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Sample records for activated caspase-3 immunoreactivity

  1. Blockade of processing/activation of caspase-3 by hypoxia

    SciTech Connect

    Han, Sang Hee; Kim, Moonil; Park, Kyoungsook; Kim, Tae-Hyoung; Seol, Dai-Wu

    2008-10-31

    Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells. However, it is largely unknown how hypoxia-inhibited Bax translocation attenuates TRAIL-induced apoptosis. Here, we demonstrate that despite its inhibitory activity in TRAIL-induced apoptosis, hypoxia does not affect TRAIL-triggered proximal apoptotic signaling events, including caspase-8 activation and Bid cleavage. Instead, hypoxia inhibited processing of caspase-3, leading to incomplete activation of the caspase. Importantly, hypoxia-blocked translocation of Bax to the mitochondria significantly inhibited releasing the mitochondrial factors, such as cytochrome c and Smac/DIABLO, to the cytosol in response to TRAIL. It is well-known that complete processing/activation of caspase-3 requires Smac/DIABLO released from mitochondria. Therefore, our data indicate that an engagement of the apoptotic mitochondrial events leading to caspase-3 activation is blocked by hypoxia. Our data shed new light on understanding of the apoptotic signal transduction and targets regulated by tumor hypoxia.

  2. FRET analysis demonstrates a rapid activating of caspase-3 during PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Wu, Yunxia; Chen, Qun

    2006-09-01

    Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. In this study, a recombinant caspase-3 substrate was used as a fluorescence resonance energy transfer (FRET) probe to detect the activation of caspase-3, and to monitor apoptosis in human lung adenocarcinoma (ASTC-a- 1) cells. With laser scanning confocal microscopy, we found that Photofrin were localized primarily in mitochondria, the primary targets of Photofrin-PDT. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the onset and completion of caspase-3 activation induced by PDT is more rapidly than that by tumor necrosis factor-α (TNF-α). The activation of caspase-3 by PDT started 20 minutes after treatment and completed in about 15 minutes. In comparison, the onset of caspase-3 activation by TNF-a was delayed by 3 hours and the completion of caspase-3 activation required a significantly longer time (approximately 90 minutes). These results indicated that the initiation and process of caspase-3 activation are different corresponding to different treatment methods. Our data suggest that caspase-3 activation mediated by the cell surface death receptors is slower than that of the mitochondrial pathway and the mitochondria is an efficient target to induce apoptosis.

  3. Fluorogenic Substrates for In Situ Monitoring of Caspase-3 Activity in Live Cells.

    PubMed

    Pérez-López, Ana M; Soria-Gila, M Lourdes; Marsden, Emma R; Lilienkampf, Annamaria; Bradley, Mark

    2016-01-01

    The in situ detection of caspase-3 activity has applications in the imaging and monitoring of multiple pathologies, notably cancer. A series of cell penetrating FRET-based fluorogenic substrates were designed and synthesised for the detection of caspase-3 in live cells. A variety of modifications of the classical caspase-3 and caspase-7 substrate sequence Asp-Glu-Val-Asp were carried out in order to increase caspase-3 affinity and eliminate caspase-7 cross-reactivity. To allow cellular uptake and good solubility, the substrates were conjugated to a cationic peptoid. The most selective fluorogenic substrate 27, FAM-Ahx-Asp-Leu-Pro-Asp-Lys(MR)-Ahx, conjugated to the cell penetrating peptoid at the C-terminus, was able to detect and quantify caspase-3 activity in apoptotic cells without cross-reactivity by caspase-7. PMID:27168077

  4. Fluorogenic Substrates for In Situ Monitoring of Caspase-3 Activity in Live Cells

    PubMed Central

    Pérez-López, Ana M.; Soria-Gila, M. Lourdes; Marsden, Emma R.; Lilienkampf, Annamaria; Bradley, Mark

    2016-01-01

    The in situ detection of caspase-3 activity has applications in the imaging and monitoring of multiple pathologies, notably cancer. A series of cell penetrating FRET-based fluorogenic substrates were designed and synthesised for the detection of caspase-3 in live cells. A variety of modifications of the classical caspase-3 and caspase-7 substrate sequence Asp-Glu-Val-Asp were carried out in order to increase caspase-3 affinity and eliminate caspase-7 cross-reactivity. To allow cellular uptake and good solubility, the substrates were conjugated to a cationic peptoid. The most selective fluorogenic substrate 27, FAM-Ahx-Asp-Leu-Pro-Asp-Lys(MR)-Ahx, conjugated to the cell penetrating peptoid at the C-terminus, was able to detect and quantify caspase-3 activity in apoptotic cells without cross-reactivity by caspase-7. PMID:27168077

  5. The proteasome is responsible for caspase-3-like activity during xylem development.

    PubMed

    Han, Jia-Jia; Lin, Wei; Oda, Yoshihisa; Cui, Ke-Ming; Fukuda, Hiroo; He, Xin-Qiang

    2012-10-01

    Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin β-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.

  6. Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer

    PubMed Central

    Tyas, Lorraine; Brophy, Victoria A.; Pope, Andrew; Rivett, A. Jennifer; Tavaré, Jeremy M.

    2000-01-01

    Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP–DEVD–YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level. PMID:11256610

  7. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  8. ROFA INCREASES CASPASE-3 ACTIVITY IN HUMAN ALVEOLAR MACRAPHAGE

    EPA Science Inventory

    Exposure to air pollution particles produces pulmonary inflammation and injury, but the mechanisms of this injury are unclear. Apoptosis, involving activation of caspases, may be one potential mechanism. In this study, we hypothesized that ROFA, a constituent of air pollution...

  9. Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction.

    PubMed

    Gilbert, Kim; Godbout, Roger; Rousseau, Guy

    2016-01-01

    Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala - peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 °C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases. PMID:26862955

  10. Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

    PubMed Central

    Gilbert, Kim; Godbout, Roger; Rousseau, Guy

    2016-01-01

    Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala – peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 °C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases. PMID:26862955

  11. Natural activation of caspase-3 is required for the development of operant behavior in postnatal ontogenesis.

    PubMed

    Kudryashova, I V; Stepanichev, M Yu; Gulyaeva, N V

    2009-01-01

    We have previously demonstrated transient increases in caspase-3 activity in the hippocampus of rat pups from age 17 days. We report here our studies on the effects of inhibition of caspase-3 during this period on the acquisition of a two-way avoidance reaction. Rat pups received intracerebroventricular doses of the caspase-3 inhibitor Z-DEVD-FMK On postnatal day 18. Control animals of the same age received the inactive peptide Z-FA-FMK or isotonic saline solution. Inhibition of caspase-3 during the period of its natural activation in the hippocampus during early ontogenesis was found to impair the development of operant behavior in rats. This was apparent as a reduction in the efficiency of learning during acquisition of active avoidance reactions and decreases in the numbers of intersignal reactions. Administration of the inhibitor had no specific action on the types of conditioned reflex activity less associated with operant learning. Thus, there were no differences between the experimental and control groups in the numbers of emotional reactions to the conditioned stimulus. The number of orientational-investigative conditioned reactions also showed no change after administration of Z-DEVD-FMK. On the background of the reduction in the efficiency of the acquisition of the conditioned active avoidance reflex, the number of incomplete acts, in contrast to other types of conditioned reactions, increased significantly after administration of Z-DEVD-FMK, which is evidence for the persistence of the ability to form associative connections between activation of the conditioned signal and the need to move to the other sector. The difficulty in these animals arose at the decision-taking stage on choosing the appropriate form of behavior. Changes in orientational-investigative behavior were not associated with inhibition of caspase-3 during the critical period of development, as the effects of Z-DEVD-FMK and Z-FA-FMK were similar.

  12. Trichomonas vaginalis promotes apoptosis of human neutrophils by activating caspase-3 and reducing Mcl-1 expression.

    PubMed

    Kang, J H; Song, H O; Ryu, J S; Shin, M H; Kim, J M; Cho, Y S; Alderete, J F; Ahn, M H; Min, D Y

    2006-09-01

    Neutrophils are the predominant inflammatory cells found in the vaginal discharge of patients with Trichomonas vaginalis infection. However, it is not known whether neutrophil apoptosis is induced by live T. vaginalis. Therefore, we examined whether T. vaginalis can influence neutrophil apoptosis, and also whether caspase-3 and the Bcl-2 family members are involved in the apoptosis. Thus, human neutrophils were incubated with live T. vaginalis and neutrophil apoptosis was evaluated by Giemsa, annexin V-PI, and DiOC6 stainings. The neutrophil apoptosis was significantly higher in those incubated with T. vaginalis than in the control group. When trichomonads were pre-treated with mAb to AP65 (adhesin protein), or when trophozoites were separated from neutrophils using a Transwell chamber, neutrophil apoptosis was significantly reduced. The activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis but was markedly enhanced during T. vaginalis-induced apoptosis. Moreover, the inhibition of caspase-3 effectively reduced T. vaginalis-induced apoptosis. Trichomonad-induced apoptosis was also associated with reduced expression of the neutrophil anti-apoptotic protein, Mcl-1. These results indicate that T. vaginalis alters Mcl-1 expression and caspase-3 activation, thereby inducing apoptosis of human neutrophils. PMID:16916367

  13. Trichomonas vaginalis promotes apoptosis of human neutrophils by activating caspase-3 and reducing Mcl-1 expression

    PubMed Central

    KANG, J. H.; SONG, H. O.; RYU, J. S.; SHIN, M. H.; KIM, J. M.; CHO, Y. S.; ALDERETE, J. F.; AHN, M. H.; MIN, D. Y.

