanisms for Seizures and Hippocampal Neuronal Degeneration Induced by Quinolinic Acid.

PubMed

Constantino, Leandra C; Binder, Luisa B; Vandresen-Filho, Samuel; Viola, Giordano G; Ludka, Fabiana K; Lopes, Mark W; Leal, Rodrigo B; Tasca, Carla I

2018-04-20

N-methyl D-aspartate (NMDA) preconditioning is evoked by the administration of a subtoxic dose of NMDA and is protective against neuronal excitotoxicity. This effect may involve a diversity of targets and cell signaling cascades associated to neuroprotection. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases (MAPKs) such as extracellular regulated protein kinase 1/2 (ERK1/2) and p38 MAPK pathways play a major role in neuroprotective mechanisms. However, their involvement in NMDA preconditioning was not yet fully investigated. The present study aimed to evaluate the effect of NMDA preconditioning on PI3K/Akt, ERK1/2, and p38 MAPK pathways in the hippocampus of mice and characterize the involvement of PI3K on NMDA preconditioning-evoked prevention of seizures and hippocampal cell damage induced by quinolinic acid (QA). Thus, mice received wortmannin (a PI3K inhibitor) and 15 min later a subconvulsant dose of NMDA (preconditioning) or saline. After 24 h of this treatment, an intracerebroventricular QA infusion was administered. Phosphorylation levels and total content of Akt, glycogen synthase protein kinase-3β (GSK-3β), ERK1/2, and p38 MAPK were not altered after 24 h of NMDA preconditioning with or without wortmmanin pretreatment. Moreover, after QA administration, behavioral seizures, hippocampal neuronal degeneration, and Akt activation were evaluated. Inhibition of PI3K pathway was effective in abolishing the protective effect of NMDA preconditioning against QA-induced seizures, but did not modify neuronal protection promoted by preconditioning as evaluated by Fluoro-Jade B staining. The study confirms that PI3K participates in the mechanism of protection induced by NMDA preconditioning against QA-induced seizures. Conversely, NMDA preconditioning-evoked protection against neuronal degeneration is not altered by PI3K signaling pathway inhibition. These results point to differential mechanisms regarding protection against a behavioral and cellular manifestation of neural damage.

Fully quantum-state resolved study of NO{sub 2} photodissociation. Correlated NO({sup 2}{Pi}{sub {Omega}}, {nu} = 0 J,A) + O({sup 3}P{sub j}) distributions

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

Sanov, A.; Bieler, C.R.; Reisler, H.

1995-09-14

Relative O({sup 3}P{sub j} = 2.1.0) spin-orbit populations correlated with specific NO[{sup 2}{Pi}{sub {Omega}} = {1/2}, 3/2; {nu} = 0; f; {Lambda} = {Pi}(A{prime}), {Pi}(A{double_prime})] product states were obtained following photolysis of NO{sub 2} at excess energies E{sup {+-}} = 390, 425, and 1054 cm{sup -1}. These fully quantum state-resolved measurements were carried out by recording spatial profiles of recoiling NO({sup 2}{Pi}{sub {Omega}}, J, {Lambda}) products using polarized radiation for photolysis and state-selective laser ionization detection. The relative O({sup 3}P{sub j}) populations correlated with each NO({sup 2}{Pi}{sub {Omega}}, J, {Lambda}) state show marked fluctuations at each excess energy as amore » function of rotational state and {Lambda}-doublet component. The relative populations also fluctuate as a function of excess energy. The O({sup 3}P{sub j}) spin-orbit population ratios, when averaged over all measurements, exhibit distributions that are colder than statistical, in agreement with previous results. In particular, we find that, on average, O({sup 3}P{sub 1}):O({sup 3}P{sub 2}) population ratios correlated with the ground NO({sup 2}{Pi}{sub {1/2}}) state are colder than the corresponding ratios correlated with the excited NO({sup 2}{Pi}{sub 3/2}) spin-orbit state. These results are in agreement with the state-specific calculations of Katigiri and Kato. 45 refs., 12 figs., 3 tabs.« less

The 20-hydroxyecdysone-induced signalling pathway in G2/M arrest of Plodia interpunctella imaginal wing cells.

PubMed

Siaussat, David; Bozzolan, Françoise; Porcheron, Patrick; Debernard, Stéphane

2008-05-01

The mechanisms involved in the control of cellular proliferation by the steroid hormone 20-hydroxyecdysone (20E) in insects are not known. We dissected the 20E signalling pathway responsible for G2/M arrest of imaginal cells from the IAL-PID2 cells of the Indian meal moth Plodia interpunctella. We first used a 5'-3' RACE-based strategy to clone a 4479bp cDNA encoding a putative P. interpunctella HR3 transcription factor named PiHR3. The deduced amino acid sequence of PiHR3 was highly similar to those of HR3 proteins from other lepidopterans, e.g. Manduca sexta and Bombyx mori. Using double-stranded RNA-mediated interference (dsRNAi), we then succeeded in blocking the ability of 20E to induce the expression of PiEcR-B1, PiUSP-2 and PiHR3 genes that encode the P. interpunctella ecdysone receptor B1-isoform, Ultraspiracle-2 isoform, the insect homologue of the vertebrate retinoid X receptor, and the HR3 transcription factor. We showed that inhibiting the 20E induction of PiEcR-B1, PiUSP-2 and PiHR3 mRNAs prevented the decreased expression of B cyclin and consequently the G2/M arrest of IAL-PID2 cells. Using this functional approach, we revealed the participation of EcR, USP and HR3 in a 20E signalling pathway that controls the proliferation of imaginal cells by regulating the expression of B cyclin.

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration.

PubMed

Kaźmierczak, Beata; Kuźma-Kozakiewicz, Magdalena; Usarek, Ewa; Barańczyk-Kuźma, Anna

2011-01-01

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

High-latitude Pi2 pulsations associated with kink-like neutral sheet oscillations

NASA Astrophysics Data System (ADS)

Wang, G. Q.; Volwerk, M.; Zhang, T. L.; Schmid, D.; Yoshikawa, A.

2017-03-01

A kink-like neutral sheet oscillation event observed by Cluster between 1436 and 1445 UT on 15 October 2004 has been investigated. The oscillations with periods between 40 and 60 s, observed at (-13.1, 8.7, -0.5) RE, are dominant in BX and BY. And they propagate mainly duskward with a velocity of (86, 147, 46) km/s. Their periods and velocity can be explained by the magnetic double-gradient instability. These oscillations are accompanied by strong field-aligned currents (FACs), which prefer to occur near the strongly tilted current sheet, and local maximum FAC tends to occur near the neutral sheet. The FACs show one-to-one correlated with a high-latitude Pi2 pulsation event recorded by KTN and TIK stations with a delay time of 60 and 90 s, respectively. Both the Pi2 and oscillations propagate westward with a comparative conjunctive speed. These findings suggest a strong relation between the FACs and Pi2, and we infer that the Pi2 is caused by the FACs. The periods of the FACs are modulated by the oscillations but not exactly equal, which is one possible reason that the period of the Pi2 caused by the FACs could be different from the oscillations. We speculate that a current circuit between the plasma sheet and ionosphere can be formed during strongly tilted current sheet, and successive tilted current sheet could generate quasiperiodic multiple FAC systems, which can generate high-latitude Pi2 pulsations and control their periods.

ent-Kaurane Diterpenoids with Neuroprotective Properties from Corn Silk ( Zea mays).

PubMed

Qi, Xiao-Li; Zhang, Ying-Ying; Zhao, Peng; Zhou, Le; Wang, Xiao-Bo; Huang, Xiao-Xiao; Lin, Bin; Song, Shao-Jiang

2018-05-25

Thirteen new ent-kaurane diterpenoids, stigmaydenes A-M (1-13), together with two known compounds (14, 15), were isolated from the crude extract of corn silk ( Zea mays). The structures of the compounds were confirmed by comprehensive spectroscopic analyses. The absolute configuration of compound 1 was defined by single-crystal X-ray diffraction. The absolute configurations of the compounds were also confirmed by comparison of experimental and calculated specific rotations. The compounds were evaluated for their neuroprotective effects against H 2 O 2 -induced SH-SY5Y cell injury, and compound 8 was active at 100 μM, as determined by flow cytometry (annexin V-FITC/PI staining) and Hoechst 33258 staining. The results suggested that compound 8 could protect neuronal cells from H 2 O 2 -induced injury by inhibiting apoptosis in SH-SY5Y cells.

The effect of hydrostatic pressure on staurosporine-induced neural differentiation in mouse bone marrow‑derived mesenchymal stem cells.

PubMed

Javanmard, F; Azadbakht, M; Pourmoradi, M

2016-01-01

In this study, the role of hydrostatic pressure on staurosporine-induced neural differentiation in mouse bone marrow mesenchymal stem cells were investigated. The cells were cultured in treatment medium containing 100 nM of staurosporine for 4 hours; then the cells were affected by hydrostatic pressure (0, 25,50, 100 mmHg). The percentage of cell viability by trypan blue staining and the percentage of cell death by Hoechst/PI differential staining were assessed. We obtained the total neurite length. Expression of β-tubulin III and GFAP (Glial fibrillary acidic protein) proteins were also analyzed by immunocytochemistry. The percentage of cell viability in treatments decreased relative to the increase in hydrostatic pressure and time (p Keywords: bone marrow mesenchymal stem cell, hydrostatic pressure, immunocytochemistry, neural differentiation, neurite length, cell differentiation.

Neutral endopeptidase (EC 3.4.24.11) downregulates the onset of intestinal inflammation in the nematode infected mouse.

PubMed

Barbara, G; De Giorgio, R; Stanghellini, V; Corinaldesi, R; Cremon, C; Gerard, N; Gerard, C; Grady, E F; Bunnett, N W; Blennerhassett, P A; Collins, S M

2003-10-01

Substance P (SP) release from sensory nerves induces neurogenic inflammation. Neutral endopeptidase (NEP) degrades SP, thereby limiting its proinflammatory effects. Intestinal inflammation following Trichinella spiralis infection markedly downregulates NEP, resulting in diminished SP degradation, with unknown functional consequences. We hypothesised that diminished expression of NEP would exacerbate T spiralis induced enteritis. NEP knockout (NEP-/-) and wild-type (NEP+/+) mice were infected with T spiralis and studied at 6, 12, 24, and 48 hours post infection (PI). Tissue inflammation was quantified by computerised cell counting and myeloperoxidase activity (MPO). The leucocyte adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), and SP were assessed by immunohistochemistry. Before infection, the lack of NEP was not associated with changes in mucosal cellularity or MPO activity. Twelve hours PI, NEP-/- mice showed a 2.5-fold increase in MPO activity at a time when values in NEP+/+ mice were still within normal limits. MPO activity and cellularity peaked at 24 hours PI. This was accompanied by increased staining for both ICAM-1 and SP in NEP-/- mice. Infusion of rhNEP to NEP-/- mice significantly reduced MPO activity 24 hours PI. These findings demonstrate that NEP downregulates the early onset of nematode intestinal inflammation and that increased bioavailability of SP and overexpression of ICAM-1 in NEP-/- mice likely play a role in the earlier onset of intestinal inflammation.

Corneal Biofilms: From Planktonic to Microcolony Formation in an Experimental Keratitis Infection with Pseudomonas Aeruginosa.

PubMed

Saraswathi, Padmanabhan; Beuerman, Roger W

2015-10-01

Microbial biofilms commonly comprise part of the infectious scenario, complicating the therapeutic approach. The purpose of this study was to determine in a mouse model of corneal infection if mature biofilms formed and to visualize the stages of biofilm formation. A bacterial keratitis model was established using Pseudomonas aeruginosa ATCC 9027 (1 × 10(8) CFU/ml) to infect the cornea of C57BL/6 black mouse. Eyes were examined post-infection (PI) on days 1, 2, 3, 5, and 7, and imaged by slit lamp microscopy, and light, confocal, and electron microscopy to identify the stages of biofilm formation and the time of appearance. On PI day 1, Gram staining showed rod-shaped bacteria adherent on the corneal surface. On PI days 2 and 3, bacteria were seen within webs of extracellular polymeric substance (EPS) and glycocalyx secretion, imaged by confocal microscopy. Scanning electron microscopy demonstrated microcolonies of active infectious cells bound with thick fibrous material. Transmission electron microscopy substantiated the formation of classical biofilm architecture with P. aeruginosa densely packed within the extracellular polymeric substances on PI days 5 and 7. Direct visual evidence showed that biofilms routinely developed on the biotic surface of the mouse cornea. The mouse model can be used to develop new approaches to deal therapeutically with biofilms in corneal infections. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Weak anti-HIV CD8+ T-cell effector activity in HIV primary infection

PubMed Central

Dalod, Marc; Dupuis, Marion; Deschemin, Jean-Christophe; Goujard, Cécile; Deveau, Christiane; Meyer, Laurence; Ngo, Nicole; Rouzioux, Christine; Guillet, Jean-Gérard; Delfraissy, Jean-François; Sinet, Martine; Venet, Alain

1999-01-01

HIV-specific CD8+ T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-γ enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8+ T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0–6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1–13). The frequency of HIV-specific CD8+ T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8+CD28– terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination. J. Clin. Invest. 104:1431–1439 (1999). PMID:10562305

Sedanolide induces autophagy through the PI3K, p53 and NF-κB signaling pathways in human liver cancer cells.

PubMed

Hsieh, Shu-Ling; Chen, Chi-Tsai; Wang, Jyh-Jye; Kuo, Yu-Hao; Li, Chien-Chun; Hsieh, Lan-Chi; Wu, Chih-Chung

2015-12-01

Sedanolide (SN), a phthalide-like compound from celery seed oil, possesses antioxidant effects. However, the effect of SN on cell death in human liver cancer cells has yet to be determined. In this study, cell viability determination, monodansylcadaverine (MDC) fluorescent staining and immunoblot analysis were performed to determine autophagy induction and autophagy-induced protein expression changes via molecular examination after human liver cancer (J5) cells were treated with SN. Our studies demonstrate that SN suppressed J5 cell viability by inducing autophagy. Phosphoinositide 3-kinase (PI3K)-I, mammalian target of rapamycin (mTOR) and Akt protein levels decreased, whereas PI3K-III, LC3-II and Beclin-1 protein levels increased following SN treatment in J5 cells. In addition, SN treatment upregulated nuclear p53 and damage-regulated autophagy modulator (DRAM) and downregulated cytosolic p53 and Tp53-induced glycolysis and apoptosis regulator (TIGAR) expression in J5 cells. Furthermore, the cytosolic phosphorylation of inhibitor of kappa B (IκB) and nuclear p65 and the DNA-binding activity of NF-κB increased after SN treatment. These results suggest that SN induces J5 cell autophagy by regulating PI3K, p53 and NF-κB autophagy-associated signaling pathways in J5 cells.

Role of phosphoinositide 3-kinase IA (PI3K-IA) activation in cardioprotection induced by ouabain preconditioning.

PubMed

Duan, Qiming; Madan, Namrata D; Wu, Jian; Kalisz, Jennifer; Doshi, Krunal Y; Haldar, Saptarsi M; Liu, Lijun; Pierre, Sandrine V

2015-03-01

Acute myocardial infarction, the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death worldwide. Like ischemic preconditioning (IPC) induced by brief episodes of ischemia and reperfusion, ouabain preconditioning (OPC) mediated by Na/K-ATPase signaling protects the heart against IR injury. Class I PI3K activation is required for IPC, but its role in OPC has not been investigated. While PI3K-IB is critical to IPC, studies have suggested that ouabain signaling is PI3K-IA-specific. Hence, a pharmacological approach was used to test the hypothesis that OPC and IPC rely on distinct PI3K-I isoforms. In Langendorff-perfused mouse hearts, OPC was initiated by 4 min of ouabain 10 μM and IPC was triggered by 4 cycles of 5 min ischemia and reperfusion prior to 40 min of global ischemia and 30 min of reperfusion. Without affecting PI3K-IB, ouabain doubled PI3K-IA activity and Akt phosphorylation at Ser(473). IPC and OPC significantly preserved cardiac contractile function and tissue viability as evidenced by left ventricular developed pressure and end-diastolic pressure recovery, reduced lactate dehydrogenase release, and decreased infarct size. OPC protection was blunted by the PI3K-IA inhibitor PI-103, but not by the PI3K-IB inhibitor AS-604850. In contrast, IPC-mediated protection was not affected by PI-103 but was blocked by AS-604850, suggesting that PI3K-IA activation is required for OPC while PI3K-IB activation is needed for IPC. Mechanistically, PI3K-IA activity is required for ouabain-induced Akt activation but not PKCε translocation. However, in contrast to PKCε translocation which is critical to protection, Akt activity was not required for OPC. Further studies shall reveal the identity of the downstream targets of this new PI3K IA-dependent branch of OPC. These findings may be of clinical relevance in patients at risk for myocardial infarction with underlying diseases and/or medication that could differentially affect the integrity of cardiac PI3K-IA and IB pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

The inhibitory mechanism of Cordyceps sinensis on cigarette smoke extract-induced senescence in human bronchial epithelial cells

PubMed Central

Liu, Ailing; Wu, Jinxiang; Li, Aijun; Bi, Wenxiang; Liu, Tian; Cao, Liuzhao; Liu, Yahui; Dong, Liang

2016-01-01

Objectives Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or stress. The bronchial epithelial cell is often injured by inhaled toxic substances, such as cigarette smoke. In the present study, we investigated whether exposure to cigarette smoke extract (CSE) induces senescence of bronchial epithelial cells; and Cordyceps sinensis mechanism of inhibition of CSE-induced cellular senescence. Methods Human bronchial epithelial cells (16HBE cells) cultured in vitro were treated with CSE and/or C. sinensis. p16, p21, and senescence-associated-galactosidase activity were used to detect cellular senescence with immunofluorescence, quantitative polymerase chain reaction, and Western blotting. Reactive oxygen species (ROS), PI3K/AKT/mTOR and their phosphorylated proteins were examined to testify the activation of signaling pathway by ROS fluorescent staining and Western blotting. Then, inhibitors of ROS and PI3K were used to further confirm the function of this pathway. Results Cellular senescence was upregulated by CSE treatment, and C. sinensis can decrease CSE-induced cellular senescence. Activation of ROS/PI3K/AKT/mTOR signaling pathway was enhanced by CSE treatment, and decreased when C. sinensis was added. Blocking ROS/PI3K/AKT/mTOR signaling pathway can attenuate CSE-induced cellular senescence. Conclusion CSE can induce cellular senescence in human bronchial epithelial cells, and ROS/PI3K/AKT/mTOR signaling pathway may play an important role in this process. C. sinensis can inhibit the CSE-induced senescence. PMID:27555762

Highly active antiretroviral therapy including protease inhibitors does not confer a unique CD4 cell benefit. The AVANTI and INCAS Study Groups.

PubMed

2000-07-07

To determine if triple combination therapy, particularly including HIV protease inhibitors (PI), confers an unique immunological benefit that is independent of reductions of plasma viral load (pVL). The correlation between changes from baseline in CD4 cell count and pVL was examined at all time points up to 52 weeks in three randomized clinical trials (AVANTI-2, AVANTI-3 and INCAS) that compared dual nucleoside therapy with triple combination therapy. Individual pVL and CD4 cell counts changes from baseline were entered into multivariate linear regression models for patients receiving double therapy and for those receiving triple therapy including a PI and/or a non-nucleoside reverse transcriptase inhibitor (NNRTI), and the null hypothesis was tested. After 52 weeks of therapy, the relationship between changes from baseline CD4 cell count and pVL was independent of whether patients were assigned double or triple therapy (P = 0.23 and 0.69 for intercept and slope, respectively), or whether patients were assigned triple therapy including a PI or triple therapy including an NNRTI (P = 0.92 and 0.95, respectively). Less than 5% of patients ever had 'discordant' increases in both CD4 cell count and pVL compared with baseline, and this proportion was unrelated to the class of therapy used. 'Discordant' decreases from baseline in both parameters were observed in up to 35% of individuals. The correlation between pVL and CD4 cell count changes from baseline improved over time on therapy, regardless of the therapeutic regimen involved. The data provide no evidence for a CD4 cell count benefit of highly active antiretroviral therapy (HAART) unique to triple therapy or PI-containing regimens.

Spontaneous switching among multiple spatio-temporal patterns in three-oscillator systems constructed with oscillatory cells of true slime mold

NASA Astrophysics Data System (ADS)

Takamatsu, Atsuko

2006-11-01

Three-oscillator systems with plasmodia of true slime mold, Physarum polycephalum, which is an oscillatory amoeba-like unicellular organism, were experimentally constructed and their spatio-temporal patterns were investigated. Three typical spatio-temporal patterns were found: rotation ( R), partial in-phase ( PI), and partial anti-phase with double frequency ( PA). In pattern R, phase differences between adjacent oscillators were almost 120 ∘. In pattern PI, two oscillators were in-phase and the third oscillator showed anti-phase against the two oscillators. In pattern PA, two oscillators showed anti-phase and the third oscillator showed frequency doubling oscillation with small amplitude. Actually each pattern is not perfectly stable but quasi-stable. Interestingly, the system shows spontaneous switching among the multiple quasi-stable patterns. Statistical analyses revealed a characteristic in the residence time of each pattern: the histograms seem to have Gamma-like distribution form but with a sharp peak and a tail on the side of long period. That suggests the attractor of this system has complex structure composed of at least three types of sub-attractors: a “Gamma attractor”-involved with several Poisson processes, a “deterministic attractor”-the residence time is deterministic, and a “stable attractor”-each pattern is stable. When the coupling strength was small, only the Gamma attractor was observed and switching behavior among patterns R, PI, and PA almost always via an asynchronous pattern named O. A conjecture is as follows: Internal/external noise exposes each pattern of R, PI, and PA coexisting around bifurcation points: That is observed as the Gamma attractor. As coupling strength increases, the deterministic attractor appears then followed by the stable attractor, always accompanied with the Gamma attractor. Switching behavior could be caused by regular existence of the Gamma attractor.

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.

[RITA combined with temozolomide inhibits the proliferation of human glioblastoma U87 cells].

PubMed

He, Xiao-Yan; Feng, Xiao-Li; Song, Xin-Pei; Zeng, Huan-Chao; Cao, Zhong-Xu; Xiao, Wei-Wei; Zhang, Bao; Wu, Qing-Hua

2016-10-20

To observe the effect of RITA, a small molecule that targets p53, combined with temozolomide (TMZ) on proliferation, colony formation and apoptosis of human glioblastoma U87 cells and explore the underlying mechanism. Cultured U87 cells were treated with RITA (1, 5, 10, 20 µmol/L), TMZ, or RITA+TMZ (half dose) for 24, 48 or 72 h. MTS assay were used to detect the cell proliferation, and the cell proliferation rate and inhibitory rate were calculated. The effect of combined treatments was evaluated by the q value. The expressions of p53, p21 and other apoptosis-associated genes were detected by qRT-PCR and Western blotting; cell apoptosis was assayed using flow cytometry with Annexin V/PI double staining; colony formation of the cells was detected with crystal violet staining. MTS assay showed that RITA at the 4 doses more potently inhibited U87 cell viability than TMZ at 72 h (P=0.000) with inhibitory rates of 25.94%-41.38% and 3.84%-8.20%, respectively. RITA combined with TMZ caused a more significant inhibition of U87 cells (29.21%-52.11%) than RITA (P<0.01) and TMZ (P=0.000) alone. At the doses above 5 µmol/L, the combined treatments with RITA+TMZ for 48 h resulted in q values exceeding 1.2 and showed an obvious synergistic effect of the drugs. Both RITA and TMZ, especially the latter, significantly increased the expressions of p53, p21, puma, and other apoptosis-associated genes to accelerate apoptosis and inhibit the growth and colony formation of U87 cells, and the effect was more obvious with a combined treatment. RITA inhibits the growth of human glioblastoma cells and enhance their sensitivity to TMZ by up-regulating p53 expression, and when combined, RITA and TMZ show a synergistic effect to cause a stronger cell inhibition.

Apoptotic activities of cardenolide glycosides from Asclepias subulata.

PubMed

Rascón-Valenzuela, L A; Velázquez, C; Garibay-Escobar, A; Vilegas, W; Medina-Juárez, L A; Gámez-Meza, N; Robles-Zepeda, R E

2016-12-04

Asclepias subulata Decne. (Apocynaceae) is a shrub occurring in Sonora-Arizona desert. The ethnic groups of Sonora, Mexico, Seris and Pimas, use this plant for the treatment of sore eyes, gastrointestinal disorders and cancer. To determine the cell death pathways that the cardenolide glycosides with antiproliferative activity found in the methanol extract of A. subulata are able to activate. The effect of cardenolide glycosides isolated of A. subulata on induction of apoptosis in cancer cells was evaluated through the measuring of several key events of apoptosis. A549 cells were treated for 12h with doses of 3.0, 0.2, 3.0 and 1.0µM of 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively. Apoptotic and necrotic cell levels were measured by double staining with annexin V-FITC/PI. Mitochondrial membrane depolarization was examined through JC-1 staining. Apoptosis cell death and the apoptosis pathways activated by cardenolide glycosides isolated of A. subulata were further characterized by the measurement of caspase-3, caspase-8 and caspase-9 activity. Apoptotic assays showed that the four cardenolide glycosides isolated of A. subulata induced apoptosis in A549 cells, which was evidencing by phosphatidylserine externalization in 18.2%, 17.0%, 23.9% and 22.0% for 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively, compared with 4.6% of control cells. Cell death was also associated with a decrease in mitochondrial membrane potential, which was more than 75% in the treated cultures respect to control. The activation of caspase-3 was observed in all cardenolide glycosides-treated cancer cells indicating the caspase-dependent apoptosis of A549 cells. Extrinsic and intrinsic apoptosis pathways were activated by cardenolide glycosides treatment at the doses tested. In this study was found that cardenolide glycosides, 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, isolated from A. subulata induced the cell death trough caspase-dependent apoptosis, which was activated, preferably, by extrinsic pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A contribution to examination of propidium iodide and annexin V plasma cells indices in multiple myeloma.

PubMed

Scudla, V; Ordeltova, M; Bacovsky, J; Vytrasova, M; Sumna, E; Martinek, A; Horak, P

2003-01-01

The aim of this study was a contemporaneous measurement and a mutual comparison of plasma cells proliferative activity and grade of apoptosis in patients with monoclonal gammopathy of undetermined significance (MGUS) and various phases of MM i.e. smoldering (SMM), stable/plateau and active (progression/relapse) forms of this disease. The analyzed group of 197 patients consisted of 30 MGUS, 21 SMM, 82 patients examined at the time of MM diagnosis and 64 patients analyzed during various phases of the disease after previous chemotherapy. Plasma cell proliferative activity was measured by means of a propidium iodide index (PC-PI) examined by flow cytometry using a DNA/CD138 double staining technique. For detection of plasma cells entering apoptosis (PC-AI) flow cytometry method with annexin V FITC and MoAb CD138 was used. The individuals with MGUS, SMM and stable/plateau form of MM had overall low levels of PC-PI (M-1.8, 1.7% and 2.1%) and relatively high levels of PC-AI (M-9.1, 10.8 and 9.0%). The correlation between PC-PI and PC-AI was in all the groups mutually highly statistically significant (p=0.000). Analysis of plasma cells proliferative activity (PC-PI) was statistically significant in comparison of MGUS or SMM and versus: patients examined at the time of MM diagnosis (p=0.018 or 0.016); patients evaluated during various phases of MM after previous chemotherapy (p=0.021 or 0.019); stable/plateau MM phase in the cohort of all patients (p=0.017 or 0.040); in the plateau phase after chemotherapy (p=0.008 or 0.024) but insignificant in comparison of MGUS and SMM and with the stable group examined at the time of MM diagnosis. Analysis of the apoptotic process revealed significant differences when comparing PC-AI of SMM but not MGUS group versus all cohort of stable/plateau MM patients (p=0.045); there were also insignificant differences in comparison of MGUS and SMM groupsand versus the stable form of MM measured at the time of MM diagnosis or plateau phase after chemotherapy. There was observed a statistically significant difference in the PC-AI in comparison of SMM group versus group of all patients examined at the time of MM diagnosis (p=0.001) or in various phases of this disease (p=0.015) and the group of MGUS patients compared with patients evaluated at the time of MM diagnosis (p=0.03). Very significant statistical differences of plasma cell proliferative (PC-PI) and apoptotic (PC-AI) activity were found when comparing the levels of both the indices of MGUS, SMM and stable/plateau MM group versus the active (progression/relapse) form of MM marked by a higher level of PC-PI (3.2%, p=0.000) and PC-AI (4.8%, p=0.000) in the whole cohort of MM patients, but also in comparison with both the active forms at the time of MM diagnosis or active forms evaluated during various phases of the disease after chemotherapy. Highly significant inverse relationship between PC-PI versus PC-AI was also revealed in the group of patients in the active (progression/relapse) phase of MM (p=0.000). These results revealed importance of measurement not only of proliferative but also of apoptotic plasma cells indices for a complex evaluation of the cells kinetics of plasma cells compartments in patients with MGUS or MM. This study confirmed the initial hypothesis of a common 'inverse relationship between the proliferative (PC-PI) and the apoptosis activity (PC-AI) in plasma cells compartments in patients with MGUS, smoldering, stable/plateau and active (progression/ relapse) forms of MM'.

Short-term effects of povidone iodine and sodium fluoride therapy on plaque levels and microbiome diversity.

PubMed

Reilly, C; Goettl, M; Steinmetz, M; Nikrad, J; Jones, R S

2016-03-01

The objective of this study is to evaluate the effect of short-term changes in the oral microbial ecology of dental plaque and plaque levels after topical treatment of a combination of 10% povidone iodine (PI) and 5% sodium fluoride varnish (FV). A single group design intervention study on 12 pediatric patients, who underwent two baseline plaques samplings before the intervention, were enrolled in the study. A modified mixed dentition Silness-Löe plaque index score was used to assess plaque accumulation and microbial composition was assessed by amplicon sequencing analysis of the 16S rRNA V4 region. Dental plaque accumulation (P = 0.0424) was reduced after 1 week using PI/FV application. This reduction was not observed between the two double-baseline visits. 16S rRNA analysis showed that the single PI/FV therapy did not have dramatic shifts in the plaque microbiome community depicted by hierarchical cluster and principle component analysis. More subtle changes were found when analyzing the Shannon diversity index after the application of PI/FV vs two baselines prior to combination therapy. The bacteria within the dental biofilms showed resilience in maintaining the overall community diversity but reduced biofilm accumulation following PI/FV therapy. Repeated uses of PI/FV may augment plaque control during dental rehabilitation in children. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Differential roles of resistance to proactive interference and suppression of prepotent responses in overgeneral memory.