    2007-01-01

    SUMMARY Neutrophils are the predominant inflammatory cells found in the vaginal discharge of patients with Trichomonas vaginalis infection. However, it is not known whether neutrophil apoptosis is induced by live T. vaginalis. Therefore, we examined whether T. vaginalis can influence neutrophil apoptosis, and also whether caspase-3 and the Bcl-2 family members are involved in the apoptosis. Thus, human neutrophils were incubated with live T. vaginalis and neutrophil apoptosis was evaluated by Giemsa, annexin V-PI, and DiOC6 stainings. The neutrophil apoptosis was significantly higher in those incubated with T. vaginalis than in the control group. When trichomonads were pre-treated with mAb to AP65 (adhesin protein), or when trophozoites were separated from neutrophils using a Transwell chamber, neutrophil apoptosis was significantly reduced. The activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis but was markedly enhanced during T. vaginalis-induced apoptosis. Moreover, the inhibition of caspase-3 effectively reduced T. vaginalis-induced apoptosis. Trichomonad-induced apoptosis was also associated with reduced expression of the neutrophil anti-apoptotic protein, Mcl-1. These results indicate that T. vaginalis alters Mcl-1 expression and caspase-3 activation, thereby inducing apoptosis of human neutrophils. PMID:16916367

  14. Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage.

    PubMed

    Zhang, Jiao; Wang, Xin; Cui, Wenjing; Wang, Wenwen; Zhang, Huamei; Liu, Lu; Zhang, Zicheng; Li, Zheng; Ying, Guoguang; Zhang, Ning; Li, Binghui

    2013-01-01

    Cytosolic caspase-3-like proteases, such as caspase-3 and caspase-7, have a central role in mediating the progress of apoptosis. Here to conveniently monitor caspase-3-like activity in the multicellular environment, we have developed genetically encoded switch-on fluorescence-base indicators that are cyclized chimeras containing a caspase-3 cleavage site as a switch. When cleaved by caspase-3-like proteases, the non-fluorescent indicator rapidly becomes fluorescent, and thus detects in real-time the activation of such caspases. We generate cultured cells constitutively expressing these chimeras, and all the healthy cells are non-fluorescent. When these cells are exposed to apoptotic stimuli, dead cells show strong fluorescence depending on caspase activation. With these tools, we monitor in real-time caspase-3-like activity in each cell under various conditions, and show for the first time that the environment of cancer cells affects their sensitivity to chemotherapeutic drugs in a modified soft agar assay. These biosensors should enable better understanding of the biological relevance of caspase-3-like proteases.

  15. Application of the FRET method for monitoring the dynamics of caspase-3 activation during apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Chen, Tongsheng; Xing, Da

    2005-01-01

    Activation of caspase-3 is a central event in apoptosis. A fluorescence techniques, fluorescence resonance energy transfer (FRET), was used to study the dynamic of caspase-3 activation during apoptosis induced by tumor necrosis factor TNF-α in living cells. The FRET probe consists a CFP (cyan fluorescent protein) and a Venus (YFP mutant, yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD (Luo et al., 2001). Human lung adenocarcinoma cell line (ASTC-a-1) were stably expressed with the FRET probe and then were treated by TNF-α, respectively. Experimental results showed that FRET could monitor more insensitively the dynamic of caspase-3 activation in real-time in vivo, and this technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.

  16. Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells

    NASA Astrophysics Data System (ADS)

    Xiao, Feng-Lian; Chen, Tong-Sheng; Qu, Jun-Le; Liu, Cheng-Yi

    2010-02-01

    Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

  17. Gamma secretase activating protein is a substrate for caspase-3: implications for Alzheimer’s disease

    PubMed Central

    Chu, Jin; Li, Jian-Guo; Joshi, Yash B.; Giannopoulos, Phillip F.; Hoffman, Nicholas E.; Madesh, Muniswamy; Praticò, Domenico

    2014-01-01

    SUMMARY Background A major hallmark feature of Alzheimer’s disease (AD) is the accumulation of amyloid β (Aβ), whose formation is regulated by the γ-secretase complex and its activating protein (also known as GSAP). Because GSAP interacts with the γ-secretase without affecting the cleavage of Notch, it is an ideal target for a viable anti-Aβ therapy. However, despite much interest in this protein, the mechanisms involved in its neurobiology are not known. Methods Post-mortem brain tissues from AD patients, transgenic mouse models of AD and neuronal cells were used to investigate the molecular mechanism involved in GSAP formation and subsequent amyloidogenesis. Results We identify a caspase-3 processing domain in the GSAP sequence and provide experimental evidence that this caspase is essential for GSAP activation and biogenesis of Aβ peptides. Furthermore, we demonstrate that caspase-3-dependent GSAP formation occurs in brains of individuals with AD and two different mouse models of AD, and that the process is biologically relevant since its pharmacological blockade reduces Aβ pathology in vivo. Interpretation Our data by identifying caspase-3 as the endogenous modulator of GSAP and Aβ production establish it as a novel, attractive and viable Aβ lowering therapeutic target for AD. PMID:25052851

  18. Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain.

    PubMed

    Konno, Ayumi; Nishimura, Akiko; Nakamura, Shiro; Mochizuki, Ayako; Yamada, Atsushi; Kamijo, Ryutaro; Inoue, Tomio; Iijima, Takehiko

    2016-06-01

    The neurotoxicity of anesthetics on the developing brain has drawn the attention of anesthesiologists. Several studies have shown that apoptosis is enhanced by exposure to anesthesia during brain development. Although apoptosis is a physiological developmental step occurring before the maturation of neural networks and the integration of brain function, pathological damage also involves apoptosis. Previous studies have shown that prolonged exposure to anesthetics causes apoptosis. Exactly when the apoptotic cascade starts in the brain remains uncertain. If it starts during the early stage of anesthesia, even short-term anesthesia could harm the brain. Therefore, apoptogenesis should be continuously monitored to elucidate when the apoptotic cascade is triggered by anesthesia. Here, we describe the development of a continuous monitoring system to detect caspase-3 activation using an in vivo model. Brain slices from postnatal days 0-4 SCAT3 transgenic mice with a heterozygous genotype (n=20) were used for the monitoring of caspase-3 cleavage. SCAT3 is a fusion protein of ECFP and Venus connected by a caspase-3 cleavable peptide, DEVD. A specimen from the hippocampal CA1 sector was mounted on a confocal laser microscope and was continuously superfused with artificial cerebrospinal fluid, propofol (2,6-diisopropylphenol, 1μM or 10μM), and dimethyl sulfoxide. Images were obtained every hour for five hours. A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio>1 indicated the number of pixels from caspase-3-activated CA1 neurons. We observed a shift in the histogram toward the right over time, indicating caspase-3 activation. This right-ward shift dramatically changed at five hours in the propofol 1μM and 10μM groups and was obviously different from that in the control group. Thus, real-time fluorescence energy transfer (FRET) imaging

  19. Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation.

    PubMed

    Tellone, Ester; Ficarra, Silvana; Russo, Annamaria; Bellocco, Ersilia; Barreca, Davide; Laganà, Giuseppina; Leuzzi, Ugo; Pirolli, Davide; De Rosa, Maria Cristina; Giardina, Bruno; Galtieri, Antonio

    2012-02-01

    The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation-deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine-haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO(2) thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme-protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the "caspase silence"; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power.

  20. Cytoprotection against Hypoxic and/or MPP⁺ Injury: Effect of δ-Opioid Receptor Activation on Caspase 3.

    PubMed

    Xu, Yuan; Zhi, Feng; Shao, Naiyuan; Wang, Rong; Yang, Yilin; Xia, Ying

    2016-01-01

    The pathological changes of Parkinson's disease (PD) are, at least partially, associated with the dysregulation of PTEN-induced putative kinase 1 (PINK1) and caspase 3. Since hypoxic and neurotoxic insults are underlying causes of PD, and since δ-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults, we sought to determine whether DOR activation could protect the cells from damage induced by hypoxia and/or MPP⁺ by regulating PINK1 and caspase 3 expressions. We exposed PC12 cells to either severe hypoxia (0.5%-1% O₂) for 24-48 h or to MPP⁺ at different concentrations (0.5, 1, 2 mM) and then detected the levels of PINK1 and cleaved caspase 3. Both hypoxia and MPP⁺ reduced cell viability, progressively suppressed the expression of PINK1 and increased the cleaved caspase 3. DOR activation using UFP-512, effectively protected the cells from hypoxia and/or MPP⁺ induced injury, reversed the reduction in PINK1 protein and significantly attenuated the increase in the cleaved caspase 3. On the other hand, the application of DOR antagonist, naltrindole, greatly decreased cell viability and increased cleaved caspase 3. These findings suggest that DOR is cytoprotective against both hypoxia and MPP⁺ through the regulation of PINK1 and caspase 3 pathways. PMID:27517901

  1. Cytoprotection against Hypoxic and/or MPP+ Injury: Effect of δ–Opioid Receptor Activation on Caspase 3

    PubMed Central

    Xu, Yuan; Zhi, Feng; Shao, Naiyuan; Wang, Rong; Yang, Yilin; Xia, Ying

    2016-01-01

    The pathological changes of Parkinson’s disease (PD) are, at least partially, associated with the dysregulation of PTEN-induced putative kinase 1 (PINK1) and caspase 3. Since hypoxic and neurotoxic insults are underlying causes of PD, and since δ-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults, we sought to determine whether DOR activation could protect the cells from damage induced by hypoxia and/or MPP+ by regulating PINK1 and caspase 3 expressions. We exposed PC12 cells to either severe hypoxia (0.5%–1% O2) for 24–48 h or to MPP+ at different concentrations (0.5, 1, 2 mM) and then detected the levels of PINK1 and cleaved caspase 3. Both hypoxia and MPP+ reduced cell viability, progressively suppressed the expression of PINK1 and increased the cleaved caspase 3. DOR activation using UFP-512, effectively protected the cells from hypoxia and/or MPP+ induced injury, reversed the reduction in PINK1 protein and significantly attenuated the increase in the cleaved caspase 3. On the other hand, the application of DOR antagonist, naltrindole, greatly decreased cell viability and increased cleaved caspase 3. These findings suggest that DOR is cytoprotective against both hypoxia and MPP+ through the regulation of PINK1 and caspase 3 pathways. PMID:27517901

  2. A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis

    SciTech Connect

    Walters, Jad; Pop, Cristina; Scott, Fiona L.; Drag, Marcin; Swartz, Paul; Mattos, Carla; Salvesen, Guy S.; Clark, A.Clay

    2010-03-12

    The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 {angstrom} (1 {angstrom} = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

  3. Cooperation of bisphenol A and leptin in inhibition of caspase-3 expression and activity in OVCAR-3 ovarian cancer cells.

    PubMed

    Ptak, Anna; Rak-Mardyła, Agnieszka; Gregoraszczuk, Ewa L

    2013-09-01

    This study was designed to investigate the effect of bisphenol A and leptin on caspase-3 expression and activity in OVCAR-3 ovarian cancer cells. Caspase-3 and survivin expression was measured at the transcript level by real-time PCR and at the protein level by Western blotting. In addition, caspase-3 activity was measured, using a fluorometric assay, upon exposure to bisphenol A (40 nM) alone, leptin (2.5 nM) alone, and the combination of both agents. 17β-estradiol (40 nM) was used as a positive control for estrogenic properties of bisphenol A. Results showed that the interaction between bisphenol A and leptin, which was similar to that observed between 17β-estradiol and leptin, led to the inhibition of caspase-3 expression and activity in OVCAR-3 cells. Surprisingly, survivin was found to not be involved in the anti-apoptotic activity of either agent. Also, results showed that leptin inhibits caspase-3 activity by acting on the signal transducers and activators of transcription 3 (STAT3) pathway, but bisphenol A and 17β-estradiol by the extracellular-signal-regulated kinases 1/2 (ERK1/2) pathway. In conclusion, the study reveals that bisphenol A and leptin interact to inhibit caspase-3 expression and activity by modulating STAT3 and ERK1/2 signaling pathways in OVCAR-3 cells.

  4. Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

    2005-01-01

    Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

  5. 1'-Acetoxychavicol acetate promotes caspase 3-activated glioblastoma cell death by overcoming enhanced cytokine expression.

    PubMed

    Williams, Musa; Tietzel, Illya; Quick, Quincy A

    2013-06-01

    The brain consumes ∼20% of the oxygen utilized in the human body, meaning that brain tumors are vulnerable to paradoxical physiological effects from free radical generation. In the present study, 1'-acetoxychavicol acetate (ACA), a naturally derived antioxidant that inhibits xanthine oxidase, was evaluated for its role as an anti-tumorigenic agent in glioblastomas. The study revealed that ACA inhibited glioblastoma cell proliferation as a consequence of promoting apoptotic cell death by enhancing caspase 3 activity. It was also shown that ACA impaired the migratory ability of glioblastoma cells by decreasing their adhesive properties. Additionally, ACA increased the protein expression levels of the pro-survival signaling cytokines, IL-6 and IL-1α, established cell protectors and survival molecules in brain tumors. Together, these results demonstrate that, despite enhanced expression of compensatory signaling molecules that contribute to tumor cell survival, ACA is an effective pro-apoptotic inducing agent in glioblastomas.

  6. CasExpress reveals widespread and diverse patterns of cell survival of caspase-3 activation during development in vivo

    PubMed Central

    Ding, Austin Xun; Sun, Gongping; Argaw, Yewubdar G; Wong, Jessica O; Easwaran, Sreesankar; Montell, Denise J

    2016-01-01

    Caspase-3 carries out the executioner phase of apoptosis, however under special circumstances, cells can survive its activity. To document systematically where and when cells survive caspase-3 activation in vivo, we designed a system, CasExpress, which drives fluorescent protein expression, transiently or permanently, in cells that survive caspase-3 activation in Drosophila. We discovered widespread survival of caspase-3 activity. Distinct spatial and temporal patterns emerged in different tissues. Some cells activated caspase-3 during their normal development in every cell and in every animal without evidence of apoptosis. In other tissues, such as the brain, expression was sporadic both temporally and spatially and overlapped with periods of apoptosis. In adults, reporter expression was evident in a large fraction of cells in most tissues of every animal; however the precise patterns varied. Inhibition of caspase activity in wing discs reduced wing size demonstrating functional significance. The implications of these patterns are discussed. DOI: http://dx.doi.org/10.7554/eLife.10936.001 PMID:27058168

  7. Electrochemiluminescent Sensing for Caspase-3 Activity Based on Ru(bpy)3(2+)-Doped Silica Nanoprobe.

    PubMed

    Dong, Yong-Ping; Chen, Gang; Zhou, Ying; Zhu, Jun-Jie

    2016-02-01

    Caspase-3 is one of the most frequently activated cysteine proteases during the apoptosis process and has been identified as a well-established cellular marker of apoptosis. In this study, a novel approach for the sensitive determination of caspase-3 activity was proposed using electrochemiluminescence (ECL) of Ru(bpy)3(2+)-doped silica (Ru@SiO2) with tripropylamine (TPA) as coreactant. A nanocomposite containing gold nanoparticles (AuNPs), poly(dimethyldiallyl ammonium chloride) (PDDA), and multiwalled carbon nanotubes (CNTs) was fabricated as an ECL platform. The biotinylated DEVD-peptide (biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Gly-Cys) was immobilized on the nanocomposite surface via the strong bonding interaction between AuNPs and the thiol group. Then the streptavidin-modified Ru(bpy)3(2+)-doped silica (Ru@SiO2-SA) was immobilized on the ECL platform via the specific interaction between biotin and streptavidin to generate ECL signal. Caspase-3 can specifically recognize and cleave the N-terminus of DEVD, leading to the loss of the biotin label and the decrease of ECL intensity to determine the activity of caspase-3. The results revealed a new ECL avenue for the sensitive and specific monitor of caspase-3, and the platform could be utilized to evaluate anticancer drugs. PMID:26730888

  8. Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

    NASA Astrophysics Data System (ADS)

    Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

    2009-07-01

    Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

  9. Coated chitosan nanoparticles encapsulating caspase 3 activator for effective treatment of colorectral cancer.

    PubMed

    Jain, Aakanchha; Jain, Sourabh; Jain, Richa; Kohli, Dharm Veer

    2015-12-01

    Cancer is the second leading cause of death worldwide, the deaths are projected to continue rising, with an estimated 12 million deaths in 2030. The aim of the present investigation is to prepare and compare the uncoated (U-CH NP) and eudragit S 100-coated (E-U-CH NP) chitosan nanoparticles encapsulating a caspase 3 activator (UCN 01), by ionic gelation method. The prepared formulations were studied for various parameters like particles size, zeta potential, transmission electron microscopy, atomic force microscopy, in vitro release study, ex vivo study using Caco 2 colon cancer cell line, and in vivo studies. The particle size and zeta potential of developed formulation was found to be particle size of 168 ± 3.7 nm and +35.8 ± 3.7 for U-CH NP and 265 ± 4.1 nm and +22.3 ± 1.1 for E-U-CH NP. TEM and AFM images revealed that U-CH NPs were round in shape and smoother at surface as compared to E-U-CH NP which have irregular surface due to coating. The E-U-CH NP showed better in vitro release than uncoated formulation in SCF (pH 6.8) than in SGF (pH 1.2). The cytotoxicity was performed by MTT assay. U-CH NP showed enhanced cytotoxicity as compared to blank (without drug) formulation. There was an increase in caspase 3 activity of U-CH NP as compared to UCN 01 alone. E-U-CH NP showed better tumor regression ability than U-CH NP. The results of plasma profile and tumor regression study demonstrated that E-U-CH NP has continuous release profile of UCN 01 and comprehensive residence time. Thus, it is better acceptable than free UCN 01 and may be a potential delivery system for the targeting and treatment of colon cancer.