PubMed

Comas, Michelle; Valentino, Kristin; Johnson, Anne F; Gibson, Bradley S; Taylor, Courtney

2018-06-12

Overgeneral memory (OGM), difficulty in retrieving specific autobiographical memories, is a robust phenomenon related to the onset and course of depressive and posttraumatic stress disorders. Inhibitory mechanisms are theorized to underlie OGM; however, empirical support for this link is equivocal. The current study examines the differential roles of two aspects of inhibitory control in association with OGM: suppression of prepotent responses and resistance to proactive interference (PI). Only resistance to PI was expected to be negatively related to OGM, whereby individuals with greater ability to resist PI would have reduced OGM. Participants (n = 49) completed a self-report measure of depressive symptoms and engaged in two tasks aimed at assessing resistance to PI and suppression of prepotent responses. Participants also completed a task assessing overgeneral autobiographical memory. As hypothesized, resistance to PI, but not suppression of prepotent responses negatively predicted OGM above and beyond the influence of depressive symptoms. Because a double dissociation was not examined, we cannot address the potential independence of the submechanisms of inhibitory control that we assessed. Results exemplify the differential associations of two components of inhibition and OGM, suggesting that resistance to PI, in particular, may contribute to the development and/or maintenance of OGM and associated depressive disorders. Directions for future research are discussed. Copyright © 2018. Published by Elsevier Ltd.

Target and beam-target spin asymmetries in exclusive π + and π - electroproduction with 1.6- to 5.7-GeV electrons

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

Bosted, P. E.; Biselli, A. S.; Careccia, S.

Beam-target double-spin asymmetries and target single-spin asymmetries in exclusive pi(+) and quasiexclusive pi(-) electroproduction were obtained from scattering of 1.6- to 5.7-GeV longitudinally polarized electrons from longitudinally polarized protons (for pi(+)) and deuterons (for pi(-)) using the CEBAF Large Acceptance Spectrometer (CLAS) at Jefferson Lab. The kinematic range covered is 1.1 < W < 2.6 GeV and 0.05 < Q(2) < 5 GeV2, with good angular coverage in the forward hemisphere. The asymmetry results were divided into approximately 40 000 kinematic bins for pi(+) from free protons and 15 000 bins for pi(-) production from bound nucleons in the deuteron.more » The present results are found to be in reasonable agreement with fits to previous world data for W < 1.7 GeV and Q(2) < 0.5 GeV2, with discrepancies increasing at higher values of Q(2), especially for W > 1.5 GeV. Very large target-spin asymmetries are observed for W > 1.6 GeV. When combined with cross-section measurements, the present results can provide powerful constraints on nucleon resonance amplitudes at moderate and large values of Q(2), for resonances with masses as high as 2.3 GeV.« less

PR01 Molecular Pathogenesis of Rickettsioses and Development of Anti-Rickettsial Treatment by Combinatorial Peptide-Based Libraries

DTIC Science & Technology

2006-02-01

likely reflecting similar cell death rates in all monolayers at late time points. By the end of the experiment at 120 hours, all monolayers showed a...50-55% increase in permeability when compared to the controls. 2. Cell death rates in rickettsiae-infected SV-HCEC monolayers In order to...necrotic cell death. Quantification of cell death was performed by determining the percent of total cells staining positive for PI. Cell death rates did

Aldosterone Induces Apoptosis in Rat Podocytes: Role of PI3-K/Akt and p38MAPK Signaling Pathways

PubMed Central

Chen, Cheng; Liang, Wei; Jia, Junya; van Goor, Harry; Singhal, Pravin C.; Ding, Guohua

2009-01-01

Background Podocytes play a critical role in the pathogenesis of glomerulosclerosis. Increasing evidence suggests that aldosterone (ALD) is involved in the initiation and progression of glomerular damage. It is, however, unknown whether there is a direct injurious effect of ALD on podocytes. Therefore, in the present study, we evaluated the effect of ALD on podocyte apoptosis and studied the role of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in this process. Methods Podocytes were incubated in media containing either buffer or increasing concentrations of ALD (10–9∼10–5M) for variable time periods. The cells were also treated with either wortmannin (inhibitor of PI3-K, 100 nM), SB202190 (SB20, inhibitor of p38MAPK, 10 μM) or buffer. All treatments were performed with or without ALD (10–7M) for 24 h. At the end of the incubation period, apoptosis was evaluated by cell nucleus staining and flow cytometric analyses. Activation of PI3-K/Akt and p38MAPK phosphorylation of cultured rat podocytes was evaluated by performing Akt kinase assay and Western blot, respectively. Results Apoptosis of cultured rat podocytes was induced by ALD in a dose- and time-dependent manner. ALD inhibited the activity of PI3-K/Akt and increased the activation of p38MAPK. PI3-K/Akt activity was further inhibited by the addition of wortmannin to the cells in the presence of ALD. This was accompanied by a significant increase in apoptosis. ALD-induced p38MAPK phosphorylation and apoptosis were inhibited when the cells were pretreated with SB20. Furthermore, treatment with spironolactone not only attenuated the proapoptotic effect of ALD, but also significantly reversed its effects on PI3-K/Akt and p38MAPK signaling pathways. Conclusion ALD induces apoptosis in rat podocytes through inhibition of PI3-K/Akt and stimulation of p38 MAPK signaling pathways. Spironolactone attenuates ALD-induced podocyte apoptosis, thereby positioning this compound as a potential promising target of intervention in human renal damage. PMID:19590239

Immunohistochemical detection of a potential molecular therapeutic target for canine hemangiosarcoma

PubMed Central

ADACHI, Mami; HOSHINO, Yuki; IZUMI, Yusuke; TAKAGI, Satoshi

2015-01-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm of dogs for which there is currently no effective treatment. A recent study suggested that receptor tyrosine kinases (RTKs), the PI3K/Akt/m-TOR and MAPK pathways are all activated in canine and human HSA. The aim of the present study was to investigate the overexpression of these proteins by immunohistochemistry in canine splenic HSA to identify potential molecular therapeutic targets. A total of 10 splenic HSAs and two normal splenic samples surgically resected from dogs were sectioned and stained with hematoxylin and eosin for histological diagnosis or analyzed using immunohistochemistry. The expression of RTKs, c-kit, VEGFR-2 and PDGFR-2, as well as PI3K/Akt/m-TOR and MEK was higher in canine splenic HSAs compared to normal spleens. These proteins may therefore be potential therapeutic targets in canine splenic HSA. PMID:26685984

Immunohistochemical detection of a potential molecular therapeutic target for canine hemangiosarcoma.

PubMed

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Takagi, Satoshi

2016-05-03

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm of dogs for which there is currently no effective treatment. A recent study suggested that receptor tyrosine kinases (RTKs), the PI3K/Akt/m-TOR and MAPK pathways are all activated in canine and human HSA. The aim of the present study was to investigate the overexpression of these proteins by immunohistochemistry in canine splenic HSA to identify potential molecular therapeutic targets. A total of 10 splenic HSAs and two normal splenic samples surgically resected from dogs were sectioned and stained with hematoxylin and eosin for histological diagnosis or analyzed using immunohistochemistry. The expression of RTKs, c-kit, VEGFR-2 and PDGFR-2, as well as PI3K/Akt/m-TOR and MEK was higher in canine splenic HSAs compared to normal spleens. These proteins may therefore be potential therapeutic targets in canine splenic HSA.

The antiproliferative effect of C2-ceramide on lung cancer cells through apoptosis by inhibiting Akt and NFκB.

PubMed

Lin, I-Ling; Chou, Han-Lin; Lee, Jin-Ching; Chen, Feng-Wei; Fong, Yao; Chang, Wei-Chiao; Huang, Hurng Wern; Wu, Chang-Yi; Chang, Wen-Tsan; Wang, Hui-Min David; Chiu, Chien-Chih

2014-01-06

The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C2-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C2-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G1 arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C2-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells.

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection

PubMed Central

Kim, Tae Yun; Song, Kye-Yong; Sohn, Woon-Mok; Kang, Shin-Yong

2001-01-01

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection. PMID:11775331

Improved method for combination of immunocytochemistry and Nissl staining.

PubMed

Kádár, Andrea; Wittmann, Gábor; Liposits, Zsolt; Fekete, Csaba

2009-10-30

Nissl staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after 3 days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry allow the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content.

Improved method for combination of immunocytochemistry and Nissl staining

PubMed Central

Kádár, Andrea; Wittmann, Gábor; Liposits, Zsolt; Fekete, Csaba

2009-01-01

Nissl-staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl-staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl-staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after three days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry, allows the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content. PMID:19615409

Use of DNA stains in immunophenotyping by slide-based cytometry

NASA Astrophysics Data System (ADS)

Gerstner, Andreas O. H.; Laffers, Wiebke; Bootz, Friedrich; Tarnok, Attila

2003-06-01

Immunophenotyping of peripheral blood leukocytes (PBL) is a very well documented application of Slide Based Cytometry (SBC). As for any other assay it is of highest importance to ensure that all cells which are relevant for an analysis are recognized. Unlike assays for cultured cells which have homogenous morphology immunophenotyping of PBLs is performed on cells with heterogeneous size and shape. Therefore, triggering on parameters related to cell morphology might lead to an incomplete analysis of just a subset of cells especially in pathological conditions. Several dyes stain DNA specifically in a wide variety of emission spectra. Many of them show some influence of the chromatin condensation and organization on the staining intensity. DNA dyes therefore can be used to differentiate between cell types having the same ploidy. This can be exploited for immunophenotyping since some dyes therefore can partially replace antibody staining. The concept of using DNA dyes in the setting of immunostaining has the following advantages: (1) nuclear staining provides a stable and easy triggering signal that guarantees both, that neither cells are excluded nor that debris or polluting particles are included into the analysis; (2) some DNA dyes differentiate between mononuclear and polymorphonuclear cells. A disadvantage of DNA dyes is that mostly cells have to be permeabilized. Because of this only one set of immunophenotypic markers can be stained, cells are fixed and permeabilized, and then nuclei are stained with the appropriate DNA dye. In the study we demonstrate the use of the most commonly available DNA dyes (7-AAD, To-Pro, To-To, PI etc.) in their applicability in immunophenotyping. An overview of spectral properties, fluorescence spill-over and optimal combinations with surface antigen staining will be shown. As in general for SBC only very small sample volumes are needed. This allows to serially analyze PBL in clinical settings that up to now could not be studied in detail such as in the critical ill patient, during major surgery, and in new-borns and infants.

Rapamycin Prevents cyclophosphamide-induced Over-activation of Primordial Follicle pool through PI3K/Akt/mTOR Signaling Pathway in vivo.

PubMed

Zhou, Linyan; Xie, Yanqiu; Li, Song; Liang, Yihua; Qiu, Qi; Lin, Haiyan; Zhang, Qingxue

2017-08-16

Primordial follicular depletion has thought to be a common adverse effect of chemotherapy especially for female of reproductive age. The study aimed to evaluate the protective effect of rapamycin on the primordial follicles and its potential mechanism for patients receiving chemotherapy. 8-week old BALB/c female mice were randomly assigned into four groups (control; rapamycin; cyclophosphamide; and rapamycin combined with cyclophosphamide). Hematoxylin staining, immunohistochemical, TUNEL, western blotting and ELISA were employed to assess inter-group differences using Student's t-test and Mann-Whitney test. Cyclophosphamide depleted the follicular reserve and induced the phosphorylation of the key proteins of PI3K/Akt/mTOR pathway in mice in a dose-dependent manner. Co-treatment with rapamycin significantly reduced primordial follicle loss at all cyclophosphamide dose groups and prevent the follicle growth wave caused by cyclophosphamide treatment (P < 0.05). TUNEL staining showed that no apoptosis occured in the primordial follicles in all groups and fewer apoptosis in large growing follicles were observed in ovaries from rapamycin + cyclophosphamide group compared to that received cyclophosphamide alone. Serum anti-Müllerian hormone (AMH) was significantly reduced in cyclophosphamide alone group, in contrast to the normal level in rapamycin + cyclophosphamide group. Compared to p-Akt/Akt and p-mtor/mtor, p-rps6/rps6 was significantly decreased in rapamycin + cyclophosphamide group (P < 0.05), indicating that rapamycin attenuated the increased level of phosphorylation of rpS6 after cyclophosphamide treatment. Rapamycin can prevent the primordial follicle activation induced by cyclophosphamide through PI3K/Akt/mTOR signaling pathway and thus plays a role in preserving the follicle pool. These results suggest that rapamycin may be an effective protection for ovarian function during chemotherapy, which means a new nonsurgical application for protection of ovarian reserve and prevention of POF.

Induction of apoptosis by pistachio (Pistacia vera L.) hull extract and its molecular mechanisms of action in human hepatoma cell line HepG2.

PubMed

Fathalizadeh, J; Bagheri, V; Khorramdelazad, H; Kazemi Arababadi, M; Jafarzadeh, A; Mirzaei, M R; Shamsizadeh, A; Hajizadeh, M R

2015-11-30

Several important Pistacia species such as P. vera have been traditionally used for treating a wide range of diseases (for instance, liver-related disorders). There is a relative lack of research into pharmacological aspects of pistachio hull. Hence, this study was aimed at investigating whether pistachio rosy hull (PRH) extract exerts apoptotic impacts on HepG2 liver cancer cell line. In order to evaluate cell viability and apoptosis in response to treatment with the extract, MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining were performed, respectively. Moreover, molecular mechanism of apoptosis induced by the extract was determined using human apoptosis PCR array. Our findings showed that PRH extract treatment reduced cell viability (IC50 ~ 0.3 mg/ml) in a dose-dependent manner. Flow cytometric analysis revealed that the extract significantly induced apoptosis in HepG2 cells. In addition, quantitative PCR array results demonstrated the regulation of a considerable number of apoptosis-related genes belonging to the TNF, BCL2, IAP, TRAF, and caspase families. We observed altered expression of both pro-apoptotic and anti-apoptotic genes associated with the extrinsic and intrinsic apoptosis signaling pathways. These results suggest that the aqueous extract of PRH possesses apoptotic activity through cytotoxic and apoptosis-inducing effects on HepG2 cells.

The relation between doses or post-plasma time points and apoptosis of leukemia cells induced by dielectric barrier discharge plasma

NASA Astrophysics Data System (ADS)

Wang, Chao; Zhang, Haixia; Xue, Zhixiao; Yin, Huijuan; Niu, Qing; Chen, Hongli

2015-12-01

The dielectric barrier discharge (DBD) plasma was applied to induce apoptosis of LT-12 leukemia cells. Plasma effects on cell death was evaluated by MTT assay and FCM apoptosis assay with Annexin V/PI double staining, suggesting that plasma killing cells rate and inducing cell apoptosis rate both positively were related to the plasma doses or the post-plasma time points. The cell death rates increased from 15.2% to 33.1% and the apoptosis rate raise from 23.8% to 28% when the dose raise from 60s to 120 s at 8 h post-plasma, while they increased from 15.4% to 34.9% and from 48% to 55.3% respectively at the same doses at 12 h post-plasma. Furthermore, the production of reactive oxygen species (ROS), gene and protein expression for Caspases and Bcl-2 family members were measured for exploring the related apoptotic mechanisms phenomenon. We found ROS immediately increased to 1.24 times of the original amount, then increasing to 5.39-fold at 20 h after treatment. The gene and protein expression for Caspases and Bcl-2 family members are very active at 8-12 h post-plasma. Our results demonstrate that DBD plasma can effectively induce tumor cell death through primarily related apoptotic mechanisms.

Effect of salt on cell viability and membrane integrity of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum as observed by flow cytometry.

PubMed

Gandhi, Akanksha; Shah, Nagendra P

2015-08-01

The aim of the current study was to investigate the effect of varying sodium chloride concentrations (0-5%) on viability and membrane integrity of three probiotic bacteria, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum, using conventional technique and flow cytometry. Double staining of cells by carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) enabled to evaluate the effect of NaCl on cell esterase activity and membrane integrity. Observations from conventional culture technique were compared with findings from flow cytometric analysis on the metabolic activities of the cells and a correlation was observed between culturability and dye extrusion ability of L. casei and B. longum. However, a certain population of L. acidophilus was viable as per the plate count method but its efflux activity was compromised. Esterase activity of most bacteria reduced significantly (P < 0.05) during one week storage at NaCl concentrations greater than 3.5%. The study revealed that L. casei was least affected by higher NaCl concentrations among the three probiotic bacteria, as opposed to B. longum where the cF extrusion performance was greatly reduced during 1 wk storage. The metabolic activity and salt resistance of L. casei was found to be highest among the bacteria studied. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Four transition metal complexes with a semicarbazone ligand bearing pyrazine unit

NASA Astrophysics Data System (ADS)

Chen, Hong; Ma, Xiu-qin; Lv, Yan-yun; Jia, Lei; Xu, Jun; Wang, Yuan; Ge, Zhi-jun

2016-04-01

Four new complexes based on L (where L = 3-ethyl-2-acetylpyrazine semicarbazone), namely [CoL2]Cl2·0.5H2O (1), [CoL2](NO3)2 (2), [CdL(H2O)2(NO3)](NO3)·H2O (3) and [CuL(CH3OH)Cl2]·[CuLCl2] (4) have been synthesized and characterized by X-ray diffraction analyses. The results show that the semicarbazone acts as a tridentate neutral ligand in all complexes. Each of complex 1 and 2 reveals a distorted octahedral geometry around the metal ion provided by two units of the ligand, while the ratio of the ligand and metal is 1:1 in complexes 3 and 4. The effect of complexes 1-4 on cell proliferation, apoptosis of human pancreatic cancer (Patu8988), human gastric cancer (SGC7901) and human hepatic cancer (SMMC7721) cell lines have been detected by MTT assay, Annexin V/PI double staining flow cytometry and TUNEL assay. The results show that complexes 1-4 can inhibit cell proliferation of Patu8988, SGC7901 and SMMC7721 cells, significantly higher than the effect of the ligand. However, the complex 4 reveals higher apoptosis rate, and displays up-regulated expression level of caspase 3, detected by western blotting, which also indicates the complex 4 can induce caspase-dependent cell apoptosis in SMMC7721.

Effect of Agmatine Sulfate on Modulation of Matrix Metalloproteinases via PI3K/Akt-1 in HT1080 Cells.

PubMed

Kim, Hyejeong; Kim, Moon-Moo

2017-11-01

The purpose of this study was to investigate the mechanism by which agmatine sulfate induces an anti-metastatic effect in human HT1080 fibrosarcoma cells, by affecting matrix metalloproteinases (MMPs). For the experiments, we used a non-toxic concentration of agmatine, below 512 μM, that was determined using an MTT assay. The effect of agmatine sulfate on metastasis was gelatin zymography, western blot, immunofluorescence staining and cell invasion assay. Agmatine sulfate inhibited MMP-2 activity stimulated by phenazine methosulfate (PMS). Furthermore, the expression level of MMP-2 stimulated by PMS, was decreased, but the expression level of TIMP-1 was increased in the presence of agmatine sulfate. Moreover, it was observed that the expression levels of ERK and p38 were increased, but those of PI3K and Akt-1 associated with the modulation of MMP-2 were decreased in this study. Furthermore, agmatine sulfate decreased the invasion level of human fibrosarcoma cells stimulated by VEGF. These results suggest that agmatine sulfate could inhibit metastasis through inhibition of MMP-2 via the PI3K/Akt-1 signaling pathway. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

PubMed Central

Zhang, Dongdong; Wang, Zhe; Sheng, Chenxia; Peng, Weijun; Hui, Shan; Gong, Wei; Chen, Shuai

2015-01-01

Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer's disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ 25–35) induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ 25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ 25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer's disease. PMID:25705234

Enzymatic Kinetic Isotope Effects from Path-Integral Free Energy Perturbation Theory.

PubMed

Gao, J

2016-01-01

Path-integral free energy perturbation (PI-FEP) theory is presented to directly determine the ratio of quantum mechanical partition functions of different isotopologs in a single simulation. Furthermore, a double averaging strategy is used to carry out the practical simulation, separating the quantum mechanical path integral exactly into two separate calculations, one corresponding to a classical molecular dynamics simulation of the centroid coordinates, and another involving free-particle path-integral sampling over the classical, centroid positions. An integrated centroid path-integral free energy perturbation and umbrella sampling (PI-FEP/UM, or simply, PI-FEP) method along with bisection sampling was summarized, which provides an accurate and fast convergent method for computing kinetic isotope effects for chemical reactions in solution and in enzymes. The PI-FEP method is illustrated by a number of applications, to highlight the computational precision and accuracy, the rule of geometrical mean in kinetic isotope effects, enhanced nuclear quantum effects in enzyme catalysis, and protein dynamics on temperature dependence of kinetic isotope effects. © 2016 Elsevier Inc. All rights reserved.

Contact lens-induced circumlimbal staining in silicone hydrogel contact lenses worn on a daily wear basis.

PubMed

Maïssa, Cécile; Guillon, Michel; Garofalo, Renee J

2012-01-01

The principal objective of the study was to measure the conjunctival staining produced in the circumlimbal region by silicone hydrogel contact lenses with different edge designs. The secondary objective was to investigate the association between circumlimbal staining and comfort. Four silicone hydrogel contact lenses: ACUVUE OASYS (knife edge design), AIR OPTIX, Biofinity (chisel edge rounded edge combination), and PureVision (rounded edge design), and 1 hydrogel contact lens, ACUVUE 2 (knife edge design), were tested. The study was conducted on a cohort population of 27 established soft contact lens wearers, who wore each contact lens type, in a random order, for a period of 10 (±2) days. Circumlimbal staining was measured in a double-masked fashion through image analysis of digital photographs of lissamine green taken under controlled experimental conditions. The results obtained showed that contact lens edge design was the primary factor controlling circumlimbal staining for silicone hydrogel lenses: a rounded edge away from the ocular surface produced the lowest staining (average, 0.19%) and a knife edge in close apposition to the ocular surface produced the highest staining (average, 1.34%). Contact lens material rigidity was also identified to affect circumlimbal staining and an inverse association between circumlimbal staining and contact lens comfort was demonstrated: the rounded edge design produced the lowest comfort (72 of 100) and the knife edge design produced the highest (87 out of 100). Soft contact lens wear induces circumlimbal staining, the level of staining being influenced by the contact lens edge design. However, high level of circumlimbal staining is not associated with decreased comfort.

The effect of Liuwei Dihuang decoction on PI3K/Akt signaling pathway in liver of type 2 diabetes mellitus (T2DM) rats with insulin resistance.

PubMed

Dai, Bing; Wu, Qinxuan; Zeng, Chengxi; Zhang, Jiani; Cao, Luting; Xiao, Zizeng; Yang, Menglin

2016-11-04

Liuwei Dihaung decoction (LWDHT) is a well-known classic traditional Chinese medicine formula, consists of six herbs including Rehmannia glutinosa Libosch.(family: Scrophulariaceae), Cornus officinalis Sieb.(family: Cornaceae), Dioscorea opposite Thunb.(family: Dioscoreaceae), Alisma orientale(G. Samuelsson) Juz (family: Alismataceae), Poria cocos (Schw.) Wolf (family: Polyporaceae) and Paeonia suffruticosa Andrews (family: Paeoniaceae). It has been used in the treatment of many types of diseases with signs of deficiency of Yin in the kidneys in China clinically. This study is aimed at investigating the effect of Liuwei dihuang decoction on PI3K/Akt signaling pathway in liver of T2DM rats with insulin resistance. T2DM model was induced in male Sprague-Dawley (SD) rats by high sugar and high fat diets combined with small dose of streptozocin (STZ) injection. The successful T2DM rats were randomly allocated three group--vehicle group, positive control group and Liuwei Dihuang decoction group. After 12-weeks treatment with distilled water, rosiglitazone and LWDHT by intragastric administration respectively, the rats were put to death in batches. The variance of fasting blood glucose (FBG) and fasting insulin (FINS) in serum were determined, the pathological changes of each rats' liver were observed by hematoxylin-eosin (HE) staining, the expression of insulin receptor substrate 2(IRS2), phosphatidylinositol 3-kinase (PI3K) and protein kinas B (Akt) involving the canonical PI3K/Akt signaling pathway were detected by Real-time fluorescent quantitative PCR (RT-PCR), and the expression level of IRS2, PI3K, Akt protein and phosphorylated IRS2, PI3K, Akt protein were evaluated by Western Blot. All the data were analyzed by SPSS 17.0. Four weeks of treatment with LWDHT could significantly decrease the level of FBG and FINS in serum, improve the cellular morphology of liver, kidney, pancreas tissue, and the expression of IRS2, PI3K, Akt mRNA and phosphorylated IRS2, PI3K, Akt protein involved in the canonical PI3K/Akt signaling pathway of T2DM rats in liver were significantly up-regulated, while the total IRS2, PI3K, and Akt protein had no obvious changes. The results suggest that Liuwei Dihuang decoction could intervene insulin resistance of T2DM, in part, through regulation of canonical PI3K/Akt signaling pathway of T2DM rats in liver. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

New concepts of histological changes in experimental augmentation cystoplasty: insights into the development of neoplastic transformation at the enterovesical and gastrovesical anastomosis.

PubMed

Gitlin, J S; Wu, X R; Sun, T T; Ritchey, M L; Shapiro, E

1999-09-01

To our knowledge the pathogenesis of malignancy associated with ileal cystoplasty, ureterosigmoidostomy and ileal conduits is currently unknown. To gain further insights into the mechanism of neoplastic transformation we studied histological changes in a canine augmentation cystoplasty model. Enterocystoplasty and gastrocystoplasty were performed using a 5 to 7 cm. patch of ileum in 8 dogs and gastric antrum in 6. Specimens were harvested 4 months postoperatively. Representative 3 microm sections of the enterovesical and gastrovesical junctions were stained with hematoxylin and eosin. Uroplakin expression was assessed using an indirect peroxidase method subjected to double staining with alcian blue and periodic acid-Schiffreagent. The bladder portion of the augmentation cystoplasty had 3 to 4 stratified cell layers covered with a distinctive umbrella cell layer. Strong uroplakin staining was visible in all cell layers except the basal layer. At the enterovesical and gastrovesical junctions 6 to 10 layers of hyperplastic, urothelial appearing cells covered the glandular epithelium of the ileal and gastric segments. These cells expressed uroplakins. At this junction zone there was a marked decrease of underlying enteric glands, which had atrophied in proportion to the degree of urothelial hyperplasia. Double staining of uroplakin stained sections with alcian blue and periodic acid-Schiff reagent revealed mucosubstances in hyperplastic urothelial cells covering the enteral segments, indicating that the cells co-expressed uroplakins and mucins. Histological changes in this experimental canine model of augmentation cystoplasty indicated that the overgrowth of hyperplastic transitional epithelium develops at the enterovesical and gastrovesical junctions. These cells express not only uroplakins, but also mucosubstances. Our results suggest that the migrated hyperplastic urothelial cells have undergone changes characteristic of the enteric and gastric epithelium, which may have important implications in the pathogenesis of malignancy in bladder augmentations.

Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

PubMed Central

Müller, Brigitte; Ellinwood, N. M.; Lorenz, Birgit; Stieger, Knut

2018-01-01

Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted. PMID:29765300

Accurate ab initio calculations which demonstrate a 3 Pi u ground state for Al2

NASA Technical Reports Server (NTRS)

Bauschlicher, Charles W., Jr.; Partridge, Harry; Langhoff, Stephen R.; Taylor, Peter R.; Walch, Stephen P.

1986-01-01

The spectroscopic parameters and separations between the three low-lying X 3 Pi u, A 3 Sigma g -, and a 1 Sigma g + states of Al2 are studied as a function of both the one-particle and n-particle basis set. Approximate correlation treatments are calibrated against full Cl calculations correlating the six valence electrons in a double-zeta plus two d-function basis set. Since the CASSCF/MRCI 3 Pi u to 3 Sigma g - separation is in excellent agreement wtih the FCI value, the MRCI calculations were carried out in an extended (20s13p6d4f)/(6s5p3d2f) gaussian basis. Including a small correction for relativistic effects, the best estimate is that 3 Sigma g - state lies 174/cm above the 3 Pi u ground state. The 1 Sigma g + state lies at least 2000/cm higher in energy. At the CPF level, inclusion of 2s and 2p correlation has little effect on D sub e, reduces T sub e by only 26/cm, and shortens the bond lengths by about 0.02 a sub o. Further strong support for a 3 Pi u ground state comes from the experimental absorption spectra, since both observed transitions can be convincingly assigned as 3 Pi u yields 3 Pi g. The (2) 3 Pi g state is observed to be sensitive to the level of correlation treatment, and to have its minimum shifted to shorter rho values, such that the strongest experimental absorption peak probably corresponds to the 0 yields 2 transition.

Ulex europaeus I lectin as a marker for vascular endothelium in human tissues.

PubMed

Holthöfer, H; Virtanen, I; Kariniemi, A L; Hormia, M; Linder, E; Miettinen, A

1982-07-01

Ulex europaeus I agglutinin, a lectin specific for some alpha-L-fucose-containing glycocompounds, was used in fluorescence microscopy to stain cryostat sections of human tissues. The endothelium of vessels of all sizes was stained ubiquitously in all tissues studied as judged by double staining with a known endothelial marker, antibodies against human clotting factor VIII. Cultured human umbilical vein endothelial cells, but not fibroblasts, also bound Ulex lectin. The staining was not affected by the blood group type of the tissue donor. In some tissues Ulex lectin presented additional binding to epithelial structures. Also, this was independent on the blood group or the ability of the tissue donor to secrete soluble blood group substances. Lotus tetragonolobus agglutinin, another lectin specific for some alpha-L-fucose-containing moieties failed to react with endothelial cells. Our results suggest that Ulex europaeus I agglutinin is a good histologic marker for endothelium in human tissues.

Uncaria rhynchophylla Ameliorates Parkinson's Disease by Inhibiting HSP90 Expression: Insights from Quantitative Proteomics.

PubMed

Lan, Yu-Long; Zhou, Jun-Jun; Liu, Jing; Huo, Xiao-Kui; Wang, Ya-Li; Liang, Jia-Hao; Zhao, Jian-Chao; Sun, Cheng-Peng; Yu, Zhen-Long; Fang, Lin-Lin; Tian, Xiang-Ge; Feng, Lei; Ning, Jing; Zhang, Bao-Jing; Wang, Chao; Zhao, Xin-Yu; Ma, Xiao-Chi

2018-06-21

Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice. MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting. URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein. URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment. © 2018 The Author(s). Published by S. Karger AG, Basel.

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

PubMed

Pelzl, Lisann; Elsir, Bhaeldin; Sahu, Itishri; Bissinger, Rosi; Singh, Yogesh; Sukkar, Basma; Honisch, Sabina; Schoels, Ludger; Jemaà, Mohamed; Lang, Elisabeth; Storch, Alexander; Hermann, Andreas; Stournaras, Christos; Lang, Florian

2017-01-01

The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

Calcium- and polyphosphate-containing acidocalcisomes in chicken egg yolk.

PubMed

Ramos, Isabela B; Miranda, Kildare; Ulrich, Paul; Ingram, Peter; LeFurgey, Ann; Machado, Ednildo A; de Souza, Wanderley; Docampo, Roberto

2010-04-09

Poly P (inorganic polyphosphate) is a polymer formed by P(i) residues linked by high-energy phosphoanhydride bonds. The presence of poly P in bacteria, fungi, algae and protists has been widely recognized, but the distribution of poly P in more complex eukaryotes has been poorly studied. Poly P accumulates, together with calcium, in acidic vesicles or acidocalcisomes in a number of organisms and possesses a diverse array of functions, including roles in stress response, blood clotting, inflammation, calcification, cell proliferation and apoptosis. We report here that a considerable amount of phosphorus in the yolk of chicken eggs is in the form of poly P. DAPI (4',6-diamidino-2-phenylindole) staining showed that poly P is localized mainly in electron-dense vesicles located inside larger vacuoles (compound organelles) that are randomly distributed in the yolk. These internal vesicles were shown to contain calcium, potassium, sodium, magnesium, phosphorus, chlorine, iron and zinc, as detected by X-ray microanalysis and elemental mapping. These vesicles stain with the acidophilic dye Acridine Orange. The presence of poly P in organellar fractions of the egg yolk was evident in agarose gels stained with Toluidine Blue and DAPI. Of the total phosphate (Pi) of yolk organelles, 16% is present in the form of poly P. Total poly P content was not altered during the first 4 days of embryogenesis, but poly P chain length decreased after 1 day of development. The results of the present study identify a novel organelle in chicken egg yolk comprising acidic vesicles with a morphology, physiology and composition similar to those of acidocalcisomes, within larger acidic vacuoles. The elemental composition of these acidocalcisomes is proportionally similar to the elemental composition of the yolk, suggesting that most of these elements are located in these organelles, which might be an important storage compartment in eggs.

Effect of lingual gauze swab placement on pulse oximeter readings in anaesthetised dogs and cats.

PubMed

Mair, A; Martinez-Taboada, F; Nitzan, M

2017-01-14

This study aimed to evaluate the effect of lingual gauze swab placement on pulse oximeter readings in anaesthetised dogs and cats. Following anaesthetic induction, the following pulse oximeter probe configurations were performed: no gauze swab (control), placement of a gauze swab between the tongue and the probe, placement of different thicknesses of gauze swab, placement of red cotton fabric, placement of a sheet of white paper and placement of the probe and gauze swab on different locations on the tongue. Oxygen saturation (SpO 2 ) and peripheral perfusion index (PI) were recorded. Placement of a gauze swab between the pulse oximeter probe and the tongue in anaesthetised dogs and cats resulted in significantly higher SpO 2 values compared with the control group. In dogs, PI values were significantly higher than the control in all groups except the quarter thickness swab group. In cats, PI was significantly higher in the double thickness swab and white paper groups compared with the control. Cats had significantly higher SpO 2 and lower PI values than dogs. The authors propose that increased contact pressure is responsible for significantly higher SpO 2 and PI readings with the use of a lingual gauze swab resulting from changes in transmural pressure and arterial compliance. British Veterinary Association.

Membrane status of boar spermatozoa after cooling or cryopreservation.

PubMed

Maxwell, W M; Johnson, L A

1997-07-15

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.

The effect of PDT on H. influenzae biofilm in vitro

NASA Astrophysics Data System (ADS)

Rhee, C.-K.; Bae, S. H.; Lee, J. W.; Ahn, J. C.; Jung, J. Y.; Suh, M.-W.

2009-02-01

Biofilm formation has been demonstrated for many mucosal pathogens such as Haemophilus influenzae. The presence of mucosal biofilms with chronic otitis media with effusion (COME) suggests that bacteria do not clear by antibiotics. Aim: To test the effect of photodynamic therapy (PDT) on H. influenzae biofilm in vitro. Methods: Sixteen biofilms of H. influenzae were maintained on culture chamber with continuous flow cell system. The biofilms were divided into control, laser, photofrin, and PDT groups. For culture group, the biofilms were cultured. For laser group, 7.2 J/cm2 of 632 nm diode laser was irradiated to the biofilms. For photofrin group, photofrins 5 and 25ug/ml were added to the media. For PDT group, photofrins 5 and 25 ug/ml were added to the media following 632 nm diode laser was irradiated (7.2 J/cm2) to the biofilms. Live/Dead (DAPI/PI) stain was performed and biofilms were examined under confocal laser microscope for thickness and density of biofilms. Results: By DAPI/PI staining, significant reduction of biofilms thickness and complete killing of H. influenzae in PDT group with 25µg photofrin was noted while the biofilms were well maintained in the other groups. Conclusion: The results of this study demonstrated that PDT appears to be effective to photoinactivate experimental H. influenzae biofilms in vitro. Clinical implication: PDT can be a possible alternative treatment to antiobiotic treatment on otitis media with biofilm formation.

Immunomodulatory activity of interleukin-27 in human chronic periapical diseases

PubMed Central

Li, Juan; Wang, Rong; Huang, Shi-Guang

2017-01-01

This study aims to observe expression of IL-27 on different cells in periapical tissues of different types of human chronic periapical diseases. Periapical tissue specimens of 60 donors, including healthy control (n=20), periapical granuloma group (n=20) and radicular cysts group (n=20), were fixed in 10% buffered formalin, stained with hematoxylin and eosin for histopathology. Then specimens were stained with double- immuno-fluorescence assay for identification of IL-27-tryptase (mast cells, MCs), IL-27-CD14 (mononuclear phagocyte cells, MPs) and IL-27-CD31 (endothelial cells, ECs) double-positive cells in periapical tissues. The results indicated that compared with healthy control, the densities (cells/mm2) of IL-27-tryptase, IL-27-CD14 and IL-27-CD31 double-positive cells were significantly increased in human chronic periapical diseases (periapical granuloma group and radicular cysts group) (P<0.001). The density of IL-27-tryptase double positive cells in radicular cysts group was significantly higher than those in periapical granuloma group (P<0.001). Densities of IL-27-CD14 and IL-27-CD31 double-positive cells in periapical granuloma group had no significant difference with those in radicular cysts group (P=0.170 and 0.138, respectively). IL-27-CD14 double positive cells density achieved to peak among three cell groups in radicular cysts groups. In conclusion, IL-27 expressed in MCs, MPs and ECs of human chronic periapical diseases with different degrees. IL-27-tryptase double-positive cells may participate in pathogenic mechanism of chronic periapical diseases, especially for formation of fibrous in periapical cysts. IL-27-CD14 and IL-27-CD31 double-positive cells may participate in immunologic response to resist periapical infection, and they may play an dual role in pathogenesis and localization of periapical diseases. PMID:28386371

Regional imbalanced activation of the calcineurin/BAD apoptotic pathway and the PI3K/Akt survival pathway after myocardial infarction.

PubMed

Li, Tieluo; Kilic, Ahmet; Wei, Xufeng; Wu, Changfu; Schwartzbauer, Gary; Yankey, G Kwame; DeFilippi, Christopher; Bond, Meredith; Wu, Zhongjun J; Griffith, Bartley P

2013-06-05

The underlying molecular mechanisms of the remodeling after myocardial infarction (MI) remain unclear. The purpose of this study was to investigate the role of a survival pathway (PI3K/Akt) and an apoptosis pathway (calcineurin/BAD) in the remodeling after MI in a large animal model. Ten Dorset hybrid sheep underwent 25% MI in the left ventricle (LV, n=10). Five sheep were used as sham control. The regional strain was calculated from sonomicrometry. Apoptosis and the activation of the PI3K/Akt and calcineurin/BAD pathways were evaluated in the non-ischemic adjacent zone and the remote zone relative to infarct by immunoblotting, immunoprecipitation, and immunofluorescence staining. Dilation and dysfunction of LV were present at 12 weeks after MI. The regional strain in the adjacent zone was significantly higher than in the remote zone at 12 weeks (36.6 ± 4.0% vs 9.5 ± 3.6%, p<0.05). Apoptosis was more severe in the adjacent zone than in the remote zone. The PI3K/Akt and calcineurin/BAD pathways were activated in the adjacent zone. Dephosphorylation and translocation of BAD were evident in the adjacent zone. Regional correlation between the strain and the expression of calcineurin/BAD indicated that the activation was strain-related (R(2)=0.46, 0.48, 0.39 for calcineurin, BAD, mitochondrial BAD, respectively, p<0.05). The PI3K/Akt survival and calcineurin/BAD apoptotic pathways were concomitantly activated in the non-ischemic adjacent zone after MI. The calcineurin/BAD pathway is strain related and its imbalanced activation may be one of the causes of progressive remodeling after MI. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Suppression of the PI3K pathway in vivo reduces cystitis-induced bladder hypertrophy and restores bladder capacity examined by magnetic resonance imaging.

PubMed

Qiao, Zhongwei; Xia, Chunmei; Shen, Shanwei; Corwin, Frank D; Liu, Miao; Guan, Ruijuan; Grider, John R; Qiao, Li-Ya

2014-01-01

This study utilized magnetic resonance imaging (MRI) to monitor the real-time status of the urinary bladder in normal and diseased states following cyclophosphamide (CYP)-induced cystitis, and also examined the role of the phosphoinositide 3-kinase (PI3K) pathway in the regulation of urinary bladder hypertrophy in vivo. Our results showed that under MRI visualization the urinary bladder wall was significantly thickened at 8 h and 48 h post CYP injection. The intravesical volume of the urinary bladder was also markedly reduced. Treatment of the cystitis animals with a specific PI3K inhibitor LY294002 reduced cystitis-induced bladder wall thickening and enlarged the intravesical volumes. To confirm the MRI results, we performed H&E stain postmortem and examined the levels of type I collagen by real-time PCR and western blot. Inhibition of the PI3K in vivo reduced the levels of type I collagen mRNA and protein in the urinary bladder ultimately attenuating cystitis-induced bladder hypertrophy. The bladder mass calculated according to MRI data was consistent to the bladder weight measured ex vivo under each drug treatment. MRI results also showed that the urinary bladder from animals with cystitis demonstrated high magnetic signal intensity indicating considerable inflammation of the urinary bladder when compared to normal animals. This was confirmed by examination of the pro-inflammatory factors showing that interleukin (IL)-1α, IL-6 and tumor necrosis factor (TNF)α levels in the urinary bladder were increased with cystitis. Our results suggest that MRI can be a useful technique in tracing bladder anatomy and examining bladder hypertrophy in vivo during disease development and the PI3K pathway has a critical role in regulating bladder hypertrophy during cystitis.

Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

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

Kang, Myoung Hee; Oh, Sang Cheul; Lee, Hyun Joo

2011-07-15

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B.more » Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.« less

Regional imbalanced activation of the calcineurin/BAD apoptotic pathway and the PI3K/Akt survival pathway after myocardial infarction

PubMed Central

Li, Tieluo; Kilic, Ahmet; Wei, Xufeng; Wu, Changfu; Schwartzbauer, Gary; Yankey, G. Kwame; DeFilippi, Christopher; Bond, Meredith; Wu, Zhongjun J; Griffith, Bartley P

2011-01-01

Background The underlying molecular mechanisms of the remodeling after myocardial infarction (MI) remain unclear. The purpose of this study was to investigate the role of a survival pathway (PI3K/Akt) and an apoptosis pathway (calcineurin/BAD) in the remodeling after MI in a large animal model. Methods Ten Dorset hybrid sheep underwent 25% MI in the left ventricle (LV, n=10). Five sheep were used as sham control. The regional strain was calculated from sonomicrometry. Apoptosis and the activation of the PI3K/Akt and calcineurin/BAD pathways were evaluated in the non-ischemic adjacent zone and the remote zone relative to infarct by immunoblotting, immunoprecipitation, and immunofluorescence staining. Results Dilation and dysfunction of LV were present at 12 weeks after MI. The regional strain in the adjacent zone was significantly higher than in the remote zone at 12 weeks (36.6 ± 4.0% vs 9.5 ± 3.6%, p < 0.05). Apoptosis was more severe in the adjacent zone than in the remote zone. The PI3K/Akt and calcineurin/BAD pathways were activated in the adjacent zone. Dephosphorylation and translocation of BAD were evident in the adjacent zone. Regional correlation between the strain and the expression of calcineurin/BAD indicated that the activation was strain-related (R2 = 0.46, 0.48, 0.39 for calcineurin, BAD, mitochondrial BAD, respectively, p < 0.05). Conclusions The PI3K/Akt survival and calcineurin/BAD apoptotic pathways were concomitantly activated in the non-ischemic adjacent zone after MI. The calcineurin/BAD pathway is strain related and its imbalanced activation may be one of the causes of progressive remodeling after MI. PMID:22088220

Magnesium prevents phosphate-induced calcification in human aortic vascular smooth muscle cells.

PubMed

Louvet, Loïc; Büchel, Janine; Steppan, Sonja; Passlick-Deetjen, Jutta; Massy, Ziad A

2013-04-01

Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease. Factors promoting calcification include abnormalities in mineral metabolism, particularly high phosphate levels. Inorganic phosphate (Pi) is a classical inducer of in vitro VC. Recently, an inverse relationship between serum magnesium concentrations and VC has been reported. The present study aimed to investigate the effects of magnesium on Pi-induced VC at the cellular level using primary HAVSMC. Alive and fixed HAVSMC were assessed during 14 days in the presence of Pi with increasing concentrations of magnesium (Mg(2+)) chloride. Mineralization was measured using quantification of calcium, von Kossa and alizarin red stainings. Cell viability and secretion of classical VC markers were also assessed using adequate tests. Involvement of transient receptor potential melastatin (TRPM) 7 was assessed using 2-aminoethoxy-diphenylborate (2-APB) inhibitor. Co-incubation with Mg(2+) significantly decreased Pi-induced VC in live HAVSMC, no effect was found in fixed cells. At potent concentrations in Pi-induced HAVSMC, Mg(2+) significantly improved cell viability and restored to basal level increased secretions of osteocalcin and matrix gla protein, whereas a decrease in osteopontin secretion was partially restored. The block of TRPM7 with 2-APB at 10(-4) M led to the inefficiency of Mg(2+) to prevent VC. Increasing Mg(2+) concentrations significantly reduced VC, improved cell viability and modulated secretion of VC markers during cell-mediated matrix mineralization clearly pointing to a cellular role for Mg(2+) and 2-APB further involved TRPM7 and a potential Mg(2+) entry to exert its effects. Further investigations are needed to shed light on additional cellular mechanism(s) by which Mg(2+) is able to prevent VC.

Down-regulation of Long Noncoding RNA MALAT1 Protects Hippocampal Neurons Against Excessive Autophagy and Apoptosis via the PI3K/Akt Signaling Pathway in Rats with Epilepsy.

PubMed

Wu, Qiang; Yi, Xuewei

2018-06-01

Epilepsy is a common chronic brain disorder and is characterized by an enduring predisposition to generate seizures. The hippocampus is especially vulnerable to seizure-induced damage. In this study, we explore the ability of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) to influence the autophagy and apoptosis of hippocampal neurons in epilepsy and the underlying mechanism involving the PI3K/Akt signaling pathway. Seventy-two Sprague-Dawley rats were assigned to normal, sham, Ep, Ep + si-NC, Ep + si-MALAT1, and Ep + si-MALAT1 + LY groups. Fluorescence in situ hybridization kit was employed to determine the MALAT1 in the brain tissues. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine the expression of MALAT1, mRNAs, and proteins. The autophagy of hippocampal neurons was evaluated under a transmission electron microscope and their apoptosis was evaluated using TUNEL staining. We found that MALAT1 and c-Met were enriched while microRNA-101 (miR-101) decreased in rats with epilepsy. The demonstration showed that MALAT1 binds to miR-101, thus regulating c-Met. In rats with epilepsy, MALAT1 depletion mediated by anti-MALAT1 siRNA resulted in activation of PI3K/Akt signaling pathway and loss of hippocampal neurons. LY294002, an inhibitor of PI3K/Akt signaling pathway, could reverse the events caused by MALAT1 knockdown. Taken together, these findings indicate that down-regulation of MALAT1 activates the PI3K/Akt signaling pathway to protect hippocampal neurons against autophagy and apoptosis in rats with epilepsy.

Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways.

PubMed

Kornasio, Reut; Riederer, Ingo; Butler-Browne, Gillian; Mouly, Vincent; Uni, Zehava; Halevy, Orna

2009-05-01

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.

Neutral endopeptidase (EC 3.4.24.11) downregulates the onset of intestinal inflammation in the nematode infected mouse

PubMed Central

Barbara, G; De Giorgio, R; Stanghellini, V; Corinaldesi, R; Cremon, C; Gerard, N; Gerard, C; Grady, E F; Bunnett, N W; Blennerhassett, P A; Collins, S M

2003-01-01

Background and aims: Substance P (SP) release from sensory nerves induces neurogenic inflammation. Neutral endopeptidase (NEP) degrades SP, thereby limiting its proinflammatory effects. Intestinal inflammation following Trichinella spiralis infection markedly downregulates NEP, resulting in diminished SP degradation, with unknown functional consequences. We hypothesised that diminished expression of NEP would exacerbate T spiralis induced enteritis. Methods: NEP knockout (NEP−/−) and wild-type (NEP+/+) mice were infected with T spiralis and studied at 6, 12, 24, and 48 hours post infection (PI). Tissue inflammation was quantified by computerised cell counting and myeloperoxidase activity (MPO). The leucocyte adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), and SP were assessed by immunohistochemistry. Results: Before infection, the lack of NEP was not associated with changes in mucosal cellularity or MPO activity. Twelve hours PI, NEP−/− mice showed a 2.5-fold increase in MPO activity at a time when values in NEP+/+ mice were still within normal limits. MPO activity and cellularity peaked at 24 hours PI. This was accompanied by increased staining for both ICAM-1 and SP in NEP−/− mice. Infusion of rhNEP to NEP−/− mice significantly reduced MPO activity 24 hours PI. Conclusions: These findings demonstrate that NEP downregulates the early onset of nematode intestinal inflammation and that increased bioavailability of SP and overexpression of ICAM-1 in NEP−/− mice likely play a role in the earlier onset of intestinal inflammation. PMID:12970139

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of posttraumatic epilepsy

PubMed Central

Berdichevsky, Yevgeny; Dryer, Alexandra M.; Saponjian, Yero; Mahoney, Mark M.; Pimentel, Corrin A.; Lucini, Corrina A.; Usenovic, Marija; Staley, Kevin J.

2013-01-01

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an antiepileptogenic, or merely anti-convulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with LDH release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well-correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are: (1), the organotypic hippocampal culture model of posttraumatic epilepsy comprises a rapid assay of antiepileptogenic and neuroprotective activities and, in this model (2), mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy. PMID:23699517

Pulsed dye laser double-pass treatment of patients with resistant capillary malformations.

PubMed

Rajaratnam, Ratna; Laughlin, Sharyn A; Dudley, Denis

2011-07-01

The pulsed dye laser is an effective and established treatment for port-wine stains and has become the generally accepted standard of care. However, in many cases, complete clearance cannot be achieved as a significant proportion of lesions become resistant to treatment. Multiple passes or pulse-stacking techniques have been used to improve the extent and rate of fading, but concerns over increased adverse effects have limited this clinical approach. In this work, a double-pass technique with the pulsed dye laser has been described, which may allow for increased depth of vascular injury, greater efficacy, and an acceptable risk profile. Our aim was to determine the efficacy and the rate of side-effects for a double-pass protocol with a pulsed dye laser (PDL) to treat patients previously treated with PDL and/or other laser modalities. A retrospective chart review was conducted of 26 patients treated with a minimum of three double-pass treatments alone, or in combination, with single pass conventional PDL. Almost half of the patients (n = 12) showed either a moderate or significant improvement in fading compared to pre-treatment photographs with the double-pass technique. In a further 12 patients, there was a mild improvement. In two patients, there was no change. Sixteen patients developed mild side-effects: blisters (n = 5), dry scabs (n = 11) and transient hyperpigmentation (n = 4). This preliminary experience suggests that a double-pass technique at defined intervals between the first and second treatment with PDL can further lighten some port-wine stains, which are resistant to conventional single-pass treatments. This technique may be a useful addition to the laser treatment of PWS and deserves further scrutiny with randomized prospective studies and histological analysis to confirm the increased depth of vascular injury.

Induction and repair of DNA double-strand breaks in rat cerebellar cortex exposed to 60Co γ-rays

NASA Astrophysics Data System (ADS)

Bulanova, T. S.; Zadneprianetc, M. G.; Ježková, L.; Kruglyakova, E. A.; Smirnova, E. V.; Boreyko, A. V.

2018-01-01

The induction and repair of DNA double-strand breaks are studied using the immunohistochemical staining procedure of paraffin-embedded rat cerebellum tissues after exposure to γ-rays of 60Co. The dose dependence of radiation-induced colocalized γH2AX/53BP1 foci is studied and its linear character is established. It is shown that these foci are efficiently eliminated 24 h after irradiation.

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy.

PubMed

Inaga, Sumire; Katsumoto, Tetsuo; Tanaka, Keiichi; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2007-04-01

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Double Pass 595 nm Pulsed Dye Laser Does Not Enhance the Efficacy of Port Wine Stains Compared with Single Pass: A Randomized Comparison with Histological Examination.

PubMed

Yu, Wenxin; Zhu, Jiafang; Wang, Lizhen; Qiu, Yajing; Chen, Yijie; Yang, Xi; Chang, Lei; Ma, Gang; Lin, Xiaoxi

2018-03-27

To compare the efficacy and safety of double-pass pulsed dye laser (DWL) and single-pass PDL (SWL) in treating virgin port wine stain (PWS). The increase in the extent of vascular damage attributed to the use of double-pass techniques for PWS remains inconclusive. A prospective, side-by-side comparison with a histological study for virgin PWS is still lacking. Twenty-one patients (11 flat PWS, 10 hypertrophic PWS) with untreated PWS underwent 3 treatments at 2-month intervals. Each PWS was divided into three treatment sites: SWL, DWL, and untreated control. Chromametric and visual evaluation of the efficacy and evaluation of side effects were conducted 3 months after final treatment. Biopsies were taken at the treated sites immediately posttreatment. Chromametric and visual evaluation suggested that DWL sites showed no significant improvement compared with SWL (p > 0.05) in treating PWS. The mean depth of photothermal damage to the vessels was limited to a maximum of 0.36-0.41 mm in both SWL and DWL sides. Permanent side effects were not observed in any patients. Double-pass PDL does not enhance PWS clearance. To improve the clearance of PWS lesions, either the depth of laser penetration should be increased or greater photothermal damage to vessels should be generated.

Acridine orange staining reaction as an index of physiological activity in Escherichia coli

NASA Technical Reports Server (NTRS)

McFeters, G. A.; Singh, A.; Byun, S.; Callis, P. R.; Williams, S.

1991-01-01

The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli. Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo. Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction. Cells from log phase appeared red, whereas those in stationary phase were green. However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used. Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction. Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green. These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions. However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction. The importance of validating the putative physiological implications of this staining reaction is stressed.

Sign Reversal of the MN-O Bond Compressibility in La{sub 1.2}Sr{sub 1.8}Mn{sub 2}O{sub 7} Below T{sub C}: Exchange Striction in the Ferromagnetic State

DOE R&D Accomplishments Database

Argyriou, D. N.; Mitchell, J. F.; Chmaissem, O.; Short, S.; Jorgensen, J. D.; Goodenough, J. B.

1997-03-01

The crystal structure of the layered perovskite La{sub 1.2}Sr{sub 1.8}Mn{sub 2}O{sub 7} has been studied under hydrostatic pressure up to {approximately} 6 kbar, in the paramagnetic and ferromagnetic states, with neutron powder diffraction. The compressibility of the Mn-O apical bonds in the double layer of MnO{sub 6} octahedra changes sign from the paramagnetic insulator (PI) to the ferromagnetic metal (FM) state; in the Fm state the Mn-O-Mn linkage between MnO{sub 2} planes expands under applied pressure, whereas they contract in the PI state. This counterintuative behavior is interpreted in terms of exchange striction, which reflect the competition between super- and double-exchange. An increase of the Mn-moment with applied pressure in the FM state is consistent with a positive dT{sub C}/dP, as well as a cant angle {theta}{sub 0} between the magnetizations of neighboring MnO{sub 2} sheets that decreases with pressure.

Single and double spin asymmetries for pion electro-production from the deuteron in the resonance region

NASA Astrophysics Data System (ADS)

Careccia, Sharon L.

The single and double spin asymmetries At and Aet have been measured in pi- electro-production off the deuteron using a longitudinally polarized electron beam and a polarized ND3 target. The electron beam was polarized using a strained GaAs cathode and the target was polarized using Dynamic Nuclear Polarization. The data were collected at beam energies of 1.6, 1.7, 2.5 and 4.2 GeV in Hall B at Jefferson Lab in the spring of 2001. The final state particles were detected in the CEBAF Large Acceptance Spectrometer (CLAS). The d(e,e'pi-p)p exclusive channel was identified using the missing mass technique and the asymmetries were extracted as a function of the momentum transfer Q2, invariant mass W, and center of mass pion angles cos(theta*) and φ*. The results are generally in agreement with the phenomenological model MAID at low energies, but there are discrepancies in the 2nd and 3rd resonance regions, as well as at forward angles.

Downregulation of PI3K/Akt/mTOR signaling pathway in curcumin-induced autophagy in APP/PS1 double transgenic mice.

PubMed

Wang, Chen; Zhang, Xiong; Teng, Zhipeng; Zhang, Tong; Li, Yu

2014-10-05

Autophagy is a lysosomal degradation pathway, which is essential for cell survival, proliferation, differentiation and homeostasis. It is well known that beta-amyloid (Aβ) aggregation is one of key characteristics for Alzheimer's disease (AD), which triggers a complex pathological cascade, leading to neurodegeneration. Recent studies have shown that Aβ peptide is generated from amyloid β precursor protein (APP) during autophagic turnover of APP-rich organelles by autophagy. Aβ generation during normal autophagy is subsequently degraded by lysosomes. Curcumin, a nature plant extraction, has been reported to inhibit the generation and deposition of Aβ; however, the underlying mechanisms are not fully understood yet. In the present study, we reported that curcumin treatment not only attenuated cognitive impairment detected by Morris water maze test, but also inhibited the generation of Aβ investigated by immunohistochemistry in APP/PS1 double transgenic AD mice. Moreover, curcumin induced autophagy in the mice, evidenced by LC3 immunofluorescence analysis and western blot assays on LC3. Furthermore, we found that curcumin significantly decreased the expression of Phosphatidylinositol 3-Kinase (PI3K), phosphorylated Akt and rapamycin (mTOR) at protein levels, respectively. Taken together, our data suggests that curcumin inhibits Aβ generation and induces of autophagy by downregulating PI3K/Akt/mTOR signaling pathway, and further shows a neuroprotective effect. Meanwhile curcumin might be a candidate neuroprotective agent for AD patients treatment by inducing autophagy. Copyright © 2014 Elsevier B.V. All rights reserved.

Double-blind placebo-controlled study of mesalamine in post-infective irritable bowel syndrome--a pilot study.

PubMed

Tuteja, Ashok K; Fang, John C; Al-Suqi, Manal; Stoddard, Gregory J; Hale, Devon C

2012-10-01

Post-infective irritable bowel syndrome (PI-IBS) is characterized by continuing symptoms of irritable bowel syndrome, typically diarrhea-predominant, following an episode of acute gastroenteritis. There is often an increase in sub-epithelial inflammatory and neuroendocrine cells on colonic mucosal biopsy. Mesalamine is an anti-inflammatory agent, effective in the treatment of inflammatory bowel disease. The goal of this study was to compare mesalamine to placebo on symptoms and quality-of-life (QOL) in PI-IBS. Twenty patients who developed diarrhea-predominant IBS after gastroenteritis were randomized to receive mesalamine (Asacol®) 1.6 gm b.i.d. or placebo for 12 weeks in a double-blind placebo-controlled study. QOL was assessed using the IBS-QOL questionnaire. Stool frequency, stool consistency, urgency, severity of abdominal pain, severity of bloating, and global-improvement scale were recorded in daily diaries for 7 days at baseline and every 4 weeks. Data were analyzed by comparing the change from baseline to last follow-up. One patient withdrew after randomization; data were incomplete in two patients. Thus, data were analyzed from 17 patients (11 men and 6 women, median age: 27 years, range 22-45 years). Mesalamine was not associated with significant improvement in global symptoms, abdominal pain, bloating, stool urgency, frequency, or consistency (all p ≥ 0.11) or QOL (p ≥ 0.16). There was no significant improvement in global symptoms or overall QOL with mesalamine in patients with PI-IBS.

Analysis of PI3K/mTOR Pathway Biomarkers and Their Prognostic Value in Women with Hormone Receptor–Positive, HER2-Negative Early Breast Cancer1

PubMed Central

Azim, Hamdy A.; Kassem, Loay; Treilleux, Isabelle; Wang, Qing; El Enein, Mona Abu; Anis, Shady E.; Bachelot, Thomas

2016-01-01

BACKGROUND: The PI3K/AKT/mTOR pathway alterations have been shown to play significant roles in the development, progression, and metastatic spread of breast cancer. Furthermore, they have been implicated in the process of drug resistance, especially endocrinal therapies. In this study, we aimed to define the correlation between the PI3K mutations and the expression of the phosphorylated forms of different downstream molecules in women with estrogen receptor (ER)–positive, human epidermal growth factor receptor 2–negative (luminal) early breast cancer treated at Cairo university hospitals. METHODS: Next-generation sequencing was used to detect mutations in the PIK3CA hotspots (in exons 9 and 20). Immunohistochemistry was performed on tissue microarray blocks prepared from samples of 35 Egyptian luminal breast cancer patients in the pathology department of Centre Léon Bérard (CLB). The intensity and the percentage of stained tumor cells were integrated to define high versus low biomarker expression. The cytoplasmic and nuclear stainings were graded separately. Patients were followed for a median of 4.7 years (2.1 to 6.9 years). Correlation was done between PI3K mutations and the immunohistochemistry expression of pAKT, LKB1, p4EBP1, and pS6 ribosomal protein (pS6RP) with the clinicopathologic features and disease free survival (DFS) of the patients. RESULTS: Median age at diagnosis was 51.3 years (range, 25 to 82 years). Tumors were larger than 20 mm in 79.2% of the cases, whereas 57.9% had axillary lymph node deposits. Only 12.3% of the patients had SBR grade I tumors, 50.8% had grade II, and 36.8% had grade III. ERs were negative in 6 patients (17%) after pathology review. Thirty-two cases were assessable for LKB1 and pAKT, 33 for p4EBP1 and pS6RP, and 24 for PI3K mutations. Nuclear LKB1, cytoplasmic LKB1, nuclear pAKT, cytoplasmic pAKT, nuclear p4EBP1, and cytoplasmic pS6RP expression was high in 65.6%, 62.5%, 62.5%, 68.8%, 42.4%, and 57.6%, respectively. PIK3CA mutations were found in 7 patients (29.2%). PI3K mutations were correlated with nuclear localization of pAKT (i.e., decreased cytoplasmic pAKT, P = .04; and increased nuclear pAKT, P = .10). There was a tendency toward an inverse correlation between PI3K mutations and the expression of pS6RP (P = .10) and p4EBP1 (P = .19). Nuclear LKB1 expression was a marker of good prognosis. It was associated with smaller tumors (P = .05), more ER (P = .08) and progesteron receptor (PgR) positivity (P = .002). In the Kaplan Meier (KM) model, patients with high nuclear LKB1 had longer DFS (hazard ratio = 0.36; 95% confidence interval, 0.15-1.10; P = .08). Nuclear pAKT high expression also carried a tendency toward longer DFS (hazard ratio = 0.51; 95% confidence interval, 0.11-1.16; P = .13). The expression of p4EBP1, pS6RP, and the PI3K mutational status did not show any prognostic significance in our cohort. CONCLUSION: Among the studied biomarkers, only nuclear expression of LKB1 and pAKT tended to predict better survival in breast cancer patients. PI3K mutation was correlated with the expression of nuclear pAKT but not pS6RP or p4EBP1. PMID:27084427

Acacia catechu Ethanolic Seed Extract Triggers Apoptosis of SCC-25 Cells.