  10. Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

    PubMed

    Cui, X; Zhang, L; Magli, A R; Catera, R; Yan, X-J; Griffin, D O; Rothstein, T L; Barrientos, J; Kolitz, J E; Allen, S L; Rai, K R; Chiorazzi, N; Chu, C C

    2016-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  11. Effects of camptothecin, etoposide and Ca2+ on caspase-3 activity and myofibrillar disruption of chicken during postmortem ageing.

    PubMed

    Chen, Lin; Feng, Xian Chao; Lu, Feng; Xu, Xing Lian; Zhou, Guang Hong; Li, Qing Yun; Guo, Xiang Ying

    2011-03-01

    Recently, a novel consideration has focused on the potential relationship of apoptosis and the protease caspases and the underlying mechanism for meat postmortem tenderization. In this study, apoptosis inducers, camptothecin and etoposide as well as Ca(2+) were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during 7 days of ageing. All three treatments resulted in significantly higher caspase-3 activities during storage (p<0.05), with the natural substrates, whereas Western blotting analysis of the α-spectrin cleavage product, 120 kDa peptide (SBDP 120), showed that Ca(2+) was more effective than either camptothecin or etopside, and all were most active up to day 3 (p<0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product, especially during the first 3 days (p<0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected, even at day 7. The findings revealed that the three apoptosis inducers could increase myofibrillar dissociation and proteolysis during the first 3 days of chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat.

  12. Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

    PubMed

    Fernández, María Belén; Daleo, Gustavo Raúl; Guevara, María Gabriela

    2015-01-01

    Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction. PMID:25486023

  13. Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

    PubMed

    Fernández, María Belén; Daleo, Gustavo Raúl; Guevara, María Gabriela

    2015-01-01

    Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction.

  14. Effect of lycopene on caspase-3 enzyme activation in liver of methanol-intoxicated rats: comparison with fomepizole.

    PubMed

    Kurcer, Mehmet Ali; Kurcer, Zehra; Koksal, Mete; Baba, Fusun; Ocak, Ali Riza; Aksoy, Nurten; Atessahin, Ahmet; Sahna, Engin

    2010-08-01

    Lycopene is one of the major carotenoids and is found almost exclusively in tomatoes and tomato products. This study was performed to evaluate the effect of lycopene on methanol-induced liver injury and to compare the results with those after fomepizole, which is used in treatment of methanol intoxication. Experiments were carried out with 30 female Wistar rats weighting 180-200 g. Rats were injected with a intraperitoneally dose of 3 g/kg methanol as a 50% solution in isotonic saline once for intoxication. Rats were pretreated with fomepizole (50 mg/kg) and/or lycopene (10 mg/kg) before methanol. After 24 hours all the drug-treated and intoxicated rats were sacrificed under anesthesia. Malondialdehyde (MDA) levels were determined in order to assess lipid peroxidation, and caspase-3 activity was determined by immunostaining of liver tissues to evaluate apoptosis. Methanol administration significantly increased the MDA level and caspase-3 activity in liver. Pretreatment with lycopene and/or fomepizole decreased the MDA levels significantly. Similarly, lycopene and fomepizole decreased methanol-induced caspase-3 activity. The findings of the present study demonstrate that methanol intoxication causes hepatic toxicity in rats and that this is likely a result of reactive oxygen species and apoptosis induction. Lycopene has protective effects against methanol-induced hepatic injury similar to fomepizole. It was demonstrated for the first time that both lycopene and fomepizole prevent methanol-induced hepatic injury by reducing the increase of lipid oxidation and caspase-3 activation. PMID:20482279

  15. Caspase-3-like activity determines the type of cell death following ionizing radiation in MOLT-4 human leukaemia cells.

    PubMed

    Coelho, D; Holl, V; Weltin, D; Lacornerie, T; Magnenet, P; Dufour, P; Bischoff, P

    2000-09-01

    Caspases, a family of cysteine proteases, play a central role in the pathways leading to apoptosis. Recently, it has been reported that a broad spectrum inhibitor of caspases, the tripeptide Z-VAD-fmk, induced a switch from apoptosis to necrosis in dexamethasone-treated B lymphocytes and thymocytes. As such a cell death conversion could increase the efficiency of radiation therapy and in order to identify the caspases involved in this cell death transition, we investigated the effects of caspase-3-related proteases inhibition in irradiated MOLT-4 cells. Cells were pretreated with Ac-DEVD-CHO, an inhibitor of caspase-3-like activity, and submitted to X-rays at doses ranging from 1 to 4 Gy. Our results show that the inhibition of caspase-3-like activity prevents completely the appearance of the classical hallmarks of apoptosis such as internucleosomal DNA fragmentation or hypodiploid particles formation and partially the externalization of phosphatidylserine. However, this was not accompanied by any persistent increase in cell survival. Instead, irradiated cells treated by this inhibitor exhibited characteristics of a necrotic cell death. Therefore, functional caspase-3-subfamily not only appears as key proteases in the execution of the apoptotic process, but their activity may also influence the type of cell death following an exposure to ionizing radiation.

  16. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    PubMed Central

    Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

    2016-01-01

    Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

  17. Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation

    SciTech Connect

    Ali, Shafaqat; Nguyen, Dang Quan; Falk, Werner; Martin, Michael Uwe

    2010-01-15

    IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

  18. Safrole oxide induces apoptosis by activating caspase-3, -8, and -9 in A549 human lung cancer cells.

    PubMed

    Du, Aiying; Zhao, Baoxiang; Yin, Deling; Zhang, Shangli; Miao, Junying

    2006-01-01

    Previously we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells. In this study, we further investigated which caspases were activated by safrole oxide during the apoptosis. The data showed that the activity of caspase-3, -8, and -9 was significantly enhanced by the compound, which suggested that safrole oxide might be used as a caspase promoter to initiate lung cancer cell apoptosis.

  19. Apoptosis in Heart Failure: Release of Cytochrome c from Mitochondria and Activation of Caspase-3 in Human Cardiomyopathy

    NASA Astrophysics Data System (ADS)

    Narula, Jagat; Pandey, Pramod; Arbustini, Eloisa; Haider, Nezam; Narula, Navneet; Kolodgie, Frank D.; dal Bello, Barbara; Semigran, Marc J.; Bielsa-Masdeu, Anna; Dec, G. William; Israels, Sara; Ballester, Manel; Virmani, Renu; Saxena, Satya; Kharbanda, Surender

    1999-07-01

    Apoptosis has been shown to contribute to loss of cardiomyocytes in cardiomyopathy, progressive decline in left ventricular function, and congestive heart failure. Because the molecular mechanisms involved in apoptosis of cardiocytes are not completely understood, we studied the biochemical and ultrastructural characteristics of upstream regulators of apoptosis in hearts explanted from patients undergoing transplantation. Sixteen explanted hearts from patients undergoing heart transplantation were studied by electron microscopy or immunoblotting to detect release of mitochondrial cytochrome c and activation of caspase-3. The hearts explanted from five victims of motor vehicle accidents or myocardial ventricular tissues from three donor hearts were used as controls. Evidence of apoptosis was observed only in endstage cardiomyopathy. There was significant accumulation of cytochrome c in the cytosol, over myofibrils, and near intercalated discs of cardiomyocytes in failing hearts. The release of mitochondrial cytochrome c was associated with activation of caspase-3 and cleavage of its substrate protein kinase C δ but not poly(ADP-ribose) polymerase. By contrast, there was no apparent accumulation of cytosolic cytochrome c or caspase-3 activation in the hearts used as controls. The present study provides in vivo evidence of cytochrome c-dependent activation of cysteine proteases in human cardiomyopathy. Activation of proteases supports the phenomenon of apoptosis in myopathic process. Because loss of myocytes contributes to myocardial dysfunction and is a predictor of adverse outcomes in the patients with congestive heart failure, the present demonstration of an activated apoptotic cascade in cardiomyopathy could provide the basis for novel interventional strategies.

  20. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    PubMed

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments.