PubMed

Lakshmi, Thangavelu; Ezhilarasan, Devaraj; Nagaich, Upendra; Vijayaragavan, Rajagopal

2017-10-01

Acacia catechu Willd ( Fabaceae ), commonly known as catechu, cachou, and black cutch, has been studied for its hepatoprotective, antipyretic, antidiarrheal, hypoglycemic, anti-inflammatory, immunomodulatory, antinociceptive, antimicrobial, free radical scavenging, and antioxidant activities. We evaluated the cytotoxic activity of ethanol extract of A. catechu seed (ACS) against SCC-25 human oral squamous carcinoma cell line. Cytotoxic effect of ACS extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, using concentrations of 0.1-1000 μg/mL for 24 h. A. catechu ethanol seed extract was treated SCC-25 cells with 25 and 50 μg/mL. At the end of treatment period, apoptotic marker gene expressions such as caspase 8, 9, Bcl-2, Bax, and cytochrome c were evaluated by semiquantitative reverse transcription-polymerase chain reaction. Morphological changes of ACS treated SCC-25 cells was evaluated by acridine orange/ethidium bromide (AO/EB) dual staining. Nuclear morphology and DNA fragmentation was evaluated by propidium iodide (PI) staining. A. catechu ethanol seed extract treatment caused cytotoxicity in SCC-25 cells with an IC 50 value of 100 μg/mL. Apoptotic markers caspases 8 and 9, cytochrome c, Bax gene expressions were significantly increased upon ACS extract treatment indicate the apoptosis induction in SCC-25 cells. This treatment also caused significant downregulation of Bcl-2 gene expression. Staining with AO/EB and PI shows membrane blebbing, and nuclear membrane distortion further confirms the apoptosis induction by ACS treatment in SCC-25 cells. The ethanol seed extracts of A. catechu was found to be cytotoxic at lower concentrations and induced apoptosis in human oral squamous carcinoma SCC-25 cells. Acacia catechu ethanolic seed extract contains phytochemicals such as epicatechin, rutin, and quercetin Acacia catechu seed (ACS) extract significantly ( P < 0.001) inhibits the active proliferation of human oral squamous carcinoma (SCC-25) cellsACS extract treatment to SCC-25 cells significantly modulated the gene expressions pertaining to apoptosis and propidium iodide and acridine orange/ethidium bromide staining also confirm the apoptosis inductionAntiproliferative and apoptosis inducing activities of ACS extract is correlated with phytochemical contents. Abbreviations used: ACS: Acacia catechu seed extract; MTT: 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide; DMSO: Dimethyl sulfoxide; AO/EO: Acridine orange/ethidium bromide; LC MS: Liquid chromatography mass spectrometry.

Mapping Large-Scale Mechanized Agriculture Across the Brazilian Cerrado Between 2001-2013

NASA Astrophysics Data System (ADS)

Spera, S. A.; Mustard, J. F.; VanWey, L.

2014-12-01

Brazil is a global commodities powerhouse. Over the last decade, dynamic changes in agricultural development and land transformations occurred within Brazil's tropical savanna region, the cerrado. This interdisciplinary study uses remote sensing tools to map land cover across more than 3.6 million km2 of cerrado and statistical methods to characterize drivers of this land-cover change. We use the MODIS Enhanced Vegetation Index 16-day data product and a decision-tree algorithm, proven highly accurate in Mato Grosso (Spera et al. 2014) and here modified for the broader cerrado region, to characterize crop type, cropping frequency, expansion, and abandonment of large-scale mechanized agriculture during the 2001-2013 period. The algorithm exploits phenological differences between forest, pasture and cerrado, and mechanized agriculture. It is parameterized to distinguish between crop rotations in Mato Grosso, Goias, and the new agricultural frontier spanning Maranhao, Tocantins, Piaui, and Bahia (MaToPiBa). Training and validation data were collected using Google's Earth Engine. We map single-cropped soy, corn, and cotton; double-cropped soy/corn and soy/cotton rotations; and irrigated agriculture across these six Brazilian cerrado states. We find that while double cropping dominates in Mato Grosso and Goias, single cropping is still the dominant form of mechanized agriculture in the burgeoning MaToPiBa region. In western Bahia alone, preliminary results show agriculture has expanded by almost 350,000 ha and double cropping has increased by almost 40,000 ha. With MaToPiBa touted as Brazil's latest and last agricultural frontier, we predict that the region will experience a transition similar to that of Mato Grosso during the 2000s—an expansion and intensification of agriculture—which may beget unprecedented ramifications on regional climate processes that can then affect ecosystem health and the economic feasibility of cultivating rain-fed export crops.

Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

DTIC Science & Technology

2017-01-01

regulated by Pim and what is the outcome of treating animals with Pim inhibitors. To explore these tasks we first stained human tumor and normal samples ...Sepharose beads. The beads were incubated with 100 ng of purified Pim-1 proteins at 37 °C for 30 minutes. Samples were analyzed by immunoblot assays using...secreted by these cells using novel technology, develop potential urine biomarkers of treatment success. 1R21 CA178324-01A1 (Kraft, PI) 06/01/15

Theoretical structures and binding energies of RNA-RNA/cyanine dyes and spectroscopic properties of cyanine dyes

NASA Astrophysics Data System (ADS)

Salaeh, Salsabila; Chong, Wei Lim; Dokmaisrijan, Supaporn; Payaka, Apirak; Yana, Janchai; Nimmanpipug, Piyarat; Lee, Vannajan Sanghiran; Dumri, Kanchana; Anh, Dau Hung

2014-10-01

Cyanine dyes have been widely used as a fluorescence probe for biomolecules and protein labeling. The mostly used cyanine dyes for nucleic acids labeling are DiSC2(3), DiSC2(5), and DiSC2(7). The possible structures and binding energies of RNA-RNA/Cyanine dyes were predicted theoretically using AutoDock Vina. The results showed that cyanine dyes and bases of RNA-RNA have the van der Waals and pi-pi interactions. The maximum absorption wavelength in the visible region obtained from the TD-DFT calculations of all cyanine dyes in the absence of the RNA-RNA double strand showed the bathochromic shift.

Perfusion index derived from a pulse oximeter can detect changes in peripheral microcirculation during uretero-renal-scopy stone manipulation (URS-SM).

PubMed

Huang, Ho-Shiang; Chu, Chun-Lin; Tsai, Chia-Ti; Wu, Cho-Kai; Lai, Ling-Ping; Yeh, Huei-Ming

2014-01-01

The objective of this study was to test the effect of removal of a ureteral obstruction (renal calculus) from anesthetized patients on the perfusion index (PI), as measured by a pulse oximeter, and on the estimated glomerular filtration rate (eGFR). This prospective study enrolled 113 patients with unilateral ureteral obstructions (kidney stones) who were scheduled for ureteroscopy (URS) laser lithotripsy. One urologist graded patient hydronephrosis before surgery. A pulse oximeter was affixed to each patient's index finger ipsilateral to the intravenous catheter, and a non-invasive blood pressure cuff was placed on the contralateral side. Ipsilateral double J stents and Foley catheters were inserted and left indwelling for 24 h. PI and mean arterial pressure (MAP) were determined at baseline, 5 min after anesthesia, and 10 min after surgery; eGFR was determined at admission, 1 day after surgery, and 14 days after surgery. Patients with different grades of hydronephrosis had similar age, eGFR, PI, mean arterial pressure (MAP), and heart rate (HR). PI increased significantly in each hydronephrosis group after ureteral stone disintegration. None of the groups had significant post-URS changes in eGFR, although eGFR increased in the grade I hydronephrosis group after 14 days. The percent change of PI correlates significantly with the percent change of MAP, but not with that of eGFR. Our results demonstrate that release of a ureteral obstruction leads to a concurrent increase of PI during anesthesia. Measurement of PI may be a valuable tool to monitor the successful release of ureteral obstructions and changes of microcirculation during surgery. There were also increases in eGFR after 14 days, but not immediately after surgery.

Source identification of PM2.5 at a port and an adjacent urban site in a coastal city of China: Impact of ship emissions and port activities.

PubMed

Xu, Lingling; Jiao, Ling; Hong, Zhenyu; Zhang, Yanru; Du, Wenjiao; Wu, Xin; Chen, Yanting; Deng, Junjun; Hong, Youwei; Chen, Jinsheng

2018-09-01

Daily PM 2.5 samples were collected simultaneously at an urban site (UB) and a nearby port-industrial site (PI) on the coast of southeastern China from April 2015 to January 2016. The PM 2.5 mass concentration at the PI (51.9μgm -3 ) was significantly higher than that at the UB. The V concentration at the PI was also significantly higher and well-correlated to the urban value, which suggests that shipping emissions had a significant impact on the PI and, to a lesser extent, on the urban area. A positive matrix factorization (PMF) analysis showed that secondary aerosols were the dominant contribution of PM 2.5 at both sites (36.4% at the PI and 27.2% at the UB), while the contribution of industry and ship emissions identified by V, Mn, and Ba at the PI (26.1%) were double those at the UB. The difference in each source contribution among the trajectory clusters that included significant differences and insignificant differences from the UB to the PI provided insight into the role of local impacts. With regards to the UB, local potential sources play important roles in industry and ship emissions, traffic emissions, fugitive dust, and in their contributions to secondary aerosols. A conditional probability function further revealed that the ship emissions and port activities distributed in the NE, E, and SSE wind sectors were responsible for the source contributions of industry and ship emissions and secondary aerosols at the UB. This study provides an example of investigating the impact of ship emissions and port activities on the surrounding air environment using land-based measurements. Copyright © 2018 Elsevier B.V. All rights reserved.

Perfusion Index Derived from a Pulse Oximeter Can Detect Changes in Peripheral Microcirculation during Uretero-Renal-Scopy Stone Manipulation (URS-SM)

PubMed Central

Huang, Ho-Shiang; Chu, Chun-Lin; Tsai, Chia-Ti; Wu, Cho-Kai; Lai, Ling-Ping; Yeh, Huei-Ming

2014-01-01

Background The objective of this study was to test the effect of removal of a ureteral obstruction (renal calculus) from anesthetized patients on the perfusion index (PI), as measured by a pulse oximeter, and on the estimated glomerular filtration rate (eGFR). Patients and Methods This prospective study enrolled 113 patients with unilateral ureteral obstructions (kidney stones) who were scheduled for ureteroscopy (URS) laser lithotripsy. One urologist graded patient hydronephrosis before surgery. A pulse oximeter was affixed to each patient's index finger ipsilateral to the intravenous catheter, and a non-invasive blood pressure cuff was placed on the contralateral side. Ipsilateral double J stents and Foley catheters were inserted and left indwelling for 24 h. PI and mean arterial pressure (MAP) were determined at baseline, 5 min after anesthesia, and 10 min after surgery; eGFR was determined at admission, 1 day after surgery, and 14 days after surgery. Results Patients with different grades of hydronephrosis had similar age, eGFR, PI, mean arterial pressure (MAP), and heart rate (HR). PI increased significantly in each hydronephrosis group after ureteral stone disintegration. None of the groups had significant post-URS changes in eGFR, although eGFR increased in the grade I hydronephrosis group after 14 days. The percent change of PI correlates significantly with the percent change of MAP, but not with that of eGFR. Conclusion Our results demonstrate that release of a ureteral obstruction leads to a concurrent increase of PI during anesthesia. Measurement of PI may be a valuable tool to monitor the successful release of ureteral obstructions and changes of microcirculation during surgery. There were also increases in eGFR after 14 days, but not immediately after surgery. PMID:25542000

A series of substituted (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-phenylprop-2-en-1-ones.

PubMed

Chopra, Deepak; Mohan, T P; Vishalakshi, B; Row, T N Guru

2007-12-01

In the molecular structures of a series of substituted chalcones, namely (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-phenylprop-2-en-1-one, C21H15FO2, (I), (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-(4-fluorophenyl)prop-2-en-1-one, C21H14F2O2, (II), (2E)-1-(4-chlorophenyl)-3-(2-fluoro-4-phenoxyphenyl)prop-2-en-1-one, C21H14ClFO2, (III), (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-(4-methylphenyl)prop-2-en-1-one, C22H17FO2, (IV), and (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one, C22H17FO3, (V), the configuration of the keto group with respect to the olefinic double bond is s-cis. The molecules pack utilizing weak C-H...O and C-H...pi intermolecular contacts. Identical packing motifs involving C-H...O interactions, forming both chains and dimers, along with C-H...pi dimers and pi-pi aromatic interactions are observed in the fluoro, chloro and methyl derivatives.

ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis.

PubMed

Nascimbeni, Anna Chiara; Giordano, Francesca; Dupont, Nicolas; Grasso, Daniel; Vaccaro, Maria I; Codogno, Patrice; Morel, Etienne

2017-07-14

The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER-plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER-plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. © 2017 The Authors.

IR-UV double resonance spectroscopic investigation of phenylacetylene-alcohol complexes. Alkyl group induced hydrogen bond switching.

PubMed

Singh, Prashant Chandra; Patwari, G Naresh

2008-06-12

The electronic transitions of phenylacetylene complexes with water and trifluoroethanol are shifted to the blue, while the corresponding transitions for methanol and ethanol complexes are shifted to the red relative to the phenylacetylene monomer. Fluorescence dip infrared (FDIR) spectra in the O-H stretching region indicate that, in all the cases, phenylacetylene is acting as a hydrogen bond acceptor to the alcohols. The FDIR spectrum in the acetylenic C-H stretching region shows Fermi resonance bands for the bare phenylacetylene, which act as a sensitive tool to probe the intermolecular structures. The FDIR spectra reveal that water and trifluoroethanol interact with the pi electron density of the acetylene C-C triple bond, while methanol and ethanol interact with the pi electron density of the benzene ring. It can be inferred that the hydrogen bonding acceptor site on phenylacetylene switches from the acetylene pi to the benzene pi with lowering in the partial charge on the hydrogen atom of the OH group. The most significant finding is that the intermolecular structures of water and methanol complexes are notably distinct, which, to the best of our knowledge, this is first such observation in the case of complexes of substituted benzenes.

Measurement of the helicity asymmetry E in ω → π + π - π 0 photoproduction

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

Akbar, Z.; Roy, P.; Park, S.

The double-polarization observablemore » $E$ was studied for the reaction $$\\gamma p\\to p\\omega$$ using the CEBAF Large Acceptance Spectrometer (CLAS) in Hall B at the Thomas Jefferson National Accelerator Facility and the longitudinally-polarized frozen-spin target (FROST). The observable was measured from the charged decay mode of the meson, $$\\omega\\to\\pi^+\\pi^-\\pi^0$$, using a circularly-polarized tagged-photon beam with energies ranging from the $$\\omega$$ threshold at 1.1 to 2.3 GeV. A partial-wave analysis within the Bonn-Gatchina framework found dominant contributions from the $3/2^+$ partial wave near threshold, which is identified with the sub-threshold $$N(1720)\\,3/2^+$$ nucleon resonance. To describe the entire data set, which consisted of $$\\omega$$ differential cross sections and a large variety of polarization observables, further contributions from other nucleon resonances were found to be necessary. Here, with respect to non-resonant mechanisms, $$\\pi$$ exchange in the $t$-channel was found to remain small across the analyzed energy range, while pomeron $t$-channel exchange gradually grew from the reaction threshold to dominate all other contributions above $$W \\approx 2$$ GeV.« less

Measurement of the helicity asymmetry E in ω → π + π - π 0 photoproduction

DOE PAGES

Akbar, Z.; Roy, P.; Park, S.; ...

2017-12-28

The double-polarization observablemore » $E$ was studied for the reaction $$\\gamma p\\to p\\omega$$ using the CEBAF Large Acceptance Spectrometer (CLAS) in Hall B at the Thomas Jefferson National Accelerator Facility and the longitudinally-polarized frozen-spin target (FROST). The observable was measured from the charged decay mode of the meson, $$\\omega\\to\\pi^+\\pi^-\\pi^0$$, using a circularly-polarized tagged-photon beam with energies ranging from the $$\\omega$$ threshold at 1.1 to 2.3 GeV. A partial-wave analysis within the Bonn-Gatchina framework found dominant contributions from the $3/2^+$ partial wave near threshold, which is identified with the sub-threshold $$N(1720)\\,3/2^+$$ nucleon resonance. To describe the entire data set, which consisted of $$\\omega$$ differential cross sections and a large variety of polarization observables, further contributions from other nucleon resonances were found to be necessary. Here, with respect to non-resonant mechanisms, $$\\pi$$ exchange in the $t$-channel was found to remain small across the analyzed energy range, while pomeron $t$-channel exchange gradually grew from the reaction threshold to dominate all other contributions above $$W \\approx 2$$ GeV.« less

[Study on the choice of functional monomer before preparation of myclobutanil molecularly imprinted polymer].

PubMed

Gao, Wen-Hui; Liu, Bo; Li, Xing-Feng; Han, Jun-Hua; Jia, Ying-Min

2014-03-01

To prepare myclobutanil molecularly imprinted polymer, a method was established for the choice of the appropriate functional monomer and its dosage. UV spectra was applied to study the combination form, the effect intensity, the optimal concentration ratio and the numbers of binding sites between myclobutanil and methyl acrylic acid (MAA) or acrylamide (AM) functional monomer. The results showed that hydrogen-bonding interaction could be formed between myclobutanil and methyl acrylic acid (MAA) or acrylamide (AM) functional monomer. The pi electron of the triazole ring conjugated double bond in my clobutanil could transit to pi* conjugate antibonding orbital when it absorbed energy. The formation of hydrogen bond could make pi-->pi* absorption band transit. Maximum absorption wavelength produced red shift with the increase in the functional monomer concentration in the system. The research revealed that the optimal concentration ratios between myclobutanil and the two monomers were c(M):c(MAA) = 1:4, c(M):c(AM) = 1:2. Myclobutanil and the both the functional monomers had the bonding ability, and strong bonding force. The prepared molecularly imprinted polymer using AM as a functional monomer had better stability and specificity of recognition for myclobutanil.

AFRRI (Armed Forces Radiobiology Research Institute) Reports, July, August and September 1987.

DTIC Science & Technology

1987-11-01

mononuclear cell layer obtained after Percol isolation contained approximately 90% mono- cytes as assessed by esterase staining. In most experiments...forming cell) were assayed using the double layer agar technique basically as described by Hagan et al. (22). The culture medium was double strength CMRL...trypticase soy broth, 20 g/ml L-asparagine. and penicillin-streptomycin. In the bottom layer of 35 mm plastic Petri dishes was 1 ml of a 1:1 mixture of culture

Influence of the charge double layer on solid oxide fuel cell stack behavior

NASA Astrophysics Data System (ADS)

Whiston, Michael M.; Bilec, Melissa M.; Schaefer, Laura A.

2015-10-01

While the charge double layer effect has traditionally been characterized as a millisecond phenomenon, longer timescales may be possible under certain operating conditions. This study simulates the dynamic response of a previously developed solid oxide fuel cell (SOFC) stack model that incorporates the charge double layer via an equivalent circuit. The model is simulated under step load changes. Baseline conditions are first defined, followed by consideration of minor and major deviations from the baseline case. This study also investigates the behavior of the SOFC stack with a relatively large double layer capacitance value, as well as operation of the SOFC stack under proportional-integral (PI) control. Results indicate that the presence of the charge double layer influences the SOFC stack's settling time significantly under the following conditions: (i) activation and concentration polarizations are significantly increased, or (ii) a large value of the double layer capacitance is assumed. Under normal (baseline) operation, on the other hand, the charge double layer effect diminishes within milliseconds, as expected. It seems reasonable, then, to neglect the charge double layer under normal operation. However, careful consideration should be given to potential variations in operation or material properties that may give rise to longer electrochemical settling times.

Protective effects of Bacopa monnieri on ischemia-induced cognitive deficits in mice: the possible contribution of bacopaside I and underlying mechanism.

PubMed

Le, Xoan Thi; Nguyet Pham, Hang Thi; Van Nguyen, Tai; Minh Nguyen, Khoi; Tanaka, Ken; Fujiwara, Hironori; Matsumoto, Kinzo

2015-04-22

Bacopa monnieri (L.) Wettst. (BM) is a medicinal plant which has been not only used as a traditional medicine to improve intelligence and memory but also taken as vegetables in Vietnam for a long time. We previously demonstrated that Bacopa monnieri (BM) alcohol extract attenuated olfactory bulbectomy-induced cognitive deficits and the deterioration of septo-hippocampal cholinergic neurons, suggesting the beneficial effects of BM for dementia patients. The present study was conducted to further clarify the anti-dementia effects of BM, using transient 2 vessels occlusion (T2VO)-induced cognitive deficits in mice, an animal model of vascular dementia, and also to investigate the constituent(s) contributing to the actions of BM, using oxygen- and glucose-deprivation (OGD)-induced hippocampal cell damage as an in vitro model of ischemia. In the in vivo experiments, T2VO mice were treated daily with a standardized BM extract (50mg/kg, p.o.) 1 week before and continuously 3 days after surgery. In the in vitro experiments, organotypic hippocampal slice cultures (OHSCs) were incubated with triterpenoid saponins from BM (bacosides) or MK-801 1h before and during a 45-min period of OGD. Neuronal cell damage in OHSCs was analyzed by measurement of propidium iodide uptake 24h after OGD. The BM treatment significantly ameliorated T2VO-induced impairments in non-spatial short term memory performance in the object recognition test. Among the bacosides tested in the in vitro experiments using OHSCs, bacopaside I (25 μM) exhibited potent neuroprotective effects against OGD-induced neuronal cell damage. Double staining with TUNEL and PI revealed that OGD caused necrosis and apoptosis and that bacopaside I attenuated the effects of OGD. The neuroprotective effects of bacopaside I were blocked by the PKC inhibitor Ro-31-8220 and PI3K inhibitor LY294002, but not by the ERK inhibitor U0126. OGD reduced the level of phospho-Akt (p-Akt), an anti-apoptotic factor, in OHSCs. This decrease was reversed by bacopaside I. Moreover, the treatment with bacopaside I itself was able to elevate the level of p-Akt in OHSCs. These results suggest that BM was beneficial for the prevention of cognitive deficits related to cerebral ischemia and also that bacopaside I, via PKC and PI3K/Akt mechanisms, played a role in the neuroprotective effects of BM observed in the mouse model. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

PubMed

Wagner, J M; Franco, M; Kephart, G M; Gleich, G J

1998-07-01

Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

Detection of endolithic spatial distribution in marble stone.

PubMed

Casanova Municchia, A; Percario, Z; Caneva, G

2014-10-01

The penetration of endolithic microorganisms, which develop to depths of several millimetres or even centimetres into the stone, and the diffusion of their extracellular substances speeds up the stone deterioration process. The aim of this study was to investigate, using a confocal laser scanning microscopy with a double-staining, a marble rock sample by observing the endolithic spatial distribution and quantifying the volume they occupied within the stone, in order to understand the real impact of these microorganisms on the conservation of stone monuments. Often the only factors taken into account by biodeterioration studies regarding endolithic microorganisms, are spread and depth of penetration. Despite the knowledge of three-dimensional spatial distribution and quantification of volume, it is indispensable to understand the real damage caused by endolithic microorganisms to stone monuments. In this work, we analyze a marble rock sample using a confocal laser scanning microscopy stained with propidium iodide and Concavalin-A conjugate with the fluorophore Alexa Fluor 488, comparing these results with other techniques (SEM microscope, microphotographs of polished cross-sections and thin-section, PAS staining methods), An image analysis approach has also been applied. The use of confocal laser scanning microscopy with double staining shows clear evidence of the presence of endolithic microorganisms (cyanobacteria and fungi) as well as the extracellular polymeric substance matrix in a three-dimensional architecture as part of the rock sample, this technique, therefore, seems very useful when applied to restoration interventions on stone monuments when endolithic growth is suspected. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

PubMed

Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

2015-08-01

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

High-flavanol and high-theobromine versus low-flavanol and low-theobromine chocolate to improve uterine artery pulsatility index: a double blind randomized clinical trial.

PubMed

Bujold, Emmanuel; Leblanc, Vicky; Lavoie-Lebel, Élise; Babar, Asma; Girard, Mario; Poungui, Lionel; Blanchet, Claudine; Marc, Isabelle; Lemieux, Simone; Belkacem, Abdous; Sidi, Elhadji Laouan; Dodin, Sylvie

2017-09-01

To evaluate the impact of high-flavanol and high-theobromine (HFHT) chocolate in women at risk of preeclampsia (PE). We conducted a single-center randomized controlled trial including women with singleton pregnancy between 11 and 14 weeks gestation who had bilateral abnormal uterine artery (UtA) waveforms (notching) and elevated pulsatility index (PI). Participants were randomized to either HFHT or low-flavanol and low-theobromine (LFLT) chocolate (30 grams daily for a total of 12 weeks). UtA PI, reported as multiple of medians (MoM) adjusted for gestational age, was assessed at baseline and 12 weeks after randomization. One hundred thirty-one women were randomized with mean gestational age of 12.4 ± 0.6 weeks and a mean UtA PI of 1.39 ± 0.31 MoM. UtA PI adjusted for gestational age significantly decreased from baseline to the second visit (12 weeks later) in the two groups (p < 0.0001) but no significant difference was observed between the groups (p = 0.16). Compared with LFLT chocolate, daily intake of HFHT chocolate was not associated with significant changes of UtA PI. Nevertheless, the improvement observed in both groups suggests that chocolate could improve placental function independently of flavanol and/or theobromine content.

Effect of sodium aescinate treatment on PCOS rat model with insulin resistance.

PubMed

Chen, L; Hu, L M; Wang, Y F; Yang, H Y; Huang, X Y; Zhou, W; Sun, H X

2017-01-01

Recent studies indicated that insulin resistance may contribute to the pathogenesis of polycystic ovary syndrome (PCOS); however, the specific mechanism is still unclear. To investigate the effect of sodium aescinate (SA) on PCOS-IR rat models. Sixty rats were randomly divided into the five groups: un-treated rats (n = 12), PCOS-IR group (n = 12), PCOS-IR group plus 50 mg/kg SA (n = 12), PCOS-IR group plus 10 mg/kg SA (n = 12), PCOS-IR group plus 150 mg/kg metformin (n = 12). On day 21, rats were sacrificed, and H(and)E staining was performed for histopathologic examination of the ovaries; moreover, the serum level of follicle-stimulating hormone (FSH), testosterone, and luteotropic hormone (LH) were measured, and the expression as well as phosphorylation of PI3K, Akt and Gsk-3β were examined using western blot assay. High dosage of SA treatment improved the morphological features of the ovaries in PCOS rats, and also induced significant decrease in serum expression of testosterone and LH/FSH ratio and significant decrease in the expression of p-PI3K, p-Akt and p-Gsk-3β. Our results demonstrated that SA treatment could alleviate the symptom of PCOS in rat model through regulating the PI3K/Akt/GSK3-β pathway (Fig. 4, Ref. 22).

Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

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

Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp

2010-06-25

Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we providemore » a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.« less

MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

PubMed

Du, Juan; Liu, Shuyan; He, Jie; Liu, Xi; Qu, Ying; Yan, Wenqing; Fan, Jianling; Li, Rong; Xi, Hao; Fu, Weijun; Zhang, Chunyang; Yang, Jing; Hou, Jian

2015-06-20

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.

Piperlongumine induces apoptosis and autophagy in leukemic cells through targeting the PI3K/Akt/mTOR and p38 signaling pathways.

PubMed

Wang, Hongfei; Wang, Yongqiang; Gao, Hongmei; Wang, Bing; Dou, Lin; Li, Yin

2018-02-01

Piperlongumine is an alkaloid compound extracted from Piper longum L. It is a chemical substance with various pharmacological effects and medicinal value, including anti-tumor, lipid metabolism regulatory, antiplatelet aggregation and analgesic properties. The present study aimed to understand whether piperlongumine induces the apoptosis and autophagy of leukemic cells, and to identify the mechanism involved. Cell viability and autophagy were detected using MTT, phenazine methyl sulfate and trypan blue exclusion assays. The apoptosis rate was calculated using flow cytometry. The protein expression levels of microtubule-associated protein 1A/1B-light chain 3, Akt and mechanistic target of rapamycin (mTOR) were measured using western blotting. The cell growth of leukemic cells was completely inhibited following treatment with piperlongumine, and marked apoptosis was also induced. Dead cells as a result of autophagy were stained using immunofluorescence and observed under a light microscope. Phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling was suppressed by treatment with piperlongumine, while p38 signaling and caspase-3 activity were induced by treatment with piperlongumine. It was concluded that piperlongumine induces apoptosis and autophagy in leukemic cells through targeting the PI3K/Akt/mTOR and p38 signaling pathways.

Experimental medium energy physics

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

Barnes, P.D.

1991-01-01

This report discusses the following topics: Search for the H Dibaryon at the AGS; Hypernuclear Weak Decay Studies at the AGS; Relativistic Proton-Nucleus and Heavy Ion-Nucleus Collisions at the SPS; Hyperon-Antihyperon Production studies at LEAR; Hyperon Photoproduction at CEBAF; Double Lambda Hypernuclei; Weak Decay of Light Hypernuclei; and {pi}{sup 0}/{gamma}Detection with the CMU Scintillator Arrays.

A novel retrotransposon inserted in the dominant Vm-B1 allele confers spring growth habit in tetraploid wheat (Triticum turgidum L.)

USDA-ARS?s Scientific Manuscript database

Wheat is traditionally divided into winter and spring wheat that either has or lacks a vernalization requirement. In this study, a doubled haploid (DH) population derived from a cross between two spring tetraploid wheat (Triticum turgidum L.) genotypes, durum ‘Lebsock’ and Persian wheat accession PI...