  1. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    PubMed

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. PMID:27130972

  2. Analysis of caspase3 activation in ChanSu-induced apoptosis of ASTC-a-1 cells by fluorescence techniques

    NASA Astrophysics Data System (ADS)

    Sun, Lei; Chen, Tongsheng; Wang, Longxiang; Wang, Huiying

    2008-02-01

    ChanSu(CS), a traditional Chinese medicine, is composed of many chemical compoments. It is isolated from the dried white secretion of the auricular and skin glands of toads, and it has been widely used for treating the heart diseases and other systemic illnesses. However, it is difficult to judge antitumor effect of agents derived from ChanSu and the underlying mechanism of ChanSu inducing cell apoptosis is still unclear. This report was performed to explore the inhibitory effect and mechanism of ChanSu on human lung adenocarcinoma cells (ASTC-a-1). Fluorescence emission spectra and fluorescence resonance energy transfer (FRET) were used to study the caspase-3 activation during the ChanSu-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. CCK-8 was used to assay the inhibition of ChanSu on the cell viability. The cells expressing stably with SCAT3 was used to examine if caspase-3 was activated by ChanSu using acceptor photobleaching technique. Our data showed that treatment of ASTC-a-1 cell with ChanSu resulted in the inhibition of viability and induction of apoptosis in a dose-dependent manner and the SCAT3 was almost cleaved 24 h after ChanSu treatment, implying that ChanSu induced cell apoptosis via a caspase-3-dependent death pathway. Our findings extend the knowledge about the cellular signaling mechanisms mediating ChanSu-induced apoptosis.

  3. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    PubMed

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  4. Regulation of caspase-3 processing by cIAP2 controls the switch between pro-inflammatory activation and cell death in microglia

    PubMed Central

    Kavanagh, E; Rodhe, J; Burguillos, M A; Venero, J L; Joseph, B

    2014-01-01

    The activation of microglia, resident immune cells of the central nervous system, and inflammation-mediated neurotoxicity are typical features of neurodegenerative diseases, for example, Alzheimer's and Parkinson's diseases. An unexpected role of caspase-3, commonly known to have executioner role for apoptosis, was uncovered in the microglia activation process. A central question emerging from this finding is what prevents caspase-3 during the microglia activation from killing those cells? Caspase-3 activation occurs as a two-step process, where the zymogen is first cleaved by upstream caspases, such as caspase-8, to form intermediate, yet still active, p19/p12 complex; thereafter, autocatalytic processing generates the fully mature p17/p12 form of the enzyme. Here, we show that the induction of cellular inhibitor of apoptosis protein 2 (cIAP2) expression upon microglia activation prevents the conversion of caspase-3 p19 subunit to p17 subunit and is responsible for restraining caspase-3 in terms of activity and subcellular localization. We demonstrate that counteracting the repressive effect of cIAP2 on caspase-3 activation, using small interfering RNA targeting cIAP2 or a SMAC mimetic such as the BV6 compound, reduced the pro-inflammatory activation of microglia cells and promoted their death. We propose that the different caspase-3 functions in microglia, and potentially other cell types, reside in the active caspase-3 complexes formed. These results also could indicate cIAP2 as a possible therapeutic target to modulate microglia pro-inflammatory activation and associated neurotoxicity observed in neurodegenerative disorders. PMID:25501826

  5. Emission spectral analysis of caspase-3 activation during artesunate (ART)-induced apoptosis of human lung adenocarcinoma cell

    NASA Astrophysics Data System (ADS)

    Pan, Wen-liang; Chen, Tong-sheng; Qu, Junle

    2009-02-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. Artemisinin-derivative combination chemotherapy is recommended by WHO since it acts rapidly and is well tolerated and particularly effective. In present investigation, we used CKK-8 assay to assess the inhibitory effects of ART on human lung adenocarcinoma (ASTC-a-1) cells. Apoptotic activity of ART in ASTC-a-1 cells was detected by means of nuclear staining with Hoechst33258. In order to monitor the activity of caspase-3 during ART-induced ASTC-a-1 cells apoptosis, the dynamical emission spectra of SCAT3, a FRET plasmid based on GFPs, were performed inside living cell expressed stably with SCAT3 after ART treatment. The results showed that (1) ART could inhibit ASTC-a-1 cells proliferation in a dose-dependent manner; (2) chromatin condensation was observed after ART treatment for 48 h; (3) the SCAT3 inside living cells were cleaved after ART treatment for 48 h, implying that caspase-3 was involved in the ART-induced apoptosis.

  6. Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa.

    PubMed

    Muñoz, Patricia Martín; Ferrusola, Cristina Ortega; Lopez, Luis Anel; Del Petre, Chiara; Garcia, Mercedes Alvarez; de Paz Cabello, Paulino; Anel, Luis; Peña, Fernando J

    2016-09-01

    Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4-HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC) = 0.820, P < 0.05] and percentages of caspase 3- and 4-HNE-positive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alpha-positive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the

  7. Mouse macrophage polarity and ROCK1 activity depend on RhoA and non-apoptotic Caspase 3.

    PubMed

    Liu, Yianzhu; Minze, Laurie J; Mumma, Lindsay; Li, Xian C; Ghobrial, Rafik M; Kloc, Malgorzata

    2016-02-15

    The macrophages have different subtypes with different functions in immune response and disease. It has been generally accepted that M1 macrophages are responsible for stimulation of immune system and inflammation while M2 macrophages play a role in tissue repair. Irrespective of the type, macrophage functions depend on actin cytoskeleton, which is under the control of small GTPase RhoA pathway and its downstream effector ROCK1. We generated RhoA-deleted macrophages and compared the effect of RhoA deletion on M0, M1 and M2 macrophage phenotype. Our studies showed that, unexpectedly, the RhoA deletion did not eliminate macrophage ROCK1 expression and increased ROCK1 activity. The RhoA deletion effect on macrophage phenotype, structure and polarity was different for each subtype. Moreover, our study indicates that the up-regulation of ROCK1 activity in RhoA-deleted macrophages and macrophage phenotype/polarity are dependent on non-apoptotic Caspase-3 and are sensitive to Caspase-3 inhibition. These novel findings will revise/complement our understanding of RhoA pathway regulation of cell structure and polarity. PMID:26875770

  8. LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

    SciTech Connect

    Russe, Otto Quintus Möser, Christine V. Kynast, Katharina L. King, Tanya S. Olbrich, Katrin Grösch, Sabine Geisslinger, Gerd Niederberger, Ellen

    2014-05-09

    Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

  9. The caspase 3 sensor Phiphilux G2D2 is activated non-specifically in S1 renal proximal tubules

    PubMed Central

    Hato, Takashi; Sandoval, Ruben; Dagher, Pierre C

    2016-01-01

    Tubular cell apoptosis is a major phenotype of cell death in various forms of acute kidney injury. Quantifying apoptosis in fixed tissues is problematic because apoptosis evolves over time and dead cells are rapidly cleared by the phagocytic system. Phiphilux is a fluorescent probe that is activated specifically by caspase 3 and does not inhibit the subsequent activity of this effector caspase. It has been used successfully to quantify apoptosis in cell culture. Here we examined the feasibility of using Phiphilux to measure renal tubular apoptosis progression over time in live animals using intravital 2-photon microscopy. Our results show that Phiphilux can detect apoptosis in S2 tubules but is activated non-specifically in S1 tubules.

  10. 1′-Acetoxychavicol acetate promotes caspase 3-activated glioblastoma cell death by overcoming enhanced cytokine expression

    PubMed Central

    WILLIAMS, MUSA; TIETZEL, ILLYA; QUICK, QUINCY A.

    2013-01-01

    The brain consumes ∼20% of the oxygen utilized in the human body, meaning that brain tumors are vulnerable to paradoxical physiological effects from free radical generation. In the present study, 1′-acetoxychavicol acetate (ACA), a naturally derived antioxidant that inhibits xanthine oxidase, was evaluated for its role as an anti-tumorigenic agent in glioblastomas. The study revealed that ACA inhibited glioblastoma cell proliferation as a consequence of promoting apoptotic cell death by enhancing caspase 3 activity. It was also shown that ACA impaired the migratory ability of glioblastoma cells by decreasing their adhesive properties. Additionally, ACA increased the protein expression levels of the pro-survival signaling cytokines, IL-6 and IL-1α, established cell protectors and survival molecules in brain tumors. Together, these results demonstrate that, despite enhanced expression of compensatory signaling molecules that contribute to tumor cell survival, ACA is an effective pro-apoptotic inducing agent in glioblastomas. PMID:23833677

  11. Caspase-3 modulates regenerative response after stroke.

    PubMed

    Fan, Wenying; Dai, Yiqin; Xu, Haochen; Zhu, Ximin; Cai, Ping; Wang, Lixiang; Sun, Chungang; Hu, Changlong; Zheng, Ping; Zhao, Bing-Qiao

    2014-02-01

    Stroke is a leading cause of long-lasting disability in humans. However, currently there are still no effective therapies available for promoting stroke recovery. Recent studies have shown that the adult brain has the capacity to regenerate neurons after stroke. Although this neurogenic response may be functionally important for brain repair after injury, the mechanisms underlying stroke-induced neurogenesis are not known. Caspase-3 is a major executioner and has been identified as a key mediator of neuronal death in the acute stage of stroke. Recently, however, accumulating data indicate that caspase-3 also participates in various biological processes that do not cause cell death. Here, we show that cleaved caspase-3 was increased in newborn neuronal precursor cells (NPCs) in the subventricular zone (SVZ) and the dentate gyrus during the period of stroke recovery, with no evidence of apoptosis. We observed that cleaved caspase-3 was expressed by NPCs and limited its self-renewal without triggering apoptosis in cultured NPCs from the SVZ of ischemic mice. Moreover, we revealed that caspase-3 negatively regulated the proliferation of NPCs through reducing the phosphorylation of Akt. Importantly, we demonstrated that peptide inhibition of caspase-3 activity significantly promoted the proliferation and migration of SVZ NPCs and resulted in a significant increase in subsequent neuronal regeneration and functional recovery after stroke. Together, our data identify a previously unknown caspase-3-dependent mechanism that constrains stroke-induced endogenous neurogenesis and should revitalize interest in targeting caspase-3 for treatment of stroke.