Environmental Impact Statement (EIS) for the Airspace Training Initiative Shaw Air Force Base, South Carolina

DTIC Science & Technology

2011-12-09

Pi -eservation Office fails to respond to ~Agency official finding of no adverse effect ~ithin the 30:.cl.ay review period, then the agency official...by a thin strip of willows (Salix spp.) and raspberry bramble (Rubus spp.). Birds observed in or around Turkey Pond were double-crested cormorant

Ectopic transgene expression in the retina of four transgenic mouse lines

PubMed Central

Gábriel, Robert; Erdélyi, Ferenc; Szabó, Gábor; Lawrence, J. Josh

2017-01-01

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/ Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/ CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR-and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/ CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression. PMID:26563404

Measurement of absolute hadronic branching fractions of D mesons and e+e- -->DD cross sections at Ec.m.=3773 MeV.

PubMed

He, Q; Muramatsu, H; Park, C S; Park, W; Thorndike, E H; Coan, T E; Gao, Y S; Liu, F; Artuso, M; Boulahouache, C; Blusk, S; Butt, J; Dambasuren, E; Dorjkhaidav, O; Li, J; Menaa, N; Mountain, R; Nandakumar, R; Randrianarivony, K; Redjimi, R; Sia, R; Skwarnicki, T; Stone, S; Wang, J C; Zhang, K; Csorna, S E; Bonvicini, G; Cinabro, D; Dubrovin, M; Briere, R A; Chen, G P; Chen, J; Ferguson, T; Tatishvili, G; Vogel, H; Watkins, M E; Rosner, J L; Adam, N E; Alexander, J P; Berkelman, K; Cassel, D G; Crede, V; Duboscq, J E; Ecklund, K M; Ehrlich, R; Fields, L; Gibbons, L; Gittelman, B; Gray, R; Gray, S W; Hartill, D L; Heltsley, B K; Hertz, D; Hsu, L; Jones, C D; Kandaswamy, J; Kreinick, D L; Kuznetsov, V E; Mahlke-Krüger, H; Meyer, T O; Onyisi, P U E; Patterson, J R; Peterson, D; Phillips, E A; Pivarski, J; Riley, D; Ryd, A; Sadoff, A J; Schwarthoff, H; Shi, X; Shepherd, M R; Stroiney, S; Sun, W M; Urner, D; Weaver, K M; Wilksen, T; Weinberger, M; Athar, S B; Avery, P; Breva-Newell, L; Patel, R; Potlia, V; Stoeck, H; Yelton, J; Rubin, P; Cawlfield, C; Eisenstein, B I; Gollin, G D; Karliner, I; Kim, D; Lowrey, N; Naik, P; Sedlack, C; Selen, M; Williams, J; Wiss, J; Edwards, K W; Besson, D; Pedlar, T K; Cronin-Hennessy, D; Gao, K Y; Gong, D T; Hietala, J; Kubota, Y; Klein, T; Lang, B W; Li, S Z; Poling, R; Scott, A W; Smith, A; Dobbs, S; Metreveli, Z; Seth, K K; Tomaradze, A; Zweber, P; Ernst, J; Mahmood, A H; Severini, H; Asner, D M; Dytman, S A; Love, W; Mehrabyan, S; Mueller, J A; Savinov, V; Li, Z; Lopez, A; Mendez, H; Ramirez, J; Huang, G S; Miller, D H; Pavlunin, V; Sanghi, B; Shipsey, I P J; Adams, G S; Chasse, M; Cravey, M; Cummings, J P; Danko, I; Napolitano, J

2005-09-16

Using 55.8 pb(-1) of e+e- collisions recorded at the psi(3770) resonance with the CLEO-c detector at CESR, we determine absolute hadronic branching fractions of charged and neutral D mesons using a double tag technique. Among measurements for three D0 and six D+ modes, we obtain reference branching fractions B(D0-->K-pi+)=(3.91+/-0.08+/-0.09)% and B(D+-->K-pi+pi+)=(9.5+/-0.2+/-0.3)%, where the uncertainties are statistical and systematic, respectively. Final state radiation is included in these branching fractions by allowing for additional, unobserved, photons in the final state. Using a determination of the integrated luminosity, we also extract the cross sections sigma(e+e- -->D0D0)=(3.60+/-0.07(+0.07)(-0.05)) nb and sigma(e+e- -->D+D-)=(2.79+/-0.07(+0.10)(-0.04)) nb.

Near-UV laser treatment of extrinsic dental enamel stains.

PubMed

Schoenly, J E; Seka, W; Featherstone, J D B; Rechmann, P

2012-04-01

The selective ablation of extrinsic dental enamel stains using a 400-nm laser is evaluated at several fluences for completely removing stains with minimal damage to the underlying enamel. A frequency-doubled Ti:sapphire laser (400-nm wavelength, 60-nanosecond pulse duration, 10-Hz repetition rate) was used to treat 10 extracted human teeth with extrinsic enamel staining. Each tooth was irradiated perpendicular to the surface in a back-and-forth motion over a 1-mm length using an ∼300-µm-diam 10th-order super-Gaussian beam with fluences ranging from 0.8 to 6.4 J/cm(2) . Laser triangulation determined stain depth and volume removed by measuring 3D surface images before and after irradiation. Scanning electron microscopy evaluated the surface roughness of enamel following stain removal. Fluorescence spectroscopy measured spectra of unbleached and photobleached stains in the spectral range of 600-800 nm. Extrinsic enamel stains are removed with laser fluences between 0.8 and 6.4 J/cm(2) . Stains removed on sound enamel leave behind a smooth enamel surface. Stain removal in areas with signs of earlier cariogenic acid attacks resulted in isolated and randomly located laser-induced, 50-µm-diam enamel pits. These pits contain 0.5-µm diam, smooth craters indicative of heat transfer from the stain to the enamel and subsequent melting and water droplet ejection. Ablation stalling of enamel stains is typically observed at low fluences (<3 J/cm(2) ) and is accompanied by a drastic reduction in porphyrin fluorescence from the Soret band. Laser ablation of extrinsic enamel stains at 400 nm is observed to be most efficient above 3 J/cm(2) with minimal damage to the underlying enamel. Unsound underlying enamel is also observed to be selectively removed after irradiation. Copyright © 2012 Wiley Periodicals, Inc.

Nucleon Resonance Structure from Exclusive Meson Electroproduction with CLAS and CLAS12

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

Philip L. Cole, Volker D. Burkert, Ralf W. Gothe, Victor I. Mokeev

2012-12-01

The CLAS detector at Jefferson Lab is a unique instrument, which has provided the lion's share of the world's data on meson photo- and electroproduction in the resonance excitation region. The electroexcitation amplitudes for the low-lying resonances P{sub 33} (1232), P{sub 11} (1440), D{sub 13} (1520), and S {sub 11} (1535) were determined over a wide range of Q{sub 2} < 5.0 GeV{sub 2} in a comprehensive analysis of exclusive single-meson ( {pi}{sup +} n, {pi}{sup 0} p) reactions in the electroproduction off protons. Further, we were able to precisely measure {pi}{sup +}{pi}{sup -}p electroproduction differential cross sections provided bymore » the nearly full kinematic coverage of the CLAS detector. And, for the first time, the electrocouplings of the P{sub 11} (1440), D{sub 13} (1520) excited states are determined from the exclusive-{pi}{sup +}{pi}{sup -}p reaction. Consistent results on the electrocouplings from two-independent analyses (single- and double-pion electroproduction) have provided compelling evidence for the reliable extraction of the N* electrocouplings. And preliminary results on the electrocouplings of the S{sub 31} (1620), S{sub 11} (1650), D{sub 33} (1700), and P{sub 13} (1720) states, which decay preferentially to the N{pi} {pi} final states, have recently become available. Theoretical analyses of these results have revealed that there are two major contributions to the resonance structure: a) an internal quark core and b) an external meson-baryon cloud. These CLAS results have had considerable impact on QCD-based studies on N* structure and in the search for manifestations of the dynamical masses of the dressed quarks. Future CLAS12 N* structure studies at high photon virtualities will considerably extend our capabilities in exploring the nature of confinement in baryons.« less

Cadmium telluride quantum dots induce apoptosis in human breast cancer cell lines.

PubMed

Naderi, Saeed; Zare, Hakimeh; Taghavinia, Nima; Irajizad, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

2018-05-01

Semiconductor quantum dots (QDs), especially those containing cadmium, have undergone marked improvements and are now widely used nanomaterials in applicable biological fields. However, great concerns exist regarding their toxicity in biomedical applications. Because of the lack of sufficient data regarding the toxicity mechanism of QDs, this study aimed to evaluate the cytotoxicity of three types of QDs: CdTe QDs, high yield CdTe QDs, and CdTe/CdS core/shell QDs on two human breast cancer cell lines MDA-MB468 and MCF-7. The breast cancer cells were treated with different concentrations of QDs, and cell viability was evaluated via MTT assay. Hoechst staining was applied for observation of morphological changes due to apoptosis. Apoptotic DNA fragmentation was visualized by the agarose gel electrophoresis assay. Flow cytometric annexin V/propidium iodide (PI) measurement was used for apoptosis detection. A significant decrease in cell viability was observed after QDs treatment ( p < 0.05). Apoptotic bodies and chromatin condensation was observed by Hoechst staining. DNA fragmentation assay demonstrated a DNA ladder profile in the exposed cells and also annexin V/PI flow cytometry confirmed apoptosis in a dose-dependent manner. Our results revealed that CdTe, high yield CdTe, and CdTe/CdS core/shell QDs induce apoptosis in breast cancer cell lines in a dose-dependent manner. This study would help realizing the underlying cytotoxicity mechanism, at least partly, of CdTe QDs and may provide information for the development of nanotoxicology and safe use of biological applications of QDs.

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

PubMed

Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

2015-12-01

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

Effects of cold atmospheric plasma on mucosal tissue culture

NASA Astrophysics Data System (ADS)

Welz, Christian; Becker, Sven; Li, Yang-Fang; Shimizu, Tetsuji; Jeon, Jin; Schwenk-Zieger, Sabina; Thomas, Hubertus M.; Isbary, Georg; Morfill, Gregor E.; Harréus, Ulrich; Zimmermann, Julia L.

2013-01-01

Thermal plasmas have been commonly used in medical applications such as plasma ablation and blood coagulation. Newer developments show that plasmas can be generated with ion temperatures close to room temperature: these non-thermal or so-called cold atmospheric plasmas (CAPs) therefore open up a wide range of further biomedical applications. Based on the understanding of the bactericidal, virucidal and fungicidal properties of CAPs, information about the effects of CAP on mucosal cells and tissue is still lacking. Therefore this study focuses on the interaction of CAP with healthy head and neck mucosal cells on a molecular level. To analyse this interaction in detail, fresh tissue samples from healthy nasal and pharyngeal mucosa were harvested during surgery, assembled to a three-dimensional tissue culture model (mini organ cultures) and treated with CAP for different treatment times. Effects on the viability, necrosis induction and mutagenic activity were evaluated with the trypan blue exclusion test, Annexin-V/PI staining and alkaline microgel electrophoresis (comet assay). Trypan blue exclusion test revealed that the CAP treatment significantly decreases the cell viability for all tested treatment times (5, 10, 30, 60 and 120 s p < 0.05), but only a treatment time of 120 s showed a cytotoxic effect as the viability dropped below 90%. Annexin-V/PI staining revealed a significant increase in necrosis in CAP treated pharyngeal tissue cultures for treatment times of 60 and 120 s (p < 0.05). For nasal tissue this effect was already detected for a 30 s treatment (p < 0.05). Comet assay analysis showed no mutagenic effects after exposure to CAP.

Kinetics of membrane damage to high (HNA) and low (LNA) nucleic acid bacterial clusters in drinking water by ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate.

PubMed

Ramseier, Maaike K; von Gunten, Urs; Freihofer, Pietro; Hammes, Frederik

2011-01-01

Drinking water was treated with ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate to investigate the kinetics of membrane damage of native drinking water bacterial cells. Membrane damage was measured by flow cytometry using a combination of SYBR Green I and propidium iodide (SGI+PI) staining as indicator for cells with permeabilized membranes and SGI alone to measure total cell concentration. SGI+PI staining revealed that the cells were permeabilized upon relatively low oxidant exposures of all tested oxidants without a detectable lag phase. However, only ozonation resulted in a decrease of the total cell concentrations for the investigated reaction times. Rate constants for the membrane damage reaction varied over seven orders of magnitude in the following order: ozone > chlorine > chlorine dioxide ≈ ferrate > permanganate > chloramine. The rate constants were compared to literature data and were in general smaller than previously measured rate constants. This confirmed that membrane integrity is a conservative and therefore safe parameter for disinfection control. Interestingly, the cell membranes of high nucleic acid (HNA) content bacteria were damaged much faster than those of low nucleic acid (LNA) content bacteria during treatment with chlorine dioxide and permanganate. However, only small differences were observed during treatment with chlorine and chloramine, and no difference was observed for ferrate treatment. Based on the different reactivity of these oxidants it was suggested that HNA and LNA bacterial cell membranes have a different chemical constitution. Copyright © 2010 Elsevier Ltd. All rights reserved.

Unfavorable effect of levetiracetam on cultured hippocampal neurons after hyperthermic injury.

PubMed

Sendrowski, Krzysztof; Sobaniec, Piotr; Poskrobko, Elżbieta; Rusak, Małgorzata; Sobaniec, Wojciech

2017-06-01

The aim of this study was to examine the viability of neurons and the putative neuroprotective effects of second-generation antiepileptic drug, levetiracetam (LEV), on cultured hippocampal neurons injured by hyperthermia. Primary cultures of rat's hippocampal neurons at 7day in vitro (DIV) were incubated in the presence or absence of LEV in varied concentrations under hyperthermic conditions. Cultures were heated in a temperature of 40°C for 24h or in a temperature of 41°C for 6h. Flow cytometry with Annexin V/PI staining as well as fluorescent microscopy assay were used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, the release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. Assessment was performed after 9DIV and 10 DIV. Incubation of hippocampal cultures in hyperthermic conditions resulted in statistically significant increase in the number of injured neurons when compared with non-heated control cultures. Intensity of neuronal destruction was dependent on temperature-value. When incubation temperature 40°C was used, over 80% of the population of neurons remained viable after 10 DIV. Under higher temperature 41°C, only less than 60% of neurons were viable after 10 DIV. Both apoptotic and necrotic pathways of neuronal death induced by hyperthermia were confirmed by Annexin V/PI staining. LEV showed no neuroprotective effects in the current model of hyperthermia in vitro. Moreover, drug, especially when used in higher concentrations, exerted unfavorable intensification of aponecrosis of cultured hippocampal neurons. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

Regulation of the Prostate Cancer Tumor Microenvironment

DTIC Science & Technology

2017-06-01

lipopolysaccharide   (LPS)   found   in   gram -­negative   bacterial  walls   recognized   by   TLR4,   double  stranded  RNA  produced  by  many  viruses  for...W81 XWH-11-1-0260 6       We  performed  further   staining  of  infiltrating  immune  populations  in  tumors  from  30   week-­old   MyD88...immunofluorescence staining , and by qPCR, to characterize tumor-infiltrating immune populations as well as activation of the downstream NF-kB pathway and

Femtosecond-assisted keratopigmentation double tunnel technique in the management of a case of Urrets-Zavalia syndrome.

PubMed

Alio, Jorge L; Rodriguez, Alejandra E; Toffaha, Bader T; El Aswad, A

2012-09-01

To describe the successful use of a double intrastromal tunnel femtosecond-assisted keratopigmentation technique to manage a case of unilateral Urrets-Zavalia syndrome. A 33-year-old man was referred with a history of trauma in his right eye due to a labor-related accident. Because of myopic anisometropia, he had been previously implanted with an angle-supported phakic intraocular lens. The patient presented iris atrophy and a fixed dilated pupil. He complained of severe and incapacitating photophobia, glare, and decreased vision. To obtain a complete iris replica, the surgery involved creation of double keratopigmented intrastromal tunnels using femtosecond laser and micronized mineral pigments. The deepest layer was stained black first and then the superficial layer was stained with a contoured greenish blue-gray color, which matched the contralateral eye. In the immediate postoperative period, the patient reported a complete elimination of photophobia associated with the corrected distance visual acuity improvement. A very adequate cosmetic outcome was also achieved. Stability was observed during the 12-month follow-up. A femtosecond-assisted keratopigmentation technique using 2 pigmented intrastromal tunnels to achieve an intracorneal pigmented replica of the iris was effective in improving the patient's severe visual function disability and cosmetic appearance. To the best of our knowledge, this is the first report of severe visual function disability caused by atrophic iris and a fixed dilated pupil treated with double intrastromal layers of keratopigmentation by means of femtosecond-created tunnels.

Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii.

PubMed

Yu, Siran; Ye, Xuewei; Chen, Lu; Xie, Xin; Zhou, Qian; Lian, Xiao-Yuan; Zhang, Zhizhen

2015-11-15

Whether sulfated saponins from Holothuria moebii inhibit the proliferation of colorectal cancer cells and have anti-colorectal tumor effects in animal model has not been investigated. To evaluate the cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii. (1) Column chromatography was used to prepare the total and individual saponins and HPLC was applied to define the components of the total saponins; (2) the activity of the total and individual saponins inhibiting the proliferation of human colorectal cancer cells was determined by SRB assay and the apoptosis induced by the saponins was qualified using cytometric analysis with Annexin V-FITC/PI double staining; and (3) the antitumor effects of the sulfated saponins on colorectal CT-26 tumor-bearing Balb/c mice were tested. The total and individual sulfated saponins significantly inhibited the proliferation of four different human colorectal cancer cells with IC50 values ranging from 1.04 to 4.08 μM (or 1.46 to 3.24 μg/ml for total saponins) and induced late apoptosis at an early treatment time in cancer cells. The total saponins (120 mg/kg) had antitumor activity in colorectal CT-26 tumor-bearing Balb/c mice. The sulfated saponins from H. moebii remarkably inhibited the proliferation of different human colorectal cancer cells and had significant anti-colorectal tumor activity in animal model. Copyright © 2015 Elsevier GmbH. All rights reserved.

Sulfated lentinan induced mitochondrial dysfunction leads to programmed cell death of tobacco BY-2 cells.

PubMed

Wang, Jie; Wang, Yaofeng; Shen, Lili; Qian, Yumei; Yang, Jinguang; Wang, Fenglong

2017-04-01

Sulphated lentinan (sLTN) is known to act as a resistance inducer by causing programmed cell death (PCD) in tobacco suspension cells. However, the underlying mechanism of this effect is largely unknown. Using tobacco BY-2 cell model, morphological and biochemical studies revealed that mitochondrial reactive oxygen species (ROS) production and mitochondrial dysfunction contribute to sLNT induced PCD. Cell viability, and HO/PI fluorescence imaging and TUNEL assays confirmed a typical cell death process caused by sLNT. Acetylsalicylic acid (an ROS scavenger), diphenylene iodonium (an inhibitor of NADPH oxidases) and protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (a protonophore and an uncoupler of mitochondrial oxidative phosphorylation) inhibited sLNT-induced H 2 O 2 generation and cell death, suggesting that ROS generation linked, at least partly, to a mitochondrial dysfunction and caspase-like activation. This conclusion was further confirmed by double-stained cells with the mitochondria-specific marker MitoTracker RedCMXRos and the ROS probe H 2 DCFDA. Moreover, the sLNT-induced PCD of BY-2 cells required cellular metabolism as up-regulation of the AOX family gene transcripts and induction of the SA biosynthesis, the TCA cycle, and miETC related genes were observed. It is concluded that mitochondria play an essential role in the signaling pathway of sLNT-induced ROS generation, which possibly provided new insight into the sLNT-mediated antiviral response, including PCD. Copyright © 2016. Published by Elsevier Inc.

Role of gap junction intercellular communication in testicular leydig cell apoptosis induced by oxaliplatin via the mitochondrial pathway.

PubMed

Tong, Xuhui; Han, Xi; Yu, Binbin; Yu, Meiling; Jiang, Guojun; Ji, Jie; Dong, Shuying

2015-01-01

Platinum agents are widely used in the chemotherapy of testicular cancer. However, adverse reactions and resistance to such agents have limited their application in antineoplastic treatment. The aim of the present study was to determine the role of gap junction intercellular communication (GJIC) composed of Cx43 on oxaliplatin‑induced survival/apoptosis in mouse leydig normal and cancer cells using MTT, Annexin V/PI double staining assays and western blot analysis. The results showed that GJIC exerted opposite effects on the mouse leydig cancer (I-10) and normal (TM3) cell apoptosis induced by oxaliplatin. In leydig cancer cells, survival of cells exposed to oxaliplatin was substantially reduced when gap junctions formed as compared to no gap junctions. Pharmacological inhibition of gap junctions by oleamide and 18-α-glycyrrhetinic acid resulted in enhanced survival/decreased apoptosis while enhancement of gap junctions by retinoic acid led to decreased survival/increased apoptosis. These effects occurred only in high‑density cultures (gap junction formed), while the pharmacological modulations had no effects when there was no opportunity for gap junction formation. Notably, GJIC played an opposite (protective) role in normal leydig cells survival/apoptosis following exposure to oxaliplatin. Furthermore, this converse oxaliplatin‑inducing apoptosis exerted through the functional gap junction was correlated with the mitochondrial pathway‑related protein Bcl-2/Bax and caspase‑3/9. These results suggested that in testicular leydig normal/cancer cells, GJIC plays an opposite role in oxaliplatin‑induced apoptosis via the mitochondrial pathway.

Cytotoxic Constituents from the Rhizomes of Curcuma zedoaria

PubMed Central

Ahmed Hamdi, Omer Abdalla; Syed Abdul Rahman, Syarifah Nur; Awang, Khalijah; Abdul Wahab, Norhanom; Looi, Chung Yeng; Thomas, Noel Francis; Abd Malek, Sri Nurestri

2014-01-01

Curcuma zedoaria also known as Temu putih is traditionally used in food preparations and treatment of various ailments including cancer. The cytotoxic activity of hexane, dichloromethane, ethyl acetate, methanol, and the methanol-soxhlet extracts of Curcuma zedoaria rhizomes was tested on two human cancer cell lines (Ca Ski and MCF-7) and a noncancer cell line (HUVEC) using MTT assay. Investigation on the chemical components in the hexane and dichloromethane fractions gave 19 compounds, namely, labda-8(17),12 diene-15,16 dial (1), dehydrocurdione (2), curcumenone (3), comosone II (4), curcumenol (5), procurcumenol (6), germacrone (7), zerumbone epoxide (8), zederone (9), 9-isopropylidene-2,6-dimethyl-11-oxatricyclo[6.2.1.01,5]undec-6-en-8-ol (10), furanodiene (11), germacrone-4,5-epoxide (12), calcaratarin A (13), isoprocurcumenol (14), germacrone-1,10-epoxide (15), zerumin A (16), curcumanolide A (17), curcuzedoalide (18), and gweicurculactone (19). Compounds (1–19) were evaluated for their antiproliferative effect using MTT assay against four cancer cell lines (Ca Ski, MCF-7, PC-3, and HT-29). Curcumenone (3) and curcumenol (5) displayed strong antiproliferative activity (IC50 = 8.3 ± 1.0 and 9.3 ± 0.3 μg/mL, resp.) and were found to induce apoptotic cell death on MCF-7 cells using phase contrast and Hoechst 33342/PI double-staining assay. Thus, the present study provides basis for the ethnomedical application of Curcuma zedoaria in the treatment of breast cancer. PMID:25126594

Effectiveness of a mouthrinse containing active ingredients in addition to chlorhexidine and triclosan compared with chlorhexidine and triclosan rinses on plaque, gingivitis, supragingival calculus and extrinsic staining.

PubMed

Kumar, S; Patel, S; Tadakamadla, J; Tibdewal, H; Duraiswamy, P; Kulkarni, S

2013-02-01

To assess the effectiveness of three different mouthrinses--chlorhexidine, triclosan + sodium fluoride and chlorhexidine + triclosan + sodium fluoride + zinc chloride--on plaque, calculus, gingivitis and stains and to evaluate the occurrence of adverse effects with these three treatments. Forty-eight healthy subjects participated in a double-blind, randomized, parallel experiment and were randomly allocated to any one of the three experimental mouthrinses: group A (0.2% chlorhexidine (CHX) gluconate), group B (0.03% triclosan + 0.025% sodium fluoride (NaF) + 12% ethyl alcohol) or group C (0.2% CHX + 0.3% triclosan + 0.3% NaF + 0.09% Zn chloride (ZnCl(2)). All the subjects were assessed for gingivitis, plaque, supragingival calculus and extrinsic stains at baseline and at the end of the 21-day experimental period. There was a significant difference (P = 0.046) in the effectiveness for the prevention of gingivitis and plaque, with subjects of group A and group C presenting least and highest gingival and plaque scores, respectively. Significant differences (P = 0.03) were observed for the accumulation of supragingival calculus where the deposition of calculus in group A was nearly double that of the group B, and group B was most effective in the prevention of supragingival calculus. Highest deposition of extrinsic stains was in the group A followed by group C and group B. There was no significant difference between the three treatments for adverse events' occurrence. CHX mouthrinse was most effective in controlling plaque and gingivitis but caused greatest deposition of extrinsic stains. Supragingival calculus deposition was least in triclosan + NaF group followed by CHX + triclosan + NaF + ZnCl(2) and CHX. More than half of the subjects reported adverse events during the experimental phase. © 2012 John Wiley & Sons A/S.

Soluble FGL2, a novel effector molecule of activated hepatic stellate cells, regulates T-cell function in cirrhotic patients with hepatocellular carcinoma.

PubMed

Sun, Ying; Xi, Dong; Ding, Wen; Wang, Faxi; Zhou, Haili; Ning, Qin

2014-10-01

To investigate the effects of soluble FGL2 (sFGL2) secreted by hepatic stellate cells (HSCs) on immune suppression in cirrhotic patients with hepatocellular carcinoma (HCC). Serum sFGL2 levels were examined by ELISA in 40 patients with HCC, liver cirrhosis (LC) or chronic HBV (CHB) infection. A double staining of the immunofluorescence analysis of α-SMA and FGL2 was performed in two cirrhotic liver specimens. The expression of FGL2 in the LX2 cell line was analyzed by immunofluorescence, Western blot and flow cytometry. T-cells purified from HCC patients using magnetic beads were cultured with LX2 cells at different ratios with anti-CD3-stimulating or FGL2-blocking antibodies. The proliferation index (PI) of CD8 + T cells was assessed by flow cytometry, and the secretion of IFN-γ was measured by ELISA. sFGL2 levels are significantly higher in patients with HCC or LC compared with those with CHB (p = 0.0039/p = 0.0020). Among HCC patients, those with cirrhosis exhibited significantly higher levels of sFGL2 compared with non-cirrhotic individuals (p = 0.0108). The expressions of FGL2 and α-SMA overlapped in HSCs in liver specimens. FGL2 protein secreted by LX2 cells inhibited T-cell proliferation of HCC patients in a dose-dependent manner in vitro. The PI of CD8 + T cells was significantly enhanced following addition of FGL2 antibody to the culture system (LX2/T-cell ratio of 1:10, p = 0.002). The level of IFN-γ in mixed cultures was inversely correlated with the number of HSCs and was reversed by incubation with FGL2 blocking antibody. sFGL2 protein is a novel effector molecule of activated HSCs, which suppresses CD8 + T cell proliferation and interferon-γ production, and it subsequently might contribute to immune suppression during fibrosis and tumorigenesis in the liver.

Bradykinin B2 receptors play a neuroprotective role in Hypoxia/reoxygenation injury related to pyroptosis pathway.

PubMed

Tang, Min; Li, Xia; Liu, Ping; Wang, Jianwen; He, Fangping; Zhu, Xiongchao

2018-05-27

Kinins are pro-inflammatory peptides that mediate numerous vascular and pain responses in tissue injury. Kinins exert their biological functions via two G-protein-coupled receptors: bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R). We previously demonstrated the up-regulation of B2R after hypoxia/reoxygenation (H/R) injury in primary cultured cortical neurons. However, the role of B2R in inflammasome-induced pyroptosis remains unknown. We induced H/R neuronal injury in primary cultured cortical neurons harvested from embryonic day 17 brains. Next, we examined the neuroprotective function of B2R in H/R-induced neuronal apoptosis or necrosis using an annexin V FITC/propidium iodide (PI) double-staining technique. The pyroptosis signaling cascade, including caspase-1, IL-1β and IL-18 levels and cleaved gasdermin D (GSDMD) expression was examined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting to explore the underlying molecular mechanism. H/R injury significantly increased B2R protein expression (P<0.05) as well as the percentage of early apoptotic and necrotic or late apoptotic neurons as verified by the annexin V FITC/PI flow cytometric analysis. Bradykinin (BK), a specific B2R agonist, caused a significant decrease in apoptotic neuronal death after H/R injury, while HOE140, a specific B2R antagonist, markedly reduced the neuoprotective effect of B2R. Following H/R injury, BK downregulated the caspase-1, IL-1β and IL-18 levels (P<0.01). In contrast, pretreatment with HOE140 significantly increased caspase-1, IL-1β, and IL-18 levels (P<0.01). Further analysis revealed that GSDMD, a key executioner of pyroptosis, is a target for B2R-mediated inhibition of neuronal pyroptosis. Cleaved GSDMD expression was significantly inhibited by BK pretreatment and significantly enhanced by HOE140 pretreatment (P<0.01). These results indicate that activation of B2R plays an important role in pyroptosis mediated by H/R injury. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Experimental mycobacteriosis in striped bass Morone saxatilis

USGS Publications Warehouse

Gauthier, David T.; Rhodes, M.W.; Vogelbein, W.K.; Kator, H.; Ottinger, C.A.

2003-01-01

Striped bass Morone saxatilis were infected intraperitoneally with approximately 105 Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21??C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from splenic tissue at all times p.i. Standard Ziehl-Neelsen staining, however, did not demonstrate acid-fast rods in most early inflammatory foci and granulomas. Large numbers of acid-fast rods were present in granulomas beginning at 8 wk p.i. Between 26 and 45 wk p.i., reactivation of disease was observed in some fish, with disintegration of granulomas, renewed inflammation, and elevated splenic bacterial densities approaching 109 colony-forming units g-1. Infection with M. shottsii or M. gordonae did not produce severe pathology. Mild peritonitis was followed by granuloma formation in the mesenteries, but, with 1 exception, granulomas were not observed in the spleen or anterior kidney. M. shottsii and M. gordonae both established persistent infections in the spleen, but were present at densities at least 2 orders of magnitude less than M. marinum at all time points observed. Granulomas in the mesenteries of M. shottsii- and M. gordonae-infected fish resolved over time, and no reactivation of disease was observed.

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.

PubMed

Berdichevsky, Yevgeny; Dryer, Alexandra M; Saponjian, Yero; Mahoney, Mark M; Pimentel, Corrin A; Lucini, Corrina A; Usenovic, Marija; Staley, Kevin J

2013-05-22

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an anti-epileptogenic, or merely anticonvulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long-term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with lactate dehydrogenase (LDH) release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are as follows: (1) the organotypic hippocampal culture model of post-traumatic epilepsy comprises a rapid assay of anti-epileptogenic and neuroprotective activities and, in this model (2) mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy.

Double insertion of homogeneously staining regions in chromosome 1 of wild Mus musculus musculus: effects on chromosome pairing and recombination.