  12. Induction of Caspase-3-like activity in Rice following release of cytochrome-f from the chloroplast and subsequent interaction with the Ubiquitin-Proteasome System

    PubMed Central

    Wang, Hongjuan; Zhu, Xiaonan; Li, Huan; Cui, Jing; Liu, Cheng; Chen, Xi; Zhang, Wei

    2014-01-01

    It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD. PMID:25103621

  13. Glucosidase II β-subunit, a novel substrate for caspase-3-like activity in rice, plays as a molecular switch between autophagy and programmed cell death.

    PubMed

    Cui, Jing; Chen, Bing; Wang, Hongjuan; Han, Yue; Chen, Xi; Zhang, Wei

    2016-01-01

    Endoplasmic reticulum (ER) stress activates unfolded protein response (UPR) and autophagy. However, prolonged, severe stresses activate programmed cell death (PCD) in both animal and plant cells. Compared to the well-studied UPR pathway, the molecular mechanisms of ER-stress-induced PCD are less understood. Here, we report the identification of Gas2, the glucosidase II β subunit in the ER, as a potential switch between PCD and autophagy in rice. MS analysis identified Gas2, GRP94, and HSP40 protein in a purified caspase-3-like activity from heat stressed rice cell suspensions. The three corresponding genes were down-regulated under DTT-induced ER stress. Gas2 and GRP94 were localized to the ER, while HSP40 localized to the cytoplasm. Compared to wild-type, a Gas2 RNAi cell line was much sensitive to DTT treatment and had high levels of autophagy. Both caspase-3 and heat-stressed cell suspension lysate could cleave Gas2, producing a 14 kDa N-terminal fragment. Conditional expression of corresponding C-terminal fragment resulted in enhanced caspase-3-like activity in the protoplasts under heat stress. We proposed that mild ER stress causes down-regulation of Gas2 and induces autophagy, while severe stress results in Gas2 cleavage by caspase-3-like activity and the cleavage product amplifies this activity, possibly participating in the initiation of PCD. PMID:27538481

  14. Glucosidase II β-subunit, a novel substrate for caspase-3-like activity in rice, plays as a molecular switch between autophagy and programmed cell death

    PubMed Central

    Cui, Jing; Chen, Bing; Wang, Hongjuan; Han, Yue; Chen, Xi; Zhang, Wei

    2016-01-01

    Endoplasmic reticulum (ER) stress activates unfolded protein response (UPR) and autophagy. However, prolonged, severe stresses activate programmed cell death (PCD) in both animal and plant cells. Compared to the well-studied UPR pathway, the molecular mechanisms of ER-stress-induced PCD are less understood. Here, we report the identification of Gas2, the glucosidase II β subunit in the ER, as a potential switch between PCD and autophagy in rice. MS analysis identified Gas2, GRP94, and HSP40 protein in a purified caspase-3-like activity from heat stressed rice cell suspensions. The three corresponding genes were down-regulated under DTT-induced ER stress. Gas2 and GRP94 were localized to the ER, while HSP40 localized to the cytoplasm. Compared to wild-type, a Gas2 RNAi cell line was much sensitive to DTT treatment and had high levels of autophagy. Both caspase-3 and heat-stressed cell suspension lysate could cleave Gas2, producing a 14 kDa N-terminal fragment. Conditional expression of corresponding C-terminal fragment resulted in enhanced caspase-3-like activity in the protoplasts under heat stress. We proposed that mild ER stress causes down-regulation of Gas2 and induces autophagy, while severe stress results in Gas2 cleavage by caspase-3-like activity and the cleavage product amplifies this activity, possibly participating in the initiation of PCD. PMID:27538481

  15. Alpha-chaconine, a potato glycoalkaloid, induces apoptosis of HT-29 human colon cancer cells through caspase-3 activation and inhibition of ERK 1/2 phosphorylation.

    PubMed

    Yang, Seun-Ah; Paek, Seung-Hwan; Kozukue, Nobuyuki; Lee, Kap-Rang; Kim, Jung-Ae

    2006-06-01

    Although alpha-chaconine, one of the two major potato trisaccharide glycoalkaloids, have shown cytotoxic effects on human cancer cells, the exact mechanism of this action of alpha-chaconine is not completely understood. In this study, we found that alpha-chaconine induced apoptosis of HT-29 cells in a time- and concentration-dependent manner by using flow cytometric analysis. We also found that caspase-3 activity and the active form of caspase-3 were increased 12 h after alpha-chaconine treatment. Caspase inhibitors, N-Ac-DEVD-CHO and Z-VAD-fmk, prevented alpha-chaconine-induced apoptosis, whereas alpha-chaconine-induced apoptosis was potentiated by PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. However, pretreatment of the cells with LY294002 and SB203580, inhibitors of PI3K and p38, respectively, BAPTA-AM, an intracellular Ca(2+) chelator, and antioxidants such as N-acetylcysteine (NAC) and Trolox had no effect on the alpha-chaconine-induced cell death. In addition, phosphorylation of ERK was reduced by the treatment with alpha-chaconine. Moreover, alpha-chaconine-induced caspase-3 activity was further increased by the pretreatment with PD98059. Thus, the results indicate that alpha-chaconine induces apoptosis of HT-29 cells through inhibition of ERK and, in turn, activation of caspase-3.

  16. Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39.

    PubMed

    Hishita, T; Tada-Oikawa, S; Tohyama, K; Miura, Y; Nishihara, T; Tohyama, Y; Yoshida, Y; Uchiyama, T; Kawanishi, S

    2001-04-01

    In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.

  17. Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.

    PubMed

    Zhang, Zhihong; Lin, Juqiang; Chu, Jun; Ma, Yan; Zeng, Shaoqun; Luo, Qingming

    2008-01-01

    Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

  18. Apoptosis induced by 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells is associated with modulation of polyamine metabolism and caspase-3 activation.

    PubMed

    Moffatt, J; Hashimoto, M; Kojima, A; Kennedy, D O; Murakami, A; Koshimizu, K; Ohigashi, H; Matsui-Yuasa, I

    2000-12-01

    The efficacy of the antitumor activity of 1'-acetoxychavicol acetate (ACA), reported to be a suppressor of chemically induced carcinogenesis, was evaluated in Ehrlich ascites tumor cells. ACA treatment resulted in changes in morphology and a dose-dependent suppression of cell viability. Apoptosis, characterized by nuclear condensation, membrane blebbing, cell shrinkage and a significant induction of caspase-3-like protease activity at 8 h in a time-course study were observed. Formation of apoptotic bodies was preceded by lowering of intracellular polyamines, particularly putrescine, and both dose- and time-dependent inhibitory and activation effect by ACA on ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT), respectively. Administration of exogenous polyamines prevented ACA-induced apoptosis represented by a reduction in the number of apoptotic bodies and also caused reduction in the induced caspase-3-like protease activity at 8 h. These findings suggest that the anticarcinogenic effects of ACA might be partly due to perturbation of the polyamine metabolic pathway and triggering of caspase-3-like activity, which result in apoptosis.

  19. Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion

    PubMed Central

    Hannan, Johanna L.; Matsui, Hotaka; Sopko, Nikolai A.; Liu, Xiaopu; Weyne, Emmanuel; Albersen, Maarten; Watson, Joseph W.; Hoke, Ahmet; Burnett, Arthur L.; Bivalacqua, Trinity J.

    2016-01-01

    Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED. PMID:27388816

  20. Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion.