PubMed

Borodin, P M; Gorlov, I P; Ladygina TYu

1990-01-01

An examination of the meiotic pattern of chromosome 1 isolated from a feral mouse population and containing a double insertion (Is) of homogeneously staining regions (HSRs) was carried out. The region delineated by the proximal breakpoint of Is(HSR;1C5) 1Icg and the distal breakpoint of Is(HSR;1E3)2Icg is desynapsed during the early pachytene stage and heterosynapsed at the midpachytene, as shown by electron microscopic analysis of synaptonemal complexes. The HSRs have no effect on the segregation of chromosome 1 in heterozygous mice. The lack of homosynapsis in the region under study causes chiasmata redistribution in heteromorphic bivalents. In normal males, single chiasmata are located in the medial part of the chromosome. In heterozygotes, this segment is heterosynapsed and unavailable for recombination. This leads to a significant decrease in the frequency of bivalents bearing single chiasmata. The total number of chiasmata per bivalent is much higher in heterozygous males than in normal ones. The recombination frequency between proximal markers fz and In also is higher in heterozygous animals. The increase in the total chiasma number in the heteromorphic bivalent is due to the addition of double chiasmata located mostly at precentromeric and pretelomeric regions of the chromosome.

Rg1 protects rat bone marrow stem cells against hydrogen peroxide-induced cell apoptosis through the PI3K/Akt pathway.

PubMed

Hu, Junzheng; Gu, Yanqing; Fan, Weimin

2016-07-01

The aim of the present study was to investigate the protective mechanism of ginsenoside Rg1 against the apoptosis of rat bone marrow stem cells (rBMSCs) under oxidative stress, and to determine the association with the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. H2O2 was used to induce oxidative injury in rBMSCs. The cells in the H2O2 model group were treated with 800 µM H2O2 for 6 h to induce oxidative injury. The cells in the ginsenoside Rg1 group were treated with 10 µM ginsenoside Rg1 for 24 h, followed by H2O2 treatment. The cells in the Akt pathway blockage group were treated with 25 µM LY294002 for 1 h, followed by ginsenoside Rg1 + H2O2 treatment. The cell counting kit-8 assay was performed to determine cell viability. Cell apoptosis was detected by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The results of flow cytometry and TUNEL staining indicated that the apoptotic rate of the H2O2 model group was significantly higher compared with that of the control group. Following the ginsenoside Rg1 pretreatment, the apoptotic rate was significantly reduced. In the Akt pathway blockage group, no significant alterations in the levels of cell apoptosis were observed compared with the H2O2 model group. Western blot analysis demonstrated that the ginsenoside Rg1 group had a significant downregulation of Bax and cleaved caspase‑3 and an upregulation of Bcl‑2 and phosphorylated Akt protein expression levels compared with the H2O2 model group and the Akt pathway blockage group. In conclusion, ginsenoside Rg1 had a protective effect against the H2O2‑induced oxidative stress of rBMSCs, and the specific mechanism may be associated with the activation of the PI3K/Akt pathway by ginsenoside Rg1.

Gold nanoparticles-conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt-mediated pathway in breast cancer cell lines (MCF-7 and MDA-MB-231).

PubMed

Balakrishnan, Solaimuthu; Mukherjee, Sudip; Das, Sourav; Bhat, Firdous Ahmad; Raja Singh, Paulraj; Patra, Chitta Ranjan; Arunakaran, Jagadeesan

2017-06-01

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles-conjugated quercetin (AuNPs-Qu-5) in MCF-7 and MDA-MB-231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs-Qu-5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V-FITC staining were performed. AuNPs and AuNPs-Qu-5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs-Qu-5 exhibited lower IC 50 value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs-Qu-5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)-mediated PI3K/Akt/GSK-3β signalling by immunoblotting and immunocytochemistry. The pro-apoptotic proteins (Bax, Caspase-3) were found to be up regulated and anti-apoptotic protein (Bcl-2) was down regulated on treatment with AuNPs-Qu-5. Additionally, AuNPs-Qu-5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK-3β. In conclusion, administration of AuNPs-Qu-5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs-Qu-5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Low-frequency ultrasound-mediated microvessel disruption combined with docetaxel to treat prostate carcinoma xenografts in nude mice: A novel type of chemoembolization

PubMed Central

Yang, Yu; Bai, Wenkun; Chen, Yini; Nan, Shuliang; Lin, Yanduan; Ying, Tao; Hu, Bing

2016-01-01

The aim of the present study was to investigate whether low-frequency ultrasound (US)-mediated microvessel disruption combined with docetaxel (DTX) can be used as a novel type of chemoembolization. Mice were assigned to four groups: i) The USMB group, treated with low-frequency US combined with microbubbles (USMB); ii) the DTX group, treated with DTX; iii) the USMB + DTX group, treated with combined therapy; and iv) the control group, which was untreated. Immediately after the first treatment, the average peak intensity (API) on contrast-enhanced US was calculated, and tumors were excised for hematoxylin and eosin (HE) staining. At 2 weeks post-treatment, the tumor volumes and wet weights were calculated, and tumors were excised for immunohistochemistry to calculate apoptotic index (AI), proliferative index (PI) and microvessel density (MVD) values. Immediately after the first treatment, in the DTX and control groups, the tumors demonstrated abundant perfusion enhancement, while in the USMB + DTX and USMB groups, blood perfusion of the tumors was interrupted. Compared with that of the control group, the API was significantly lower in the USMB + DTX USMB groups (all P<0.001). HE staining showed that tumor microvasculature was disrupted into flaky hematomas and severely dilated microvessels in the USMB + DTX and USMB groups. In the DTX and control groups, there was no distinct evidence of the disruption and dilation of blood microvessels. At the end of the treatment, the mean tumor inhibition ratio was 73.33, 46.67 and 33.33% for the USMB + DTX, DTX and USMB groups, respectively. The USMB + DTX group had the highest AI, and the lowest PI and MVD compared with the other groups, although the difference between the USMB + DTX and DTX groups with regard to PI and MVD was not significant (USMB + DTX vs. DTX group, P=0.345 and P=0.059, respectively). In conclusion, as a novel type of chemoembolization, USMB combined with DTX is more effective than USMB or DTX alone in inhibiting tumor growth via the enhancement of apoptosis, and the suppression of proliferation and angiogenesis. PMID:27446386

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

PubMed

Li, Congfen; Takahashi, Chikara; Zhang, Liangxuan; Huseni, Mahrukh; Stankovich, Basha; Mashhedi, Haider; Lee, Joanna; French, Dorothy; Anderson, Jeff Eastham; Kim, Doris; Howell, Kathy; Brauer, Matthew J; Kowanetz, Marcin; Yan, Yibing; Humke, Eric; Ebens, Allen; Hampton, Garret; Lackner, Mark R; Hegde, Priti; Jia, Shidong

2013-03-23

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

PubMed Central

2013-01-01

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. Results The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. Conclusions We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials. PMID:23522020

Platelet Function in Basset Hound Hereditary Thrombopathy.

DTIC Science & Technology

1986-01-01

4021, 1975. 20. DZANDU, J.K., DEH, M.E., BARRETT, D.L., AND WISE, G.E. Detec- tion of erythrocyte membrane proteins, sialoproteins , and lipids in the...Wise GE. Detection of erythrocyte membrane proteins, sialoproteins , and lipids in the same polyacrylamide gel using a double staining technique. Proc

Mapping resistance to the Ug99 race group of the stem rust pathogen in a spring wheat landrace

USDA-ARS?s Scientific Manuscript database

Wheat landrace PI 374670 has seedling and field resistance to stem rust caused by Puccinia graminis f. sp tritici Eriks. & E. Henn (Pgt) race TTKSK. To elucidate the inheritance of resistance, 216 BC1F2 families, 192 double haploid (DH) lines, and 185 recombinant inbred lines (RILs) were developed b...

Study on a Cavitating Hydrofoil having a Practical Blade Profile Shape.

DTIC Science & Technology

1980-10-31

and punched on regular IBM cards. These cards were conveniently used as input data for data reduction. The data acquisition system also has an...FIGURE 3.4 FLOW CONFIGURATION OF DOUBLE WAKE MODEL FOR PARTIALLY CAVITATING FOIL W =+ itp B C F T w St (pI FIGURE 3.5 POTENTIAL PLANE ia W T B C F W

DIRECT IMAGING OF FINE STRUCTURES IN GIANT PLANET-FORMING REGIONS OF THE PROTOPLANETARY DISK AROUND AB AURIGAE

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

Hashimoto, J.; Tamura, M.; Fukue, T.

We report high-resolution 1.6 {mu}m polarized intensity (PI) images of the circumstellar disk around the Herbig Ae star AB Aur at a radial distance of 22 AU (0.''15) up to 554 AU (3.''85), which have been obtained by the high-contrast instrument HiCIAO with the dual-beam polarimetry. We revealed complicated and asymmetrical structures in the inner part ({approx}<140 AU) of the disk while confirming the previously reported outer (r {approx}> 200 AU) spiral structure. We have imaged a double ring structure at {approx}40 and {approx}100 AU and a ring-like gap between the two. We found a significant discrepancy of inclination anglesmore » between two rings, which may indicate that the disk of AB Aur is warped. Furthermore, we found seven dips (the typical size is {approx}45 AU or less) within two rings, as well as three prominent PI peaks at {approx}40 AU. The observed structures, including a bumpy double ring, a ring-like gap, and a warped disk in the innermost regions, provide essential information for understanding the formation mechanism of recently detected wide-orbit (r > 20 AU) planets.« less

Noncovalent Pi-Pi Stacking at the Carbon-Electrolyte Interface: Controlling the Voltage Window of Electrochemical Supercapacitors.

PubMed

Li, Mengya; Westover, Andrew S; Carter, Rachel; Oakes, Landon; Muralidharan, Nitin; Boire, Timothy C; Sung, Hak-Joon; Pint, Cary L

2016-08-03

A key parameter in the operation of an electrochemical double-layer capacitor is the voltage window, which dictates the device energy density and power density. Here we demonstrate experimental evidence that π-π stacking at a carbon-ionic liquid interface can modify the operation voltage of a supercapacitor device by up to 30%, and this can be recovered by steric hindrance at the electrode-electrolyte interface introduced by poly(ethylene oxide) polymer electrolyte additives. This observation is supported by Raman spectroscopy, electrochemical impedance spectroscopy, and differential scanning calorimetry that each independently elucidates the signature of π-π stacking between imidazole groups in the ionic liquid and the carbon surface and the role this plays to lower the energy barrier for charge transfer at the electrode-electrolyte interface. This effect is further observed universally across two separate ionic liquid electrolyte systems and is validated by control experiments showing an invariant electrochemical window in the absence of a carbon-ionic liquid electrode-electrolyte interface. As interfacial or noncovalent interactions are usually neglected in the mechanistic picture of double-layer capacitors, this work highlights the importance of understanding chemical properties at supercapacitor interfaces to engineer voltage and energy capability.

Measurement of target and double-spin asymmetries for the $$\\vec e\\vec p\\to e\\pi^+ (n)$$ reaction in the nucleon resonance region at low $Q^2$

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

Zheng, X.; Adhikari, K. P.; Bosted, P.

We report measurements of target- and double-spin asymmetries for the exclusive channelmore » $$\\vec e\\vec p\\to e\\pi^+ (n)$$ in the nucleon resonance region at Jefferson Lab using the CEBAF Large Acceptance Spectrometer (CLAS). These asymmetries were extracted from data obtained using a longitudinally polarized NH$$_3$$ target and a longitudinally polarized electron beam with energies 1.1, 1.3, 2.0, 2.3 and 3.0 GeV. The new results are consistent with previous CLAS publications but are extended to a low $Q^2$ range from $0.0065$ to $0.35$ (GeV$/c$)$^2$. The $Q^2$ access was made possible by a custom-built Cherenkov detector that allowed the detection of electrons for scattering angles as low as $$6^\\circ$$. These results are compared with the unitary isobar models JANR and MAID, the partial-wave analysis prediction from SAID and the dynamic model DMT. In many kinematic regions our results, in particular results on the target asymmetry, help to constrain the polarization-dependent components of these models.« less

Measurement of target and double-spin asymmetries for the $$\\vec e\\vec p\\to e\\pi^+ (n)$$ reaction in the nucleon resonance region at low $Q^2$

DOE PAGES

Zheng, X.; Adhikari, K. P.; Bosted, P.; ...

2016-10-19

We report measurements of target- and double-spin asymmetries for the exclusive channelmore » $$\\vec e\\vec p\\to e\\pi^+ (n)$$ in the nucleon resonance region at Jefferson Lab using the CEBAF Large Acceptance Spectrometer (CLAS). These asymmetries were extracted from data obtained using a longitudinally polarized NH$$_3$$ target and a longitudinally polarized electron beam with energies 1.1, 1.3, 2.0, 2.3 and 3.0 GeV. The new results are consistent with previous CLAS publications but are extended to a low $Q^2$ range from $0.0065$ to $0.35$ (GeV$/c$)$^2$. The $Q^2$ access was made possible by a custom-built Cherenkov detector that allowed the detection of electrons for scattering angles as low as $$6^\\circ$$. These results are compared with the unitary isobar models JANR and MAID, the partial-wave analysis prediction from SAID and the dynamic model DMT. In many kinematic regions our results, in particular results on the target asymmetry, help to constrain the polarization-dependent components of these models.« less

Emodin induces apoptosis of human cervical cancer hela cells via intrinsic mitochondrial and extrinsic death receptor pathway.

PubMed

Yaoxian, Wang; Hui, Yu; Yunyan, Zhang; Yanqin, Liu; Xin, Ge; Xiaoke, Wu

2013-07-16

Emodin is a natural anthraquinone derivative isolated from the Rheum palmatum L. The aim of the present study was to investigate the effect of emodin on the apoptosis of the human cervical cancer line HeLa and to identify the mechanisms involved. Relative cell viability was assessed by MTT assay after treatment with emodin. Cell apoptosis was detected with TUNEL, Hoechst 33342 staining and quantified with flow cytometry using annexin FITC-PI staining. The percentage of apoptotic cells was 0.8, 8.2, 22.1, and 43.7%, respectively. The mRNA levels of Caspase-9, -8 and -3 detected by Real-time PCR after treatment with emodin were significantly increased. Emodin increased the protein levels of Cytochome c, Apaf-1, Fas, FasL, and FADD but decreased the protein levels of Pro-caspase-9, Pro-caspase-8 and Pro-caspase-3. We conclude that the emodin inhibited HeLa proliferation by inducing apoptosis through the intrinsic mitochondrial and extrinsic death receptor pathways.

Use of a benzimidazole derivative BF-188 in fluorescence multispectral imaging for selective visualization of tau protein fibrils in the Alzheimer's disease brain.

PubMed

Harada, Ryuichi; Okamura, Nobuyuki; Furumoto, Shozo; Yoshikawa, Takeo; Arai, Hiroyuki; Yanai, Kazuhiko; Kudo, Yukitsuka

2014-02-01

Selective visualization of amyloid-β and tau protein deposits will help to understand the pathophysiology of Alzheimer's disease (AD). Here, we introduce a novel fluorescent probe that can distinguish between these two deposits by multispectral fluorescence imaging technique. Fluorescence spectral analysis was performed using AD brain sections stained with novel fluorescence compounds. Competitive binding assay using [(3)H]-PiB was performed to evaluate the binding affinity of BF-188 for synthetic amyloid-β (Aβ) and tau fibrils. In AD brain sections, BF-188 clearly stained Aβ and tau protein deposits with different fluorescence spectra. In vitro binding assays indicated that BF-188 bound to both amyloid-β and tau fibrils with high affinity (K i  < 10 nM). In addition, BF-188 showed an excellent blood-brain barrier permeability in mice. Multispectral imaging with BF-188 could potentially be used for selective in vivo imaging of tau deposits as well as amyloid-β in the brain.

[Procyanidins enhance the chemotherapeutic sensitivity of laryngeal carcinoma cells to cisplatin through autophagy pathway].

PubMed

Yu, Feng; Liu, Wei; Gong, Xiao Rong; Zhou, Yi Bo; Lin, Ying

2018-03-01

To investigate the effect of Procyanidins (OPCs) on the autophagy of laryngeal cancer cell line TU686 and to explore the effect of OPCs on the chemosensitivity of laryngeal cancer cells to DDP in terms of autophagy and apoptosis. CCK-8 was used to detected the effect of different concentrations of OPC and DDP on TU686 cell viability. Experimental grouping: Both kinds of cells were divided into CON group, DDP group, OPC group and MIX group. Annexin-V-FITC/PI double staining of flow cytometry was used to detect the effect of each experimental group on the apoptosis. Cell immunofluorescence staining was used to detect the formation of autophagy. Western blot was used to detect the expression of autophagy-related and apoptosis-related proteins. Autophagy inhibitors (3-MA) were used to study the effect of autophagy on apoptosis. The results of CCK-8 showed that TU686 cells were inhibited by OPC and DDP in a concentration-dependent manner for 24 hours. LC3-Ⅱ protein staining showed that compared with CON group, DDP group and OPC group, MIX group significantly induced autophagy formation in TU686 cells ( P <0.05). Flow cytometry showed that compared with CON group, apoptosis of TU686 cells was induced in DDP group, OPC group and MIX group. And the effect of MIX on apoptosis was significantly higher than that of OPC and DDP groups ( P <0.05). After pretreatment with 3-MA, the apoptotic effect of OPC group and MIX group on TU686 cells was significantly decreased ( P <0.05). Western blot results showed that the expression of LC3-Ⅱ and Caspase-3 in DDP, OPC and MIX groups was significantly higher than that in CON group ( P <0.05). In MIX group, the expression of LC3-Ⅱ and Caspase-3 also had significant difference ( P <0.05) compared with single drug group. After using 3-MA to inhibit autophagy, the expression of LC3-Ⅱ was significantly decreased ( P <0.05), and the expression of Caspase-3 was decreased along with LC3-Ⅱ, but the decrease of Caspase-3 expression was only significant in OPC and MIX group ( P <0.05). OPC can induce autophagy in laryngeal carcinoma TU686 cells and promote its apoptosis, which in turn enhances sensitivity of laryngeal cancer cells to cisplatin chemotherapy. Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.

Antibiofilm efficacy of silver nanoparticles against biofilm of extended spectrum β-lactamase isolates of Escherichia coli and Klebsiella pneumoniae

NASA Astrophysics Data System (ADS)

Ansari, Mohammad Azam; Khan, Haris M.; Khan, Aijaz A.; Cameotra, Swaranjit Singh; Pal, Ruchita

2014-10-01

The ability of bacteria to develop antibiotic resistance and colonize abiotic surfaces by forming biofilms is a major cause of medical implant-associated infections and results in prolonged hospitalization periods and patient mortality. Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own demerits that include chemical-based complications; emergent antibiotic-resistant strains, and so on. Silver nanoparticles (AgNPs) are renowned for their influential antimicrobial activity. We demonstrate the biofilm formation by extended spectrum β-lactamases-producing Escherichia coli and Klebsiella spp. by direct visualization applying tissue culture plate, tube, and Congo red agar methods. Double fluorescent staining for confocal laser scanning microscopy (CLSM) consisted of propidium iodide staining to detect bacterial cells and concanavalin A-fluorescein isothiocyanate staining to detect the exopolysaccharides matrix were used. Scanning electron microscopy observations clearly indicate that AgNPs reduced the surface coverage by E. coli and Klebsiella spp. thus prevent the biofilm formations. Double-staining technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the exopolysaccharides formation. The AgNPs-coated surfaces effectively restricted biofilm formation of the tested bacteria. In our study, we could demonstrate the complete antibiofilm activity AgNPs at a concentration as low as 50 μg/ml. Our findings suggested that AgNPs can be exploited towards the development of potential antibacterial coatings for various biomedical and environmental applications. These formulations can be used for the treatment of drug-resistant bacterial infections caused by biofilms, at much lower nanosilver loading with higher efficiency.

Regulatory Role of the NF-kB Pathway in Lymphangiogenesis and Breast Cancer Metastasis

DTIC Science & Technology

2010-07-01

with anti - LYVE-1 and anti -VEGFR-3 or anti -Prox1 antibodies in serial sections of p50 KO and WT lungs, showing reduced lymphatic vessel density...3 protein as determined by MFI analysis of slides double-stained with anti -VEGFR-3 and anti -LYVE-1 antibodies (Figure 2). These data indicate that...expression of VEGFR-3 and LYVE-1 on liver endothelial cells compared with WT. (A) Livers of p50 KO and WT mice were double immunostained with anti -VEGFR

Detailed Structure of the Outer Disk Around HD 169142 with Polarized Light in H-band

NASA Technical Reports Server (NTRS)

Momose, Munetake; Morita, Ayaka; Fukagawa, Misato; Muto, Takayuki; Takeuchi, Taku; Hashimoto, Jun; Honda, Mitsuhiko; Kudo, Tomoyuki; Okamoto, Yoshiko K.; Kanagawa, Kazuhiro D.;

Effects of endoplasmic reticulum stress on the autophagy, apoptosis, and chemotherapy resistance of human breast cancer cells by regulating the PI3K/AKT/mTOR signaling pathway.

PubMed

Zhong, Jia-Teng; Yu, Jian; Wang, Hai-Jun; Shi, Yu; Zhao, Tie-Suo; He, Bao-Xia; Qiao, Bin; Feng, Zhi-Wei

2017-05-01

Nowadays, although chemotherapy is an established therapy for breast cancer, the molecular mechanisms of chemotherapy resistance in breast cancer remain poorly understood. This study aims to explore the effects of endoplasmic reticulum stress on autophagy, apoptosis, and chemotherapy resistance in human breast cancer cells by regulating PI3K/AKT/mTOR signaling pathway. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect the cell viability of six human breast cancer cell lines (MCF-7, ZR-75-30, T47D, MDA-MB-435s, MDA-MB-453, and MDA-MB-231) treated with tunicamycin (5 µM), after which MCF-7 cells were selected for further experiment. Then, MCF-7 cells were divided into the control (without any treatment), tunicamycin (8 µ), BEZ235 (5 µ), and tunicamycin + BEZ235 groups. Cell viability of each group was testified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blotting was applied to determine the expressions of endoplasmic reticulum stress and PI3K/AKT/mTOR pathway-related proteins and autophagy- and apoptosis-related proteins. Monodansylcadaverine and Annexin V-fluorescein isothiocyanate/propidium iodide staining were used for determination of cell autophagy and apoptosis. Furthermore, MCF-7 cells were divided into the control (without any treatment), tunicamycin (5 µM), cisplatin (16 µM), cisplatin (16 µM) + BEZ235 (5 µM), tunicamycin (5 µM) + cisplatin (16 µM), and tunicamycin (5 µM) + cisplatin (16 µM) + BEZ235 groups. Cell viability and apoptosis were also evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Annexin V-fluorescein isothiocyanate/propidium iodide staining. In MCF-7 cells treated with tunicamycin, cell viability decreased significantly, but PEAK, eIF2, and CHOP were upregulated markedly and p-PI3K, p-AKT, and p-MTOR were downregulated in dose- and time-dependent manners. In the tunicamycin + BEZ235 group, the cell viability was lower and the apoptosis rate was higher than those of the control and monotherapy groups. Compared with the cisplatin group, the tunicamycin + cisplatin group showed a relatively higher growth inhibition rate; the growth inhibition rate substantially increased in the tunicamycin + cisplatin + BEZ235 group than the tunicamycin + cisplatin group. The apoptosis rate was highest in tunicamycin + cisplatin + BEZ235 group, followed by tunicamycin + cisplatin group and then cisplatin group. Our study provide evidence that endoplasmic reticulum stress activated by tunicamycin could promote breast cancer cell autophagy and apoptosis and enhance chemosensitivity of MCF-7 cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

Hydrogen-Rich Saline Attenuates Brain Injury Induced by Cardiopulmonary Bypass and Inhibits Microvascular Endothelial Cell Apoptosis Via the PI3K/Akt/GSK3β Signaling Pathway in Rats.

PubMed

Chen, Keyan; Wang, Nan; Diao, Yugang; Dong, Wanwei; Sun, YingJie; Liu, Lidan; Wu, Xiuying

2017-01-01

Cardiopulmonary bypass (CPB) is prone to inducing brain injury during open heart surgery. A hydrogen-rich solution (HRS) can prevent oxidation and apoptosis, and inhibit inflammation. This study investigated effects of HRS on brain injury induced by CPB and regulatory mechanisms of the PI3K/Akt/GSK3β signaling pathway. A rat CPB model and an in vitro cell hypoxia model were established. After HRS treatment, Rat behavior was measured using neurological deficit score; Evans blue (EB) was used to assess permeability of the blood-brain barrier (BBB); HE staining was used to observe pathological changes; Inflammatory factors and brain injury markers were detected by ELISA; the PI3K/Akt/GSK3β pathway-related proteins and apoptosis were assessed by western blot, immunohistochemistry and qRT -PCR analyses of brain tissue and neurons. After CPB, brain tissue anatomy was disordered, and cell structure was abnormal. Brain tissue EB content increased. There was an increase in the number of apoptotic cells, an increase in expression of Bax and caspase-3, a decrease in expression of Bcl2, and increases in levels of Akt, GSK3β, P-Akt, and P-GSK3β in brain tissue. HRS treatment attenuated the inflammatory reaction ,brain tissue EB content was significantly reduced and significantly decreased expression levels of Bax, caspase-3, Akt, GSK3β, P-Akt, and P-GSK3β in the brain. After adding the PI3K signaling pathway inhibitor, LY294002, to rat cerebral microvascular endothelial cells (CMECs), HRS could reduce activated Akt expression and downstream regulatory gene phosphorylation of GSK3β expression, and inhibit CMEC apoptosis. The PI3K/Akt/GSK3β signaling pathway plays an important role in the mechanism of CPB-induced brain injury. HRS can reduce CPB-induced brain injury and inhibit CMEC apoptosis through the PI3K/Akt/GSK3β signaling pathway. © 2017 The Author(s). Published by S. Karger AG, Basel.

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal

PubMed Central

Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

2017-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, ΔespF), N-terminal sequence (219 bp, ΔespFN), and C-terminal sequence (528 bp, ΔespFC) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, ΔespF/pespF, ΔespFN/pespFN, and ΔespFC/pespFC by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), ΔespF, ΔespF/pespF, ΔespFC, ΔespFC/pespFC, ΔespFN, and ΔespFN/pespFN groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, ΔespF/pespF, and ΔespFC were significantly higher than that of ΔespF, ΔespFN, ΔespFC/pespFC, and ΔespFN/pespFN group (p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain. PMID:28983470

The combined transduction of copper, zinc-superoxide dismutase and catalase mediated by cell-penetrating peptide, PEP-1, to protect myocardium from ischemia-reperfusion injury.

PubMed

Huang, Guang-Qing; Wang, Jia-Ning; Tang, Jun-Ming; Zhang, Lei; Zheng, Fei; Yang, Jian-Ye; Guo, Ling-Yun; Kong, Xia; Huang, Yong-Zhang; Liu, Yong; Chen, Shi-You

2011-05-21

Our previous studies indicate that either PEP-1-superoxide dismutase 1 (SOD1) or PEP-1-catalase (CAT) fusion proteins protects myocardium from ischemia-reperfusion-induced injury in rats. The aim of this study is to explore whether combined use of PEP-1-SOD1 and PEP-1-CAT enhances their protective effects. SOD1, PEP-1-SOD1, CAT or PEP-1-CAT fusion proteins were prepared and purified by genetic engineering. In vitro and in vivo effects of these proteins on cell apoptosis and the protection of myocardium after ischemia-reperfusion injury were measured. Embryo cardiac myocyte H9c2 cells were used for the in vitro studies. In vitro cellular injury was determined by the expression of lactate dehydrogenase (LDH). Cell apoptosis was quantitatively assessed with Annexin V and PI double staining by Flow cytometry. In vivo, rat left anterior descending coronary artery (LAD) was ligated for one hour followed by two hours of reperfusion. Hemodynamics was then measured. Myocardial infarct size was evaluated by TTC staining. Serum levels of myocardial markers, creatine kinase-MB (CK-MB) and cTnT were quantified by ELISA. Bcl-2 and Bax expression in left ventricle myocardium were analyzed by western blot. In vitro, PEP-1-SOD1 or PEP-1-CAT inhibited LDH release and apoptosis rate of H9c2 cells. Combined transduction of PEP-1-SOD1 and PEP-1-CAT, however, further reduced the LDH level and apoptosis rate. In vivo, combined usage of PEP-1-SOD1 and PEP-1-CAT produced a greater effect than individual proteins on the reduction of CK-MB, cTnT, apoptosis rate, lipoxidation end product malondialdehyde, and the infarct size of myocardium. Functionally, the combination of these two proteins further increased left ventricle systolic pressure, but decreased left ventricle end-diastolic pressure. This study provided a basis for the treatment or prevention of myocardial ischemia-reperfusion injury with the combined usage of PEP-1-SOD1 and PEP-1-CAT fusion proteins.

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK-3β and CREB through PI3-K/Akt pathways.

PubMed

Simão, Fabrício; Matté, Aline; Pagnussat, Aline S; Netto, Carlos A; Salbego, Christianne G

2012-10-01

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK-3β) and cAMP response element-binding protein (CREB) through phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four-vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK-3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3-K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK-3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3-K, compromised the neuroprotective effect of resveratrol and decreased the level of p-Akt, p-GSK-3β and p-CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt, GSK-3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3-K/Akt signaling pathway, subsequently downregulating expression of GSK-3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

PubMed Central

Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

2004-01-01

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286]. PMID:14521509

Antitumor Activity and Induction of TP53-Dependent Apoptosis toward Ovarian Clear Cell Adenocarcinoma by the Dual PI3K/mTOR Inhibitor DS-7423

PubMed Central

Kashiyama, Tomoko; Oda, Katsutoshi; Ikeda, Yuji; Shiose, Yoshinobu; Hirota, Yasuhide; Inaba, Kanako; Makii, Chinami; Kurikawa, Reiko; Miyasaka, Aki; Koso, Takahiro; Fukuda, Tomohiko; Tanikawa, Michihiro; Shoji, Keiko; Sone, Kenbun; Arimoto, Takahide; Wada-Hiraike, Osamu; Kawana, Kei; Nakagawa, Shunsuke; Matsuda, Koichi; McCormick, Frank; Aburatani, Hiroyuki; Yano, Tetsu; Osuga, Yutaka; Fujii, Tomoyuki

2014-01-01

DS-7423, a novel, small-molecule dual inhibitor of phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR), is currently in phase I clinical trials for solid tumors. Although DS-7423 potently inhibits PI3Kα (IC50 = 15.6 nM) and mTOR (IC50 = 34.9 nM), it also inhibits other isoforms of class I PI3K (IC50 values: PI3Kβ = 1,143 nM; PI3Kγ = 249 nM; PI3Kδ = 262 nM). The PI3K/mTOR pathway is frequently activated in ovarian clear cell adenocarcinomas (OCCA) through various mutations that activate PI3K-AKT signaling. Here, we describe the anti-tumor effect of DS-7423 on a panel of nine OCCA cell lines. IC50 values for DS-7423 were <75 nM in all the lines, regardless of the mutational status of PIK3CA. In mouse xenograft models, DS-7423 suppressed the tumor growth of OCCA in a dose-dependent manner. Flow cytometry analysis revealed a decrease in S-phase cell populations in all the cell lines and an increase in sub-G1 cell populations following treatment with DS-7423 in six of the nine OCCA cell lines tested. DS-7423-mediated apoptosis was induced more effectively in the six cell lines without TP53 mutations than in the three cell lines with TP53 mutations. Concomitantly with the decreased phosphorylation level of MDM2 (mouse double minute 2 homolog), the level of phosphorylation of TP53 at Ser46 was increased by DS-7423 in the six cell lines with wild-type TP53, with induction of genes that mediate TP53-dependent apoptosis, including p53AIP1 and PUMA at 39 nM or higher doses. Our data suggest that the dual PI3K/mTOR inhibitor DS-7423 may constitute a promising molecular targeted therapy for OCCA, and that its antitumor effect might be partly obtained by induction of TP53-dependent apoptosis in TP53 wild-type OCCAs. PMID:24504419

Isorhamnetin Attenuates Atherosclerosis by Inhibiting Macrophage Apoptosis via PI3K/AKT Activation and HO-1 Induction

PubMed Central

Luo, Yun; Sun, Guibo; Dong, Xi; Wang, Min; Qin, Meng; Yu, Yingli; Sun, Xiaobo

2015-01-01

Background and Purpose Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet. Methods and Results Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions. Conclusion In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction. PMID:25799286

Dexamethasone protects auditory hair cells against TNFalpha-initiated apoptosis via activation of PI3K/Akt and NFkappaB signaling.

PubMed

Haake, Scott M; Dinh, Christine T; Chen, Shibing; Eshraghi, Adrien A; Van De Water, Thomas R

2009-09-01

Tumor necrosis factor alpha (TNFalpha) is associated with trauma-induced hearing loss. Local treatment of cochleae of trauma-exposed animals with a glucocorticoid is effective in reducing the level of hearing loss that occurs post-trauma (e.g., electrode insertion trauma-induced hearing loss/dexamethasone treatment). Dexamethasone (Dex) protects auditory hair cells (AHCs) from trauma-induced loss by activating cellular signal pathways that promote cell survival. Organ of Corti explants challenged with an ototoxic level of TNFalpha was the trauma model with Dex the otoprotective drug. A series of inhibitors were used in combination with the Dex treatment of TNFalpha-exposed explants to investigate the signal molecules that participate in Dex-mediated otoprotection. The otoprotective capacity of Dex against TNFalpha ototoxicity was determined by hair cell counts obtained from fixed explants stained with FITC-phalloidin labeling with investigators blinded to specimen identity. The general caspase inhibitor Boc-d-fmk prevented TNFalpha-induced AHC death. There was a significant reduction (p<0.05) in the efficacy of Dex otoprotection against TNFalpha ototoxicity when the following cellular events were blocked: (1) glucocorticoid receptors (Mif); (2) PI3K (LY294002); (3) Akt/PKB (SH-6); and (4) NFkappaB (NFkappaB-I). Dex treatment protects hair cells against TNFalpha apoptosis in vitro by activation of PI3K/Akt and NFkappaB signaling.

Neuroprotective effect of nicorandil through inhibition of apoptosis by the PI3K/Akt1 pathway in a mouse model of deep hypothermic low flow.

PubMed

Yu, Di; Fan, Changfeng; Zhang, Weiyan; Wen, Zhongyuan; Hu, Liang; Yang, Lei; Feng, Yu; Yin, Ke-Jie; Mo, Xuming

2015-10-15

Nicorandil exerts a protective effect on ischemia-reperfusion (I/R) injury in the brain and kidney through anti-apoptotic mechanisms. However, the mechanism by which nicorandil protects against I/R injury induced by deep hypothermic low flow (DHLF) remains unclear. We used a cerebral I/R model induced by DHLF to determine the neuroprotective effects and possible mechanisms of nicorandil. Hematoxylin-eosin (HE) staining and in situ terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) assay were used to detect changes in cell morphology and the number of apoptotic cells in hippocampus, respectively. The apoptotic regulators including Bcl-2, Bax, Akt, and p-Akt (the active, phosphorylated form of Akt) were examined by Western blot (WB). Histopathological findings showed that nicorandil significantly alleviated morphological damage in hippocampal and reduced the number of TUNEL-positive nuclei induced by DHLF. Nicorandil also increased the expression of Bcl-2 and decreased the expression of Bax, while increasing p-Akt level. Consistent with these results, nicorandil-mediated neuroprotection was reduced in the Akt1+/- mutant mice and inhibited by LY294002, a PI3K inhibitor. These findings showed that nicorandil provides a neuroprotective role in DHLF-induced I/R injury by inhibiting apoptosis via activation of the PI3K/Akt1 signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Super resolution imaging of HER2 gene amplification

NASA Astrophysics Data System (ADS)

Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

2016-02-01

HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

Epiretinal membrane negative staining and double peeling in a single block with Brilliant Blue G.

PubMed

Martins, David; Neves, Pedro

2018-01-01

To describe a surgical technique for combined peeling of epiretinal and internal limiting membranes. The authors present their procedure of choice for epiretinal membrane surgery: negative staining effect using Brilliant Blue G and single block removal of the epiretinal and internal limiting membranes in a single step. A total of 26 eyes were operated with the described technique. In all cases, the peeling was performed successfully and with no complications. Minimum postoperative follow-up was 12 months. There were no recurrences of epiretinal membranes. The ideal surgical approach for epiretinal membranes should attempt to reduce mechanical trauma, light exposure, and dye toxicity.

Carbon dioxide diffusion across stomata and mesophyll and photo-biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton.

PubMed

Singh, Shardendu K; Badgujar, Girish; Reddy, Vangimalla R; Fleisher, David H; Bunce, James A

2013-06-15

Nutrients such as phosphorus may exert a major control over plant response to rising atmospheric carbon dioxide concentration (CO2), which is projected to double by the end of the 21st century. Elevated CO2 may overcome the diffusional limitations to photosynthesis posed by stomata and mesophyll and alter the photo-biochemical limitations resulting from phosphorus deficiency. To evaluate these ideas, cotton (Gossypium hirsutum) was grown in controlled environment growth chambers with three levels of phosphate (Pi) supply (0.2, 0.05 and 0.01mM) and two levels of CO2 concentration (ambient 400 and elevated 800μmolmol(-1)) under optimum temperature and irrigation. Phosphate deficiency drastically inhibited photosynthetic characteristics and decreased cotton growth for both CO2 treatments. Under Pi stress, an apparent limitation to the photosynthetic potential was evident by CO2 diffusion through stomata and mesophyll, impairment of photosystem functioning and inhibition of biochemical process including the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxyganase and the rate of ribulose-1,5-bisphosphate regeneration. The diffusional limitation posed by mesophyll was up to 58% greater than the limitation due to stomatal conductance (gs) under Pi stress. As expected, elevated CO2 reduced these diffusional limitations to photosynthesis across Pi levels; however, it failed to reduce the photo-biochemical limitations to photosynthesis in phosphorus deficient plants. Acclimation/down regulation of photosynthetic capacity was evident under elevated CO2 across Pi treatments. Despite a decrease in phosphorus, nitrogen and chlorophyll concentrations in leaf tissue and reduced stomatal conductance at elevated CO2, the rate of photosynthesis per unit leaf area when measured at the growth CO2 concentration tended to be higher for all except the lowest Pi treatment. Nevertheless, plant biomass increased at elevated CO2 across Pi nutrition with taller plants, increased leaf number and larger leaf area. Copyright © 2013 Elsevier GmbH. All rights reserved.

Meiotic synapsis of homogeneously staining regions (HSRs) in chromosome 1 of Mus musculus.

PubMed

Winking, H; Reuter, C; Traut, W

1993-05-01

About 50 copies of a long-range repeat DNA family with a repeat size of roughly 100 kb and with sequence homology to mRNAs are clustered in the G-light band D of chromosome 1 of the house mouse, Mus musculus. We studied amplified versions of the cluster which are found in many wild populations of M. musculus. They are cytogenetically conspicuous as one or two C-band positive homogeneously staining regions (single- and double band HSRs) which increase the mitotic length of chromosome 1. The double band HSR was phylogenetically derived from a single band HSR by a paracentric inversion. In homozygous condition, such HSRs contribute, albeit not as much as expected from their mitotic length, to the synaptonemal complex (SC) length of chromosome 1. In HSR heterozygous animals an elongation of the SCs was not noticeable. In single band HSR heterozygous males, synapsis proceeds regularly and continuously from the distal telomere towards the centromeric end without forming buckles. Thus, the single band HSR has no adverse effect on pairing. The same straight pairing behaviour was found in the majority of double band HSR heterozygous spermatocytes. This shows that extensive nonhomologous pairing can take place in the earliest phase of synapsis. Synapsis was discontinuous, leaving the central part of the bivalent 1 asynapsed, in only 14.3% of double band HSR heterozygous cells. In such cells the chromosome 1 SC is completed at a later stage of meiosis. The delay is presumably an effect of the inversion that includes one HSR band and the segment between the two HSR bands.

Skeletal mineralization deficits and impaired biogenesis and function of chondrocyte-derived matrix vesicles in Phospho1−/− and Phospho1/Pit1 double knockout mice

PubMed Central

Yadav, Manisha C.; Bottini, Massimo; Cory, Esther; Bhattacharya, Kunal; Kuss, Pia; Narisawa, Sonoko; Sah, Robert L.; Beck, Laurent; Fadeel, Bengt; Farquharson, Colin; Millán, José Luis

2016-01-01

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis but that their chondrocyte- and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of Pi and phosphate transporter-mediated influx of Pi. To test this hypothesis, we generated mice with col2a1-driven cre-mediated ablation of Slc20a1, hereafter referred to as Pit1, alone or in combination with a Phospho1 gene deletion. Pit1col2/col2 mice did not show any major phenotypic abnormalities, while severe skeletal deformities were observed in the [Phospho1−/−; Pit1col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1−/− mice. Histological analysis of [Phospho1−/−; Pit1col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1−/−; Pit1col2/col2] skeleton displayed significantly decreases in BV/TV%, trabecular number and bone mineral density, as well as decreased stiffness, decreased strength, and increased post-yield deflection compared to Phospho1−/− mice. Using atomic force microscopy we found that ~80% of [Phospho1−/−; Pit1col2/col2] MVs were devoid of mineral in comparison to ~50 % for the Phospho1−/− MVs and ~25% for the WT and Pit1col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1−/− and [Phospho1−/−; Pit1col2/col2] chondrocytes. These data support the involvement of PiT-1 in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. PMID:26773408

TPC Proteins Are Phosphoinositide-activated Sodium-selective Ion Channels in Endosomes and Lysosomes

PubMed Central

Wang, Xiang; Zhang, Xiaoli; Dong, Xian-ping; Samie, Mohammad; Li, Xinran; Cheng, Xiping; Goschka, Andrew; Shen, Dongbiao; Zhou, Yandong; Harlow, Janice; Zhu, Michael X.; Clapham, David E.; Ren, Dejian; Xu, Haoxing

2012-01-01

Summary Mammalian Two-Pore Channels (TPC1, 2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P2, and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na+, not K+, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes, and may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles. PMID:23063126

In vitro and in vivo activation of mitochondrial membrane permeability transition pore using triiodothyronine.

PubMed

Endlicher, R; Drahota, Z; Červinková, Z

2016-06-20

Using a novel method for evaluating mitochondrial swelling (Drahota et al. 2012a) we studied the effect of calcium (Ca(2+)), phosphate (P(i)), and triiodothyronine (T(3)) on the opening of mitochondrial membrane permeability transition pore and how they interact in the activation of swelling process. We found that 0.1 mM P(i), 50 microM Ca(2+) and 25 microM T(3) when added separately increase the swelling rate to about 10 % of maximal values when all three factors are applied simultaneously. Our findings document that under experimental conditions in which Ca(2+) and P(i) are used as activating factors, the addition of T(3) doubled the rate of swelling. T(3) has also an activating effect on mitochondrial membrane potential. The T(3) activating effect was also found after in vivo application of T(3). Our data thus demonstrate that T(3) has an important role in opening the mitochondrial membrane permeability pore and activates the function of the two key physiological swelling inducers, calcium and phosphate ions.

[Research on magnetic coupling centrifugal blood pump control based on a self-tuning fuzzy PI algorithm].

PubMed

Yang, Lei; Yang, Ming; Xu, Zihao; Zhuang, Xiaoqi; Wang, Wei; Zhang, Haibo; Han, Lu; Xu, Liang

2014-10-01

The purpose of this paper is to report the research and design of control system of magnetic coupling centrifugal blood pump in our laboratory, and to briefly describe the structure of the magnetic coupling centrifugal blood pump and principles of the body circulation model. The performance of blood pump is not only related to materials and structure, but also depends on the control algorithm. We studied the algorithm about motor current double-loop control for brushless DC motor. In order to make the algorithm adjust parameter change in different situations, we used the self-tuning fuzzy PI control algorithm and gave the details about how to design fuzzy rules. We mainly used Matlab Simulink to simulate the motor control system to test the performance of algorithm, and briefly introduced how to implement these algorithms in hardware system. Finally, by building the platform and conducting experiments, we proved that self-tuning fuzzy PI control algorithm could greatly improve both dynamic and static performance of blood pump and make the motor speed and the blood pump flow stable and adjustable.

First measurement of target and double spin asymmetries for e-vectorp-vector{yields}ep{pi}{sup 0} in the nucleon resonance region above the {delta}(1232)

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

Biselli, A. S.; Burkert, V. D.; Avakian, H.

2008-10-15

The exclusive channel p-vectore-vector,e{sup '}p){pi}{sup 0} was studied in the first and second nucleon resonance regions in the Q{sup 2} range from 0.187 to 0.770 GeV{sup 2} at Jefferson Lab using the CEBAF Large Acceptance Spectrometer. Longitudinal target and beam-target asymmetries were extracted over a large range of center-of-mass angles of the {pi}{sup 0} and compared to the unitary isobar model MAID, the dynamic model by Sato and Lee, and the dynamic model DMT. A strong sensitivity to individual models was observed, in particular for the target asymmetry and in the higher invariant mass region. This data set, once includedmore » in the global fits of the above models, is expected to place strong constraints on the electrocoupling amplitudes A{sub 1/2} and S{sub 1/2} for the Roper resonance N(1400)P{sub 11} and the N(1535)S{sub 11} and N(1520)D{sub 13} states.« less

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis.

PubMed

Jiang, Xi-Wen; Wang, Jing; Chan, Leo Lai; Lam, Paul Kwan Sing; Gu, Ji-Dong

2015-08-01

Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.

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

Xie, Yuchao; McGill, Mitchell R.; Du, Kuo

3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT)more » release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.« less

Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA.

PubMed

Zhang, Wenjun; Hubbard, Antony; Baca-Parkinson, Leslie; Stanislaw, Stacey; Vladich, Frank; Robida, Mark D; Grille, James G; Maxwell, Daniel; Tsao, Tsu-Shuen; Carroll, William; Gardner, Tracie; Clements, June; Singh, Shalini; Tang, Lei

2015-09-01

The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Semen characteristics and selected biochemical markers of canine seminal plasma in various seasons of the year.

PubMed

Strzeżek, R; Szemplińska, K; Filipowicz, K; Kordan, W

2015-01-01

The aim of this study was to evaluate the influence of season on selected qualitative semen characteristics and biochemical markers of canine seminal plasma. Whole ejaculates were collected from 5 crossbred dogs aged 2-8 years. The study covered a period of one year divided into four seasons: spring (March, April, May), summer (June, July, August), autumn (September, October, November) and winter (December, January, February). Semen samples were subjected to macroscopic and microscopic analyses to determine semen volume, total sperm counts and sperm morphology parameters. The study also involved the determination of sperm motility parameters (CASA system), sperm plasma membrane integrity (SPMI, fluorescent staining SYBR-14/PI), sperm mitochondrial membrane potential (MMP, fluorescent staining JC-1/PI) and the ATP content of sperm cells. Total protein content (TPC) and the activity of alkaline phosphatase (AP) and acid phosphatase (AcP) were determined in biochemical analyses of seminal plasma. No significant differences in ejaculate volume, SMPI or ATP content of sperm cells were observed between seasons. The highest total sperm counts were reported in ejaculates acquired in summer and autumn. The lowest MMP values were determined in summer ejaculates. No significant differences in sperm motility (MOT) were observed throughout the experiment, but ejaculates collected in autumn and winter were characterized by the highest progressive motility (PMOT). AP activity and TPC were not significantly affected by season. However, AcP activity levels were significantly lower in autumn than in the remaining seasons. Seasonal variations in the analyzed macroscopic and microscopic parameters of ejaculates and biochemical markers of seminal plasma did not exert a clear negative effect on the quality of canine semen.

Induction of FGF-2 synthesis by IL-1beta in aqueous humor through P13-kinase and p38 in rabbit corneal endothelium.

PubMed

Song, Jong-Suk; Lee, Jeong Goo; Kay, EunDuck P

2010-02-01

To determine whether the elevated level of interleukin (IL)-1beta in aqueous humor after transcorneal freezing upregulates FGF-2 synthesis in rabbit corneal endothelium through PI3-kinase and p38 pathways. Transcorneal freezing was performed in New Zealand White rabbits to induce an injury-mediated inflammation. The concentration of IL-1beta was measured, and the expression of FGF-2, p38, and Akt underwent Western blot analysis. Intracellular location of FGF-2 and actin cytoskeleton was determined by immunofluorescence staining. Massive infiltration of polymorphonuclear leukocytes (PMNs) to the corneal endothelium was observed after freezing, and IL-1beta concentration in the aqueous humor was elevated in a time-dependent manner after freezing. Similarly, FGF-2 expression was increased in a time-dependent manner. When corneal endothelium was stained with anti-FGF-2 antibody, the nuclear location of FGF-2 was observed primarily in the cornea after cryotreatment, whereas FGF-2 in normal corneal endothelium was localized at the plasma membrane. Treatment of the ex vivo corneal tissue with IL-1beta upregulated FGF-2 and facilitated its nuclear location in corneal endothelium. Transcorneal freezing disrupted the actin cytoskeleton at the cortex, and cell shapes were altered from cobblestone morphology to irregular shape. Topical treatment with LY294002 and SB203580 on the cornea after cryotreatment blocked the phosphorylation of Akt and p38, respectively, in the corneal endothelium. These inhibitors also reduced FGF-2 levels and partially blocked morphologic changes after freezing. These data suggest that after transcorneal freezing, IL-1beta released by PMNs into the aqueous humor stimulates FGF-2 synthesis in corneal endothelium via PI3-kinase and p38.

Delivering growth factors through a polymeric scaffold to cell cultures containing both nucleus pulposus and annulus fibrosus.

PubMed

Akyuva, Yener; Kaplan, Necati; Yilmaz, Ibrahim; Ozbek, Hanefi; Sirin, Duygu Yasar; Karaaslan, Numan; Guler, Olcay; Ateş, Özkan

2018-04-09

The aim of this in vitro experimental study was to design a novel, polyvinyl alcohol(PVA)-basedpolymericscaffold that permits the controlled release of insulin-likegrowthfactor1(IGF-1)/bonemorphogenetic protein-2(BMP-2) following intervertebral disc administration. The drug delivery system was composed of two different solutions that formed a scaffold within seconds after coming into contact with each other. We performed swelling,pH,temperature tests and analysis of the controlled release of growth factors from this system.The release kinetics of the growth factors was determined through enzyme linked immunosorbent assay(ELISA). Cell proliferation and viability was monitored with microscopy and analyzed using an MTT assay and acridine orange/propidium iodide(AO/PI) staining. Chondroadherin(CHAD), hypoxiainduciblefactor-1alpha(HIF-1α),collagentypeII(COL2A1) gene expressions were determined with quantitative real-timepolymerasechainreaction(qRT-PCR) analysis to show the effects of IGF-1/BMP-2 administration on annulus fibrosus cell(AFC)/nucleus pulposus cell(NPC) cultures. The scaffold allowed for the controlled release of IGF-1 and BMP-2 in different time intervals. It was observed that as the application time increased, the number of cells and the degree of extracellular matrix development increased in AFC/NPC cultures. AO/PI staining and an MTT analysis showed that cells retained their specific morphology and continued to proliferate. It was observed that HIF-1α and CHAD expression increased in a time-dependent manner, and there wasn't any COL2A1 expression in the AFC/NPC cultures. The designed scaffold may be used as an alternative method for intervertebral disc administration of growth factors after further in vivo studies. We believe that such prototype scaffolds may be an innovative technology in targeted drug therapies after reconstructive neurosurgeries.

Globo H expression is associated with driver mutations and PD-L1 expressions in stage I non-small cell lung cancer.

PubMed

Yang, Ching-Yao; Lin, Mong-Wei; Chang, Yih-Leong; Wu, Chen-Tu

2017-12-12

Globo H is a tumor-associated carbohydrate antigen exclusively expressed in cancer cells rather than normal tissue. Globo H has been found on many cancers of epithelial origins, and become an attractive target for cancer vaccine. We aimed to study the expression of Globo H in non-small cell lung cancer (NSCLC) patients, and correlated its expression with common driver mutations, clinical outcomes, and status of immune checkpoint, programmed death-ligand 1 (PD-L1). The study enrolled 228 patients with surgically resected stage I NSCLC, including 139 patients with adenocarcinoma (ADC) and 89 patients with squamous cell carcinoma (SqCC). Using immunohistochemistry, tumors with moderate to strong membranous staining in ⩾ 1% tumor cells per section were scored as positive Globo H expression. Driver mutations including EGFR, KRAS, BRAF were detected by direct sequencing, while ALK, PI3KCA, FGFR1 and PD-L1 expression was detected by immunohistochemical (IHC) staining. Positive Globo H expression was detected in 88 of the 228 (38.6%) patients. These included 51 of 139 (36.7%) patients with ADC and 37 of 89 (41.6%) patients with SqCC. Positive Globo H expression was significantly associated with EGFR mutation and PD-L1 expression in the ADC group, and PI3KCA overexpression in the SqCC group. The survival analysis showed that Globo H expression was not an independent prognostic factor in stage I NSCLC. Globo H expression was correlated with specific driver mutations in ADC and SqCC NSCLC tumors, as well as PD-L1 status. Immunotherapy targeting Globo H may have potential application in lung cancer treatment.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes

PubMed Central

Mircheff, Austin K.; Wang, Yanru; Schechter, Joel E.; Li, Meng; Tong, Warren; Attar, Mayssa; Chengalvala, Murty; Harmuth, Joe; Prusakiewicz, Jeffery J.

2016-01-01

Purpose To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. Methods Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1° ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). Results Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1° ATD-eyes and exacerbated 1° ATD-LG atrophy. IMIP were evident in 2° ATD-LG as well as 1° ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. Conclusions The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms. PMID:27423911

FGF21 protects human umbilical vein endothelial cells against high glucose-induced apoptosis via PI3K/Akt/Fox3a signaling pathway.

PubMed

Guo, Dongmin; Xiao, Lele; Hu, Huijun; Liu, Mihua; Yang, Lu; Lin, Xiaolong

2018-05-25

Diabetic macroangiopathy is the main cause of morbidity and mortality in patients with diabetes. Endothelial cell injury is a pathological precondition for diabetic macroangiopathy. Fibroblast growth factor 21 (FGF21) is a key metabolic regulator which has recently been suggested to protect cardiac myocytes and vascular cells against oxidative stress-induced injury in vitro and vivo. In this study, we aimed to investigate the protective capacity of FGF21 in human umbilical vein endothelial cells (HUVECs) against high glucose (HG)-induced apoptosis via phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt)/FoxO3a pathway. The cell viability was examined by CCK-8 assay, Intracellular ROS levels were measured by the detection of the fluorescent product formed by the oxidation of DCFH-DA, Apoptosis was analyzed using Hoechst 33258 nuclear staining and Flow Cytometry Analysis (FCA), the expression of protein were detected by Western blot. Results show that pretreating HUVECs with FGF21 before exposure to HG increases cell viability, while decreasing apoptosis and the generation of reactive oxygen species. Western blot analysis shows that HG reduces the phosphorylation of Akt and FoxO3a, and induces nuclear localization of FoxO3a. The effects were significantly reversed by FGF21 pre-treatment. Furthermore, the protective effects of FGF21 were prevented by PI3K/Akt inhibitor LY294002. Our data demonstrates that FGF21 protects HUVECs from HG-induced oxidative stress and apoptosis via the activation of PI3K/Akt/FoxO3a signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Tangeretin alters neuronal apoptosis and ameliorates the severity of seizures in experimental epilepsy-induced rats by modulating apoptotic protein expressions, regulating matrix metalloproteinases, and activating the PI3K/Akt cell survival pathway.

PubMed

Guo, Xiao-Qian; Cao, Yu-Ling; Hao, Fang; Yan, Zhong-Rui; Wang, Mei-Ling; Liu, Xue-Wu

2017-09-01

Epilepsy is complex neural disarray categorized by recurring seizures. Despite recent advances in pharmacotherapies for epilepsy, its treatment remains a challenge due to the contrary effects of the drugs. As a result, the identification of novel anti-epileptic drugs (AEDs) with neuroprotective properties and few side effects is of great value. Thus, the present study assessed the treatment effects of tangeretin using a rat model of pilocarpine-induced epilepsy. Separate groups of male Wistar rats received oral administrations of tangeretin at 50, 100, or 200mg/kg for 10 days and then, on the 10th day, they received an intraperitoneal injection of pilocarpine (30mg/kg). Subsequently, neuronal degeneration and apoptosis were assessed using Nissl staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay procedures. Additionally, the expressions of phosphatidylinositol-3-kinase (PI3K/Akt) pathway proteins, cleaved caspase-3, Bad, Bcl-2, Bcl-xL, and Bax were determined using Western blot analyses. Tangeretin reduced the seizure scores and latency to first seizure of the rats and effectively activated the pilocarpine-induced suppression of PI3K/Akt signaling. Additionally, tangeretin effectively regulated the levels of apoptosis-inducing factor (AIF) in mitochondria as well as the expressions of apoptotic pathway proteins. Seizure-induced elevations in the activities and expressions of matrix metalloproteinases (MMPs)-2 and -9 were also modulated. The present results indicate that tangeretin exerted potent neuroprotective effects against pilocarpine-induced seizures via the activation of PI3K/Akt signaling and the regulation of MMPs. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Silibinin alleviates high glucose-suppressed osteogenic differentiation of human bone marrow stromal cells via antioxidant effect and PI3K/Akt signaling.

PubMed

Ying, Xiaozhou; Chen, Xiaowei; Liu, Haixiao; Nie, Pengfei; Shui, Xiaolong; Shen, Yue; Yu, Kehe; Cheng, Shaowen

2015-10-15

High glucose is one of the possible causes for osteoporosis and fracture in diabetes mellitus. Our previous study showed that silibinin can increase osteogenic effect by stimulating osteogenic genes expression in human bone marrow stem cells (hBMSCs). However, no study has yet investigated the effect of silibinin on osteogenic differentiation of hBMSCs cultured with high glucose. The aim of this study was to evaluate the influence of high glucose on osteogenic differentiation of hBMSCs and to determine if silibinin can alleviate those effects. In this study, the hBMSCs were cultured in an osteogenic medium with physiological (normal glucose, NG, 5.5mM) or diabetic (high glucose, HG, 30mM). The effects of silibinin on HG-induced osteogenic differentiation were evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction. HG-induced oxidative damage was also assessed. Western blot were performed to examine the role of PI3K/Akt pathway. We demonstrated that HG suppressed osteogenic differentiation of hBMSCs, manifested by a decrease in expression of osteogenic markers and an increase of oxidative damage markers including reactive oxygen species and lipid peroxide (MDA). Remarkably, all of the observed oxidative damage and osteogenic dysfunction induced by HG were inhibited by silibinin. Furthermore, the PI3K/Akt pathway was activated by silibinin. These results demonstrate that silibinin may attenuate HG-mediated hBMSCs dysfunction through antioxidant effect and modulation of PI3K/Akt pathway, suggesting that silibinin may be a superior drug candidate for the treatment of diabetes related bone diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Procyanidins, from Castanea mollissima Bl. shell, induces autophagy following apoptosis associated with PI3K/AKT/mTOR inhibition in HepG2 cells.

PubMed

Zhang, Haihui; Luo, Xiaoping; Ke, Jiajia; Duan, Yuqing; He, Yuanqing; Zhang, Di; Cai, Meihong; Sun, Guibo; Sun, Xiaobo

2016-07-01

Procyanidins from Castanea mollissima Bl. shell (CSPCs) induced autophagy and apoptosis in HepG2 cells and its mechanism remains to be examined. In this paper, autophagy was measured by the lipid modification of light chain-3 (LC3) and the formation of autophagosomes. Hoechst 33258 staining and flow cytometer analysis were used to measure apoptosis. The western blot analysis was used to examine the effects of CSPCs on the expression of LC3, PI3K, phosphorylation of AKT, mTOR, Bcl-2, Bad, Bax, BID and cleaved caspase 3 in HepG2 cells. The results showed that 3-methyladenine (3-MA) and apoptosis inhibitor (Z-VAD) could inhibited the death of HepG2 induced by CSPCs for 48h (150μg/mL). CSPCs induced the accumulation of autophagosomes and microtubule-associated proteins light chain 3-II (LC3-II, a marker of autophagy). P-AKT, PI3K and mTOR were significantly decreased on CSPCs exposure. However, these phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA and Z-VAD. CSPCs also induced the expression of Bad, Bax and Beclin-1 proteins and decreased the expression of Bcl-2, which was inhibited by 3-MA and Z-VAD. Moreover the apoptotic cell death could be inhibited by 3-MA. In addition, inhibition of LC3-II by siRNA-dependent knockdown attenuated the cleavage of caspase 3. These results suggested CSPCs could trigger autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway, enhanced apoptosis in HepG2 cells which may be associated with the mitochondria-dependent signaling way. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

High-fat diet amplifies renal renin angiotensin system expression, blood pressure elevation, and renal dysfunction caused by Ceacam1 null deletion.

PubMed

Li, Caixia; Culver, Silas A; Quadri, Syed; Ledford, Kelly L; Al-Share, Qusai Y; Ghadieh, Hilda E; Najjar, Sonia M; Siragy, Helmy M

2015-11-01

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis. Copyright © 2015 the American Physiological Society.

Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers.