    PubMed

    Hannan, Johanna L; Matsui, Hotaka; Sopko, Nikolai A; Liu, Xiaopu; Weyne, Emmanuel; Albersen, Maarten; Watson, Joseph W; Hoke, Ahmet; Burnett, Arthur L; Bivalacqua, Trinity J

    2016-01-01

    Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

  1. Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner

    PubMed Central

    Mnich, K; Carleton, L A; Kavanagh, E T; Doyle, K M; Samali, A; Gorman, A M

    2014-01-01

    Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner. PMID:24787014

  2. Time lapse imaging analysis of the effect of ER stress modulators on apoptotic cell assessed by caspase3/7 activation in NG108-15 cells

    PubMed Central

    Saito, Ayako; Suga, Kei; Ono-Nakagawa, Risa; Sanada, Masumi; Akagawa, Kimio

    2015-01-01

    This paper reports the data from the long term time lapse imaging of neuronal cell line NG108-15 that were treated with apoptosis inducer or various ER stress inducers. Use of the fluorescent reporter for activated caspase3/7 in combination with the conventional light microscope allowed us to investigate the time course of apoptosis induction at the single cell level. Quantitative as well as qualitative data are presented here to show the effect of two different ER stress modulating chemical compounds on caspase3/7-dependent apoptosis in neuronal cell line NG108-15 cells. Additional results and interpretation of our data concerning ER stress and apoptosis in NG108-15 cells can be found in Suga et al. (2015) [1] and in Suga et al. (2015) [2]. PMID:26759824

  3. NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - But not palmitate-induced toxicity.

    PubMed

    Anderson, Kimberley J; Russell, Aaron P; Foletta, Victoria C

    2015-01-01

    The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells. PMID:26380811

  4. Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study

    PubMed Central

    Gregoraszczuk, Ewa L; Rak-Mardyła, Agnieszka; Ryś, Janusz; Jakubowicz, Jerzy; Urbański, Krzysztof

    2015-01-01

    We aimed to develop a cost-effective and robust method to predict drug resistance in individual patients. Representative tissue fragments were obtained from tumors removed from female patients, aged 24-74 years old. The tumor tissue was taken by a histopathology’s or a surgeon under sterile conditions. Cells obtained by enzymatic dissociation from tumor after surgery, were cultured as a monolayer for 6 days. Paclitaxel, doxorubicin, carboplatin and endoxan alone or in combination were added at the beginning of culture and after 6 days, Alamar blue test was used for showing action on cell proliferation why caspase- 3 activity assays for verifying action on apoptosis. Inhibitory action on cell proliferation was noted in 2 of 12 patients tumor treated with both single and combined drugs. Using caspase-3 assay we showed that 50% of tumor cells was resistant to single chemotherapeutic drugs and 40% for combined. In 2 of 12 tumors, which did not reacted on single drugs, positive synergistic action on cell proliferation was observed in combination of D + E and C + E. This pilot study suggests: 1) monolayer culture of tumor cells, derived from individual patients, before chemotherapy could provide a suitable model for studying resistance for drugs; 2) caspase-3 activity is cheap and useful methods; 3) Alamar blue test should be taken into consideration for measuring cell proliferation. PMID:26664382

  5. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity.

    PubMed

    Goryashchenko, Alexander S; Khrenova, Maria G; Bochkova, Anna A; Ivashina, Tatiana V; Vinokurov, Leonid M; Savitsky, Alexander P

    2015-07-22

    This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb(3+) and from sensitized Tb(3+) to acceptor--the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

  6. Whole venom of Loxosceles similis activates caspases-3, -6, -7, and -9 in human primary skin fibroblasts.

    PubMed

    Dantas, Arthur Estanislau; Horta, Carolina Campolina Rebello; Martins, Thais M M; do Carmo, Anderson Oliveira; Mendes, Bárbara Bruna Ribeiro de Oliveira; Goes, Alfredo M; Kalapothakis, Evanguedes; Gomes, Dawidson A

    2014-06-01

    Spiders of the Loxosceles genus represent a risk to human health due to the systemic and necrotic effects of their bites. The main symptoms of these bites vary from dermonecrosis, observed in the majority of cases, to occasional systemic hemolysis and coagulopathy. Although the systemic effects are well characterized, the mechanisms of cell death triggered by the venom of these spiders are poorly characterized. In this study, we investigated the cell death mechanisms induced by the whole venom of the spider Loxosceles similis in human skin fibroblasts. Our results show that the venom initiates an apoptotic process and a caspase cascade involving the initiator caspase-9 and the effector caspases-3, -6, and -7.

  7. Low concentration of arsenic could induce caspase-3 mediated head kidney macrophage apoptosis with JNK-p38 activation in Clarias batrachus

    SciTech Connect

    Datta, Soma; Mazumder, Shibnath; Ghosh, Debabrata; Dey, Saibal; Bhattacharya, Shelley

    2009-12-15

    We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 muM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V-propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47{sup phox}. Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47{sup phox} translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH{sub 2}-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38-JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.

  8. Canine Notochordal Cell-Secreted Factors Protect Murine and Human Nucleus Pulposus Cells from Apoptosis by Inhibition of Activated Caspase-9 and Caspase-3/7

    PubMed Central

    Mehrkens, Arne; Karim, M. Zia; Kim, Sarah; Hilario, Raychel; Fehlings, Michael G.; Erwin, William Mark

    2013-01-01

    Introduction Effective therapies that may stop or even reverse disc degeneration remain elusive. A minimally invasive method through which nucleus pulposus (NP) cell viability could be achieved would revolutionize the treatment of degenerative disc disease (DDD). With the presented work, we have investigated if nonchondrodystrophic (NCD) canine intervertebral disc (IVD)-derived notochordal cell conditioned medium (NCCM) and chondrodystrophic (CD) canine IVD-derived conditioned medium (CDCM) are able to protect murine and human NP cells from apoptosis. Materials and Methods We developed NCCM and CDCM from hypoxic culture of freshly isolated NPs from NCD and CD canines, respectively. We obtained murine NP cells from nine different C57BL/6 mice and human NP cells from four patients who underwent surgery for discectomy. The cells were cultured with ADMEM/F-12 (control media), NCCM, or CDCM under hypoxic conditions (3.5% O2) and treated with IL-1β + FasL or Etoposide. All media were supplemented with 2% fetal bovine serum. We then determined the expression of specific apoptotic pathways in the murine and human NP cells by recording activated caspase-8, caspase-9, and caspase-3/7 activity. Results In the murine NP cells, NCCM inhibits IL-1β + FasL- and Etoposide-mediated apoptosis via suppression of activated caspase-9 and caspase-3/7, CDCM demonstrated an inhibitory effect on IL-1β + FasL-mediated apoptosis via caspase-3/7 (Fig. 1A). In the human NP cells, NCCM inhibits Etoposide- mediated apoptosis via suppression of activated caspase-8, caspase-9, and mainly caspase-3/7. CDCM demonstrated an inhibitory effect on Etoposide-mediated apoptosis via suppression of activated caspase-8, caspase-9, and mainly caspase-3/7, though not as effective as NCCM (Fig. 1B). Conclusion IL-1β + FasL are known key molecules in the progression of DDD. Here, we demonstrate that soluble factors secreted by the NCD IVD NP strongly protect murine NP cells not only

  9. Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats via Downregulation of iNOS/HO-1/Bax and Activated Caspase-3 and Inhibition of Hydroxyl Radical Formation.

    PubMed

    Yang, Po-Sheng; Lin, Po-Yen; Chang, Chao-Chien; Yu, Meng-Che; Yen, Ting-Lin; Lan, Chang-Chou; Jayakumar, Thanasekaran; Yang, Chih-Hao

    2015-01-01

    Antrodia camphorata (A. camphorata) is a fungus generally used in Chinese folk medicine for treatment of viral hepatitis and cancer. Our previous study found A. camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animal models. In this study, we sought to investigate the molecular mechanisms of neuroprotective effects of A. camphorata in middle cerebral artery occlusion (MCAO) rats. A selective occlusion of the middle cerebral artery (MCA) with whole blood clots was used to induce ischemic stroke in rats and they were orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone or combined with aspirin (5 mg/kg/day). To provide insight into the functions of A. camphorata mediated neuroprotection, the expression of Bax, inducible nitric oxide synthase (iNOS), haem oxygenase-1 (HO-1), and activated caspase-3 was determined by Western blot assay. Treatment of aspirin alone significantly reduced the expressions of HO-1 (P < 0.001), iNOS (P < 0.001), and Bax (P < 0.01) in ischemic regions. The reduction of these expressions was more potentiated when rats treated by aspirin combined with A. camphorata (0.75 g/kg/day). Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (P < 0.01). Moreover, treatment of A. camphorata significantly (P < 0.05) reduced fenton reaction-induced hydroxyl radical (OH(•)) formation at a dose of 40 mg/mL. Taken together, A. camphorata has shown neuroprotective effects in embolic rats, and the molecular mechanisms may correlate with the downregulation of Bax, iNOS, HO-1, and activated caspase-3 and the inhibition of OH(•) signals.