PubMed

Carretero, M I; Fumuso, F G; Neild, D M; Giuliano, S M; Cetica, P; Miragaya, M H

2015-09-01

The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP-BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3h at 38 °C with 5% CO2 and 100% humidity. Three aliquots were further incubated 1h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone. One without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2 ± 14.4% live+dead AR sperm) (P < 0.05). These samples showed no motility and viability was very low (0-30%). Both ionomycin and progesterone presented significantly higher (P < 0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live+dead sperm) and the acrosome-present CB pattern (r = 0.64; P = 0.000) in all the evaluated samples. the FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR. Copyright © 2015 Elsevier B.V. All rights reserved.

Combination therapy utilizing shRNA knockdown and an optimized resistant transgene for rescue of diseases caused by misfolded proteins.

PubMed

Li, Chengwen; Xiao, Pingjie; Gray, Steven James; Weinberg, Marc Scott; Samulski, R Jude

2011-08-23

Molecular knockdown of disease proteins and restoration of wild-type activity represent a promising but challenging strategy for the treatment of diseases that result from the accumulation of misfolded proteins (i.e., Huntington disease, amyotrophic lateral sclerosis, and α-1 antitrypsin deficiency). In this study we used alpha-1 antitrypsin (AAT) deficiency with the piZZ mutant phenotype as a model system to evaluate the efficiency of gene-delivery approaches that both silence the piZZ transcript (e.g., shRNA) and restore circulating wild-type AAT expression from resistant codon-optimized AAT (AAT-opt) transgene cassette using adeno-associated virus (AAV) vector delivery. After systemic injection of a self-complimentary AAV serotype 8 (scAAV8) vector encoding shRNA in piZZ transgenic mice, both mutant AAT mRNA in the liver and defected serum protein level were inhibited by 95%, whereas liver pathology, as monitored by dPAS and fibrosis staining, reversed. To restore blood AAT levels in AAV8/shRNA-treated mice, several strategies to restore functional AAT levels were tested, including using AAV AAT-opt transgene cassettes targeted to muscle and liver, or combination vectors carrying piZZ shRNA and AAT-opt transgenes separately, or a single bicistronic AAV vector. With these molecular approaches, we observed over 90% knockdown of mutant AAT with a 13- to 30-fold increase of circulating wild-type AAT protein from the shRNA-resistant AAT-opt cassette. The molecular approaches applied in this study can simultaneously prevent liver pathology and restore blood AAT concentration in AAT deficiencies. Based on these observations, similar gene-therapy strategies could be considered for any diseases caused by accumulation of misfolded proteins.

Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting

NASA Technical Reports Server (NTRS)

Allen, D. L.; Linderman, J. K.; Roy, R. R.; Bigbee, A. J.; Grindeland, R. E.; Mukku, V.; Edgerton, V. R.

1997-01-01

The role of apoptosis in the elimination of myonuclei during hindlimb unloading-induced atrophy and the inhibition of apoptosis in the prevention of muscle atrophy were examined. The number of nuclei demonstrating double-stranded DNA fragmentation seen by terminal deoxynucleotidyl transferase (TDT) histochemical staining, an indicator of apoptosis, was significantly increased after 14 days of suspension. Double staining with TDT and antilaminin immunohistochemistry revealed that some TDT-positive nuclei were within the fiber lamina and were most likely myonuclei. The number of fibers containing morphologically abnormal nuclei was also significantly greater in suspended compared with control rats. Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance exercise attenuated the increase in TDT-positive nuclei (approximately 26%, P > 0.05) and significantly decreased the number of fibers with morphologically abnormal nuclei. The data suggest that 1) "programmed nuclear death" contributes to the elimination of myonuclei and/or satellite cells from atrophying fibers, and 2) GH/IGF-I administration plus muscle loading ameliorates the apoptosis associated with hindlimb unloading.

Phase 2 Randomized, Double-Masked, Vehicle-Controlled Trial of Recombinant Human Nerve Growth Factor for Neurotrophic Keratitis.

PubMed

Bonini, Stefano; Lambiase, Alessandro; Rama, Paolo; Sinigaglia, Francesco; Allegretti, Marcello; Chao, Wendy; Mantelli, Flavio

2018-04-10

To evaluate the safety and efficacy of topical recombinant human nerve growth factor (rhNGF) for treating moderate-to-severe neurotrophic keratitis (NK), a rare degenerative corneal disease resulting from impaired corneal innervation. Phase 2 multicenter, randomized, double-masked, vehicle-controlled trial. Patients with stage 2 (moderate) or stage 3 (severe) NK in 1 eye. The REPARO phase 2 study assessed safety and efficacy in 156 patients randomized 1:1:1 to rhNGF 10 μg/ml, 20 μg/ml, or vehicle. Treatment was administered 6 drops per day for 8 weeks. Patients then entered a 48- or 56-week follow-up period. Safety was assessed in all patients who received study treatment, whereas efficacy was by intention to treat. Corneal healing (defined as <0.5-mm maximum diameter of fluorescein staining in the lesion area) was assessed by masked central readers at week 4 (primary efficacy end point) and week 8 (key secondary end point) of controlled treatment. Corneal healing was reassessed post hoc by masked central readers using a more conservative measure (0-mm staining in the lesion area and no other persistent staining). At week 4 (primary end point), 19.6% of vehicle-treated patients achieved corneal healing (<0.5-mm lesion staining) versus 54.9% receiving rhNGF 10 μg/ml (+35.3%; 97.06% confidence interval [CI], 15.88-54.71; P < 0.001) and 58.0% receiving rhNGF 20 μg/ml (+38.4%; 97.06% CI, 18.96-57.83; P < 0.001). At week 8 (key secondary end point), 43.1% of vehicle-treated patients achieved less than 0.5-mm lesion staining versus 74.5% receiving rhNGF 10 μg/ml (+31.4%; 97.06% CI, 11.25-51.49; P = 0.001) and 74.0% receiving rhNGF 20 μg/ml (+30.9%; 97.06% CI, 10.60-51.13; P = 0.002). Post hoc analysis of corneal healing by the more conservative measure (0-mm lesion staining and no other persistent staining) maintained statistically significant differences between rhNGF and vehicle at weeks 4 and 8. More than 96% of patients who healed after controlled rhNGF treatment remained recurrence free during follow-up. Treatment with rhNGF was well tolerated; adverse effects were mostly local, mild, and transient. Topical rhNGF is safe and more effective than vehicle in promoting healing of moderate-to-severe NK. Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Development of a Double Glass Mounting Method Using Formaldehyde Alcohol Azocarmine Lactophenol (FAAL) and its Evaluation for Permanent Mounting of Small Nematodes.

PubMed

Zahabiun, Farzaneh; Sadjjadi, Seyed Mahmoud; Esfandiari, Farideh

2015-01-01

Permanent slide preparation of nematodes especially small ones is time consuming, difficult and they become scarious margins. Regarding this problem, a modified double glass mounting method was developed and compared with classic method. A total of 209 nematode samples from human and animal origin were fixed and stained with Formaldehyde Alcohol Azocarmine Lactophenol (FAAL) followed by double glass mounting and classic dehydration method using Canada balsam as their mounting media. The slides were evaluated in different dates and times, more than four years. Different photos were made with different magnification during the evaluation time. The double glass mounting method was stable during this time and comparable with classic method. There were no changes in morphologic structures of nematodes using double glass mounting method with well-defined and clear differentiation between different organs of nematodes in this method. Using this method is cost effective and fast for mounting of small nematodes comparing to classic method.

Development of a Double Glass Mounting Method Using Formaldehyde Alcohol Azocarmine Lactophenol (FAAL) and its Evaluation for Permanent Mounting of Small Nematodes

PubMed Central

ZAHABIUN, Farzaneh; SADJJADI, Seyed Mahmoud; ESFANDIARI, Farideh

2015-01-01

Background: Permanent slide preparation of nematodes especially small ones is time consuming, difficult and they become scarious margins. Regarding this problem, a modified double glass mounting method was developed and compared with classic method. Methods: A total of 209 nematode samples from human and animal origin were fixed and stained with Formaldehyde Alcohol Azocarmine Lactophenol (FAAL) followed by double glass mounting and classic dehydration method using Canada balsam as their mounting media. The slides were evaluated in different dates and times, more than four years. Different photos were made with different magnification during the evaluation time. Results: The double glass mounting method was stable during this time and comparable with classic method. There were no changes in morphologic structures of nematodes using double glass mounting method with well-defined and clear differentiation between different organs of nematodes in this method. Conclusion: Using this method is cost effective and fast for mounting of small nematodes comparing to classic method. PMID:26811729

A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

PubMed

Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

2012-04-01

Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

The apoptosis induced by HMME-based photodynamic therapy in rabbit vascular smooth muscle cells

NASA Astrophysics Data System (ADS)

Yin, Huijuan; Li, Xiaoyuan; Lin, Hong; Liu, Jianzhong; Yu, Hongkui

2007-02-01

Objective To study the effects of HMME-based photodynamic therapy on proliferation and apoptosis of rabbit vascular smooth muscle cells(VSMCs). Method The cytotoxic effect of HMME-PDT on rabbit vascular smooth muscle cells was studied by means of Trypan Blue assay, HMME at 10μg/ml concentration and the light dose at 2.4~4.8 J/cm2 were selected in the studies. The morphological character 24h post-PDT was investigated by HE Staining. Annexin V and propidium iodide (PI) binding assays were performed to analyze the characteristics of cell death after HMME-PDT. Furthermore, The intracellular distributions of the HMME were measured by the confocal laser scanning microscope. Result It was showed the photocytotoxity to VSMC cells was dose related by Trypan Blue assay. Histology observing suggests HMME-PDT could induce cell death through apoptosis or necrosis, and the apoptosic rate was up to 50.5% by AnnexinV /PI assay. Moreover, the fluorescence images of HMME intracellular localization demonstrated that the HMME diffused into the mitochondria. Conclusion HMME-PDT could significantly inhibite VSMC proliferation and induce apoptosis.

Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death.

PubMed

Zamulaeva, I A; Lekakh, I V; Kiseleva, V I; Gabai, V L; Saenko, A S; Shevchenko, A S; Poverenny, A M

1997-08-18

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion-exchange chromatography and were then affinity-isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2 +/- 10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7 +/- 7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.

Hybrid Dye-Sensitized Solar Cells Consisting of Double Titania Layers for Harvesting Light with Wide Range of Wavelengths

NASA Astrophysics Data System (ADS)

Sadamasu, Kengo; Inoue, Takafumi; Ogomi, Yuhei; Pandey, Shyam S.; Hayase, Shuzi

2011-02-01

We report a hybrid dye-sensitized solar cell consisting of double titania layers (top and bottom layers) stained with two dyes. A top layer fabricated on a glass was mechanically pressed with a bottom layer fabricated on a glass cloth. The glass cloth acts as a supporter of a porous titania layer as well as a holder of electrolyte. The incident photon to current efficiency (IPCE) curve had two peaks corresponding to those of the two dyes, which demonstrates that electrons are collected from both the top and bottom layers.

Layered double hydroxide nanoparticles promote self-renewal of mouse embryonic stem cells through the PI3K signaling pathway

NASA Astrophysics Data System (ADS)

Wu, Youjun; Zhu, Rongrong; Zhou, Yang; Zhang, Jun; Wang, Wenrui; Sun, Xiaoyu; Wu, Xianzheng; Cheng, Liming; Zhang, Jing; Wang, Shilong

2015-06-01

Embryonic stem cells (ESCs) hold great potential for regenerative medicine due to their two unique characteristics: self-renewal and pluripotency. Several groups of nanoparticles have shown promising applications in directing the stem cell fate. Herein, we investigated the cellular effects of layered double hydroxide nanoparticles (LDH NPs) on mouse ESCs (mESCs) and the associated molecular mechanisms. Mg-Al-LDH NPs with an average diameter of ~100 nm were prepared by hydrothermal methods. To determine the influences of LDH NPs on mESCs, cellular cytotoxicity, self-renewal, differentiation potential, and the possible signaling pathways were explored. Evaluation of cell viability, lactate dehydrogenase release, ROS generation and apoptosis demonstrated the low cytotoxicity of LDH NPs. The alkaline phosphatase activity and the expression of pluripotency genes in mESCs were examined, which indicated that exposure to LDH NPs could support self-renewal and inhibit spontaneous differentiation of mESCs under feeder-free culture conditions. The self-renewal promotion was further proved to be independent of the leukemia inhibitory factor (LIF). Furthermore, cells treated with LDH NPs maintained the potential to differentiate into all three germ layers both in vitro and in vivo through formation of embryoid bodies and teratomas. In addition, we observed that LDH NPs initiated the activation of the PI3K/Akt pathway, while treatment with the PI3K inhibitor LY294002 could block the effects of LDH NPs on mESCs. The results confirmed that the promotion of self-renewal by LDH NPs was associated with activation of the PI3K/Akt signaling pathway. Altogether, our studies identified a new role of LDH NPs in maintaining self-renewal of mouse ES cells which could potentially be applied in stem cell research.Embryonic stem cells (ESCs) hold great potential for regenerative medicine due to their two unique characteristics: self-renewal and pluripotency. Several groups of nanoparticles have shown promising applications in directing the stem cell fate. Herein, we investigated the cellular effects of layered double hydroxide nanoparticles (LDH NPs) on mouse ESCs (mESCs) and the associated molecular mechanisms. Mg-Al-LDH NPs with an average diameter of ~100 nm were prepared by hydrothermal methods. To determine the influences of LDH NPs on mESCs, cellular cytotoxicity, self-renewal, differentiation potential, and the possible signaling pathways were explored. Evaluation of cell viability, lactate dehydrogenase release, ROS generation and apoptosis demonstrated the low cytotoxicity of LDH NPs. The alkaline phosphatase activity and the expression of pluripotency genes in mESCs were examined, which indicated that exposure to LDH NPs could support self-renewal and inhibit spontaneous differentiation of mESCs under feeder-free culture conditions. The self-renewal promotion was further proved to be independent of the leukemia inhibitory factor (LIF). Furthermore, cells treated with LDH NPs maintained the potential to differentiate into all three germ layers both in vitro and in vivo through formation of embryoid bodies and teratomas. In addition, we observed that LDH NPs initiated the activation of the PI3K/Akt pathway, while treatment with the PI3K inhibitor LY294002 could block the effects of LDH NPs on mESCs. The results confirmed that the promotion of self-renewal by LDH NPs was associated with activation of the PI3K/Akt signaling pathway. Altogether, our studies identified a new role of LDH NPs in maintaining self-renewal of mouse ES cells which could potentially be applied in stem cell research. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02339d

Theoretical studies of chemisorption and dimer model systems: Moller-Plesset and configuration interaction calculations on PdH, PdC, PdO, PdF, Pd sub 2 , and PdCO

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

Schwerdtfeger, P.; McFeaters, J.S.; Moore, J.J.

1991-01-01

Ab initio SCF studies have been performed to study the molecular properties of several single-bonded palladium compounds, PdH, PdC, PdO, PdF, Pd{sub 2}, and PdCO, which are important in surface and materials science. Electron correlation effects were evaluated by a second- and third-order Moller-Plesset (MP) perturbation theory and a size-consistency-corrected configuration interaction with single and double substitutions (CISC). Relativistic effects were investigated for PdH and PdF. The ground state of PdC has been calculated at the CISC level to be a {sup 3}{Pi} state which is only 0.26 eV below the {sup 3}{Sigma}{sup {minus}} state (previously assigned ground state) andmore » 0.51 eV below the {sup 1}{Sigma}{sup +} state. PdC is predicted to be stable in the gas phase, and the possibility of preparing this compound is investigated. The bonding in CO chemisorbed on palladium is studied by using the model Pd-CO system. The effect of d{sub {pi}}-{pi}{sup *} back-bonding, discussed at the Hartree-Fock and CI level, is compared with results from multiple-scattering {Chi}{alpha} calculations. The C-O stretching frequency shift for CO on palladium was analyzed at various levels of theory, and the results indicated that the decrease in the CO force constant associated with chemisorption is not solely the result of d{sub {pi}}-{pi}{sup *} back-bonding.« less

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

PubMed

Kim, Eunhye; Zheng, Zhong; Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

2016-01-01

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

Nuclear diacylglycerol lipase-α in rat brain cortical neurons: evidence of 2-arachidonoylglycerol production in concert with phospholipase C-β activity.

PubMed

García del Caño, Gontzal; Aretxabala, Xabier; González-Burguera, Imanol; Montaña, Mario; López de Jesús, Maider; Barrondo, Sergio; Barrio, Ramón J; Sampedro, Carmen; Goicolea, M Arantzazu; Sallés, Joan

2015-03-01

In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2-arachidonoylglycerol (2-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2-AG locally produced within the neuronal nucleus. © 2014 International Society for Neurochemistry.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

PubMed

Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki

2016-01-01

Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.

Generation of intensity covariations of the oxygen green and red lines in the nightglow

NASA Astrophysics Data System (ADS)

Misawa, K.; Takeuchi, I.; Kato, Y.; Aoyama, I.

1984-02-01

The cause of intensity covariations of the oxygen green and red lines is studied. Intensity covariations are compared with the auroral-electrojet-activity index AE, the substorm Pi2, and the magnetogram. It is suggested that intensity covariations or double-intensity maxima of the red line occur in association with intense auroral substorms, and that they are the direct experimental evidences of Testud's theory (1973).

USSR Report, Agriculture.

DTIC Science & Technology

1984-05-17

Improved 37 Winter Crop Preference 37 Winter Crop Sowing 37 Volgograd Field Operations 37 Moisture Patention Work 37 Strawberries in October 37...this work has been organized on a double shift basis. /Text/ /Moscow SEL’SKAYA ZHIZN’ in Russian 15 Jan 84 pi/ 7026 STRAWBERRIES IN OCTOBER—Ulyanovsk...neighboring forests of Ulyanovsk, strawberries have ripened for the second time during the one season. For example, a walk under the canopy of the October

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

PubMed Central

Daude, Nathalie; Westaway, David

2012-01-01

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrPC. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn0/0/Prnp0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrPC, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable. PMID:22929230

Variable thickness double-refracting plate

DOEpatents

Hadeishi, Tetsuo

1976-01-01

This invention provides an A.C., cyclic, current-controlled, phase retardation plate that uses a magnetic clamp to produce stress birefringence. It was developed for an Isotope-Zeeman Atomic Absorption Spectrometer that uses polarization modulation to effect automatic background correction in atomic absorption trace-element measurements. To this end, the phase retardation plate of the invention is a variable thickness, photoelastic, double-refracting plate that is alternately stressed and released by the magnetic clamp selectively to modulate specific components selected from the group consisting of circularly and plane polarized Zeeman components that are produced in a dc magnetic field so that they correspond respectively to Zeeman reference and transmission-probe absorption components. The polarization modulation changes the phase of these polarized Zeeman components, designated as .sigma. reference and .pi. absorption components, so that every half cycle the components change from a transmission mode to a mode in which the .pi. component is blocked and the .sigma. components are transmitted. Thus, the Zeeman absorption component, which corresponds in amplitude to the amount of the trace element to be measured in a sample, is alternately transmitted and blocked by a linear polarizer, while the circularly polarized reference components are continuously transmitted thereby. The result is a sinusoidally varying output light amplitude whose average corresponds to the amount of the trace element present in the sample.

Large-angle production of charged pions with incident pion beams on nuclear targets

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

Apollonio, M.; Chimenti, P.; Giannini, G.

2009-12-15

Measurements of the double-differential {pi}{sup {+-}} production cross section in the range of momentum 100{<=}p{<=}800 MeV/c and angle 0.35{<=}{theta}{<=}2.15 rad using {pi}{sup {+-}} beams incident on beryllium, aluminum, carbon, copper, tin, tantalum, and lead targets are presented. The data were taken with the large-acceptance hadron production (HARP) detector in the T9 beam line of the CERN Proton Synchrotron. The secondary pions were produced by beams in a momentum range from 3 to 12.9GeV/c hitting a solid target with a thickness of 5% of a nuclear interaction length. The tracking and identification of the produced particles was performed using a small-radiusmore » cylindrical time projection chamber placed inside a solenoidal magnet. Incident particles were identified by an elaborate system of beam detectors. Results are obtained for the double-differential cross sections d{sup 2}{sigma}/dp d{theta} at six incident-beam momenta. Data at 3,5,8, and 12GeV/c are available for all targets, while additional data at 8.9 and 12.9GeV/c were taken in positive particle beams on Be and Al targets, respectively. The measurements are compared with several generators of GEANT4 and the MARS Monte Carlo simulation.« less

Purification of the major endoglucanase from Aspergillus fumigatus Fresenius.

PubMed

Parry, J B; Stewart, J C; Heptinstall, J

1983-08-01

Aspergillus fumigatus (Fresenius), IMI 246651, A.T.C.C. 46324, produces two beta-glucosidase enzymes, cotton-solubilizing activity, xylanase and endoglucanase enzymes which can be separated by gel-filtration chromatography. The major endoglucanase does not bind to concanavalin A-Sepharose and does not stain with periodic acid/Schiff reagent. It is homogeneous on polyacrylamide isoelectric focusing (pI = 7.1) and has a mol.wt. of 12500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The endoglucanase produces glucose and a mixture of oligosaccharides from cellulose; the purified enzyme has a small dextranase activity. It is stable at 50 degrees C and pH 6.

Ab initio calculation of the electronic structures of the (7)Sigma+ ground and A (7)Pi and a (5)Sigma+ excited states of MnH.

PubMed

Tomonari, Mutsumi; Nagashima, Umpei; Hirano, Tsuneo

2009-04-21

Electronic structures and molecular constants of the ground (7)Sigma(+) and low-lying A (7)Pi and a (5)Sigma(+) electronic excited states of the MnH molecule were studied by multireference single and double excitation configuration interaction (MR-SDCI) with Davidson's correction (+Q) calculations under exact C(infinity v) symmetry using Slater-type basis sets. To correctly describe the (7)Sigma(+) electronic ground state, X (7)Sigma(+), at the MR-SDCI+Q calculation, we employed a large number of reference configurations in terms of the state-averaged complete active space self-consistent field (CASSCF) orbitals, taking into account the contribution from the B (7)Sigma(+) excited state. The A (7)Pi and a (5)Sigma(+) states can well be described by the MR-SDCI wave functions based on the CASSCF orbitals obtained for the lowest state only. In the MR-SDCI+Q, calculations of the X (7)Sigma(+), A (7)Pi, and a (5)Sigma(+) states required 16, 7, and 17 reference configurations, respectively. Molecular constants, i.e., r(e) and omega(e) of these states and excitation energy from the X (7)Sigma(+) state, obtained at the MR-SDCI+Q level, showed a good agreement with experimental values. The small remaining differences may be accounted for by taking relativistic effects into account.

Phosphoniums as catalysts for metal-free polymerization: Synthesis of well-defined poly(propylene oxide)

NASA Astrophysics Data System (ADS)

Zhang, Jie; Liu, Quan; Ren, Haojun; Zhang, Nanjie; Li, Pengfei; Yang, Kang

2017-11-01

The anionic ring-opening polymerization of propylene oxide (PO) was initiated with glycerol and catalyzed by three new synthetic phosphonium salts, tetrakis (pyrrolidino) phosphonium (Py4P1+), tetrakis (piperidino) phosphonium (Pi4P1+), tetrakis (morpholino) phosphonium (Mo4P1+), and the known tetrakis [cyclohexyl (methyl) amino] phosphonium (Cy4P1+) and tetrakis [tris (dimethylamino) phosphonoamino] phosphazene (P5+). The effects of substituents on the polymerization behavior, especially the molecular weight and its distribution, degree of unsaturation, and the sequential structures of poly (propylene oxide) (PPO) were investigated. The structures of these catalysts and PPOs were characterized by FT-IR, 1H and 13C NMR, and GPC. The results indicate that Cy4P1+, Py4P1+, and Pi4P1+ have lower optimum reaction temperatures at 90, 70, and 70 °C, respectively, and are better than traditional catalysts KOH and double metal cyanide. PPO samples with high molecular weight, narrow polydispersity, and high functionality were accessible when catalyzed with Cy4P1+, Pi4P1+, and P5+ at the optimum temperature. Notably, Pi4P1+ formed unimodal distribution PPO with 9000 g/mol, 2.93 of functionality, and 0.008 mmol/g degree of unsaturation. Majority segments of PPO from five catalysts adopted the stereoregular head-to-tail structure, exhibiting excellent regularity.

Involvement of Lysosome Membrane Permeabilization and Reactive Oxygen Species Production in the Necrosis Induced by Chlamydia muridarum Infection in L929 Cells.

PubMed

Chen, Lixiang; Wang, Cong; Li, Shun; Yu, Xin; Liu, Xue; Ren, Rongrong; Liu, Wenwen; Zhou, Xiaojing; Zhang, Xiaonan; Zhou, Xiaohui

2016-04-28

Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs have to be released to the surrounded cells. Therefore, the interactions between Chlamydiae and cell death pathways could greatly influence the outcomes of Chlamydia infection. However, the underlying molecular mechanisms remain elusive. Here, we investigated host cell death after Chlamydia infection in vitro, in L929 cells, and showed that Chlamydia infection induces cell necrosis, as detected by the propidium iodide (PI)-Annexin V double-staining flow-cytometric assay and Lactate dehydrogenase (LDH) release assay. The production of reactive oxygen species (ROS), an important factor in induction of necrosis, was increased after Chlamydia infection, and inhibition of ROS with specific pharmacological inhibitors, diphenylene iodonium (DPI) or butylated hydroxyanisole (BHA), led to significant suppression of necrosis. Interestingly, live-cell imaging revealed that Chlamydia infection induced lysosome membrane permeabilization (LMP). When an inhibitor upstream of LMP, CA-074-Me, was added to cells, the production of ROS was reduced with concomitant inhibition of necrosis. Taken together, our results indicate that Chlamydia infection elicits the production of ROS, which is dependent on LMP at least partially, followed by induction of host-cell necrosis. To our best knowledge, this is the first live-cell-imaging observation of LMP post Chlamydia infection and report on the link of LMP to ROS to necrosis during Chlamydia infection.

Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

PubMed

Qu, Su; Yan, Liang; Fang, Bo; Ye, Shoudong; Li, Ping; Ge, Shengyang; Wu, Jian; Qu, Di; Song, Houyan

2017-04-15

To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation. Copyright © 2017 Elsevier Inc. All rights reserved.

HCV core protein represses the apoptosis and improves the autophagy of human hepatocytes

PubMed Central

Liu, Changhong; Qu, Aihua; Han, Xiaochun; Wang, Yiguo

2015-01-01

Objectives: This study aims to investigate the influence on human hepatocytes apoptosis and autophagy by the hepatitis C virus (HCV) core protein. Methods: QSG-7701, a human-derived non-neoplastic liver cell line, was transfected with PIRES-core vector that was a eukaryotic vector to express HCV core protein. Fluorescence microscope was used to observe the changes of nuclei in apoptosis cells by Annex in V-FITC/PI double staining. Flow cytometry was applied to detect the rate of cell apoptosis. Western blotting was used to detect the expression of HCV core protein, transcription factor nuclear factor-kappa B (NF-κB), autophagic biomarker microtubule associated protein 1 light chain 3 (LC3), and Beclin-1. Results: The apoptosis rate was significantly lower (P < 0.05) in QSG7701/core group (transfected with PIRES-core vector, (1.34±0.07)%) than in QSG7701 group (no transfection, (2.35±0.11)%) and in QSG7701 QSG7701/pcDNA3.1 group (transfected with pcDNA3.1 vector, (2.58±0.1)%). NF-κB expression was up-expressed in QSG7701/core group than in QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). LC3-II expression and Beclin-1 expression was significant higher in QSG7701/core group than in the QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). Conclusion: HCV core protein can repress the apoptosis and improve the autophagy of QSG7701 through up-regulating NF-κB and Beclin-1 expression. PMID:26629077

Tanshinone IIA ameliorates apoptosis of myocardiocytes by up-regulation of miR-133 and suppression of Caspase-9.

PubMed

Song, Tao; Yao, Yuan; Wang, Teng; Huang, He; Xia, Hao

2017-11-15

To explore the potential protective effect of Tanshinone ⅡA on myocardial cell apoptosis and elucidate the underlying molecular mechanisms. The rat heart cell H9c2 was treated by either H 2 O 2 or doxorubicin (DOX) to mimic oxidative stress and DNA damage conditions in vivo. Cell growth was monitored by optical microscope observation or CCK-8 counting kit. The relative expression of miR-133 and U6 snoRNA was semi-quantitated by RT-PCR or real-time PCR. Cell apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The microRNA binding sites were predicted by online bioinformatics tools. The regulatory effect of miR-133 on caspase-9 was measured by luciferase reporter assay. Apoptosis pathway factors were analyzed by immunoblotting. Our data demonstrated that Tanshinone ⅡA significantly ameliorated myocardial apoptosis induced by either H 2 O 2 or DOX. The protective effect was likely mediated by up-regulation of miR-133. We further identified Caspase-9 as the target of miR-133. Tanshinone ⅡA treatment significantly reversed down-regulation of miR-133 under harsh conditions and in turn suppressed evoking of Caspase-9 and related apoptotic effectors, which consequently contributed to the improvement of myocardial injury. In conclusion, Tanshinone ⅡA ameliorated myocardial apoptosis via restoration of miR-133 and suppression Caspase-9 signaling cascade, which underlies its well-proven clinical benefit and warrants larger scale clinical applications. Copyright © 2017. Published by Elsevier B.V.

Chloral Hydrate Treatment Induced Apoptosis of Macrophages via Fas Signaling Pathway.

PubMed

Cai, Jun; Peng, Yanxia; Chen, Ting; Liao, Huanjin; Zhang, Lifang; Chen, Qiuhua; He, Yiming; Wu, Ping; Xie, Tong; Pan, Qingjun

2016-12-10

BACKGROUND There are recent reports on several anesthetics that have anti-inflammatory and anti-infective effects apart from their uses for pain relief and muscle relaxation. Chloral hydrate is a clinical anesthetic drug and sedative that has also been reported to attenuate inflammatory response, but the mechanisms are not clearly understood. MATERIAL AND METHODS This study investigated the effect of chloral hydrate treatment on the apoptosis of macrophages and explored the underlying mechanisms. RAW264.7 macrophages were treated with various concentrations of chloral hydrate for various lengths of time. Morphological changes were observed under a light microscope and apoptosis was detected with annexin-V-FITC/PI double-staining assay, Hochest 33258 and DNA ladder assay, the expression of Fas/FasL was detected with a flow cytometer, and the Fas signaling pathway was assessed by Western blotting. RESULTS The results showed that chloral hydrate treatment induced the morphology of RAW264.7 macrophages to change shape from typical fusiform to round in a concentration- and time-dependent manner, and was finally suspended in the supernatant. For the induction of apoptosis, chloral hydrate treatment induced the apoptosis of RAW264.7 macrophages from early-to-late stage apoptosis in a concentration- and time-dependent manner. For the mechanism, chloral hydrate treatment induced higher expression of Fas on RAW264.7 macrophages, and was also associated with changes in the expression of proteins involved in Fas signaling pathways. CONCLUSIONS Chloral hydrate treatment can induce the apoptosis of RAW264.7 macrophages through the Fas signaling pathway, which may provide new options for adjunctive treatment of acute inflammation.