  10. Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats via Downregulation of iNOS/HO-1/Bax and Activated Caspase-3 and Inhibition of Hydroxyl Radical Formation

    PubMed Central

    Yang, Po-Sheng; Lin, Po-Yen; Chang, Chao-Chien; Yu, Meng-Che; Yen, Ting-Lin; Lan, Chang-Chou; Jayakumar, Thanasekaran; Yang, Chih-Hao

    2015-01-01

    Antrodia camphorata (A. camphorata) is a fungus generally used in Chinese folk medicine for treatment of viral hepatitis and cancer. Our previous study found A. camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animal models. In this study, we sought to investigate the molecular mechanisms of neuroprotective effects of A. camphorata in middle cerebral artery occlusion (MCAO) rats. A selective occlusion of the middle cerebral artery (MCA) with whole blood clots was used to induce ischemic stroke in rats and they were orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone or combined with aspirin (5 mg/kg/day). To provide insight into the functions of A. camphorata mediated neuroprotection, the expression of Bax, inducible nitric oxide synthase (iNOS), haem oxygenase-1 (HO-1), and activated caspase-3 was determined by Western blot assay. Treatment of aspirin alone significantly reduced the expressions of HO-1 (P < 0.001), iNOS (P < 0.001), and Bax (P < 0.01) in ischemic regions. The reduction of these expressions was more potentiated when rats treated by aspirin combined with A. camphorata (0.75 g/kg/day). Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (P < 0.01). Moreover, treatment of A. camphorata significantly (P < 0.05) reduced fenton reaction-induced hydroxyl radical (OH•) formation at a dose of 40 mg/mL. Taken together, A. camphorata has shown neuroprotective effects in embolic rats, and the molecular mechanisms may correlate with the downregulation of Bax, iNOS, HO-1, and activated caspase-3 and the inhibition of OH• signals. PMID:26379739

  11. Kayeassamin A Isolated from the Flower of Mammea siamensis Triggers Apoptosis by Activating Caspase-3/-8 in HL-60 Human Leukemia Cells

    PubMed Central

    Uto, Takuhiro; Tung, Nguyen Huu; Thongjankaew, Pinjutha; Lhieochaiphant, Sorasak; Shoyama, Yukihiro

    2016-01-01

    Background: Mammea siamensis (Miq.) T. Anders. is used as a medicinal plant in Thailand and has several traditional therapeutic properties. In a previous study, we isolated eight compounds from the flower of M. siamensis and demonstrated that kayeassamin A (KA) exhibited potent antiproliferative activity against human leukemia and stomach cancer cell lines. Objective: In this study, we investigated the effect of KA on cell viability and apoptotic mechanisms in HL-60 human leukemia cells. Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Nuclear morphology and DNA fragmentation were observed using Hoechst 33258 staining and agarose gel electrophoresis, respectively. The sub-G1 phase of cells was analyzed by flow cytometry after the cellular DNA had been stained with propidium iodide. The protein levels of poly (ADP-ribose) polymerase (PARP) and caspases were determined by Western blotting. Results: KA exhibited a significant cytotoxic effect in a dose- and time-dependent manner, and induced chromatin condensation, DNA fragmentation, and sub-G1 phase DNA content, known as molecular events associated with the induction of apoptosis. In addition, KA strongly induced the activation of PARP and caspase-3 and -8, with weak caspase-9 activation. Furthermore, KA-induced DNA fragmentation was abolished by pretreatment with z-VAD-FMK (a broad caspase inhibitor), z-DEVD-FMK (a caspase-3 inhibitor), and z-IETD-FMK (a caspase-8 inhibitor), but not by z-LEHD-FMK (a caspase-9 inhibitor) pretreatment. Conclusion: These results indicate that KA triggers apoptotic cell death by activation of caspase-3 and -8 in HL-60 cells. SUMMARY Kayeassamin A (KA) isolated from the flower of Mammea siamensis exhibited a significant cytotoxic effect in HL-60 human leukemia cells. KA triggers apoptotic cell death by activating caspase-3/-8. Abbreviations Used: KA: Kayeassamin A; MTT: 3-(4,5-dimethylthiazol-2-yl)-2

  12. Kayeassamin A Isolated from the Flower of Mammea siamensis Triggers Apoptosis by Activating Caspase-3/-8 in HL-60 Human Leukemia Cells

    PubMed Central

    Uto, Takuhiro; Tung, Nguyen Huu; Thongjankaew, Pinjutha; Lhieochaiphant, Sorasak; Shoyama, Yukihiro

    2016-01-01

    Background: Mammea siamensis (Miq.) T. Anders. is used as a medicinal plant in Thailand and has several traditional therapeutic properties. In a previous study, we isolated eight compounds from the flower of M. siamensis and demonstrated that kayeassamin A (KA) exhibited potent antiproliferative activity against human leukemia and stomach cancer cell lines. Objective: In this study, we investigated the effect of KA on cell viability and apoptotic mechanisms in HL-60 human leukemia cells. Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Nuclear morphology and DNA fragmentation were observed using Hoechst 33258 staining and agarose gel electrophoresis, respectively. The sub-G1 phase of cells was analyzed by flow cytometry after the cellular DNA had been stained with propidium iodide. The protein levels of poly (ADP-ribose) polymerase (PARP) and caspases were determined by Western blotting. Results: KA exhibited a significant cytotoxic effect in a dose- and time-dependent manner, and induced chromatin condensation, DNA fragmentation, and sub-G1 phase DNA content, known as molecular events associated with the induction of apoptosis. In addition, KA strongly induced the activation of PARP and caspase-3 and -8, with weak caspase-9 activation. Furthermore, KA-induced DNA fragmentation was abolished by pretreatment with z-VAD-FMK (a broad caspase inhibitor), z-DEVD-FMK (a caspase-3 inhibitor), and z-IETD-FMK (a caspase-8 inhibitor), but not by z-LEHD-FMK (a caspase-9 inhibitor) pretreatment. Conclusion: These results indicate that KA triggers apoptotic cell death by activation of caspase-3 and -8 in HL-60 cells. SUMMARY Kayeassamin A (KA) isolated from the flower of Mammea siamensis exhibited a significant cytotoxic effect in HL-60 human leukemia cells. KA triggers apoptotic cell death by activating caspase-3/-8. Abbreviations Used: KA: Kayeassamin A; MTT: 3-(4,5-dimethylthiazol-2-yl)-2

  13. UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells

    PubMed Central

    Liu, Cailing; Vojnovic, Dijana; Kochevar, Irene E.; Jurkunas, Ula V.

    2016-01-01

    Purpose To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. Methods Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. Results Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. Conclusions Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD). PMID:27127932

  14. Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose.

    PubMed

    Hinck, L; Van Der Smissen, P; Heusterpreute, M; Donnay, I; De Hertogh, R; Pampfer, S

    2001-02-01

    Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 microM] against caspase-3 and aurin [1 microM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.

  15. Erythropoietin inhibits gamma-irradiation-induced apoptosis by upregulation of Bcl-2 and decreasing the activation of caspase 3 in human UT-7/erythropoietin cell line.

    PubMed

    Liu, Yuan-Yuan; She, Zhen-Jue; Yao, Ming-Hui

    2010-05-01

    1. Erythropoietin (EPO) can reverse radiotherapy-induced anaemia by stimulating bone marrow cells to produce erythrocytes. However, there are limited studies that address the mechanisms by which EPO exerts its beneficial effects in radiotherapy-induced anaemia. In the present study, we used a human bone marrow-derived EPO-dependent leukaemia cell line UT-7/EPO that progressed further in erythroid development to evaluate the anti-apoptotic effects of EPO on irradiated human erythroid progenitor. 2. The UT-7/EPO cells exposed to gamma-irradiation were cultured in the presence or absence of EPO at a concentration of 7 U/mL. The cell viability, cell apoptosis and the expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 were examined. 3. The results showed that EPO protected the viability of human UT-7/EPO cells exposed to gamma-irradiation. EPO significantly inhibited gamma-irradiation-induced apoptosis in human UT-7/EPO cells: a significant decrease in the percentage of apoptotic cells was observed (62, 69 and 62% at 24, 48 and 72 h, respectively). Furthermore, EPO significantly increased the expression of Bcl-2 protein and the relative Bcl-2/Bax ratio, and decreased the activation of caspase 3 and formation of the p17 and p12 cleavage in similar conditions. 4. In conclusion, EPO exerts anti-apoptotic effects on irradiated human UT-7/EPO cells through upregulation of Bcl-2 protein and the relative Bcl-2/Bax ratio, and by decreasing the activation of caspase 3. These findings may contribute to our understanding of the beneficial function of EPO in radiotherapy-induced anaemia.

  16. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

    SciTech Connect

    Chou, C.-T.; He Shiping; Jan, C.-R. . E-mail: crjan@isca.vghks.gov.tw

    2007-02-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.

  17. A pro-apoptotic 15-kDa protein from Bacopa monnieri activates caspase-3 and downregulates Bcl-2 gene expression in mouse mammary carcinoma cells.

    PubMed

    Kalyani, Manjula Ishwara; Lingaraju, Sheela Mysore; Salimath, Bharathi P

    2013-01-01

    In diseases such as cancer, induction of apoptosis has been a new target for mechanism-based drug discovery. The central component of the process of apoptosis is a proteolytic system involving a family of proteases called caspases. Apoptosis involves characteristic morphological and biochemical events ultimately leading to cell demise. Apoptotic induction is evidently central to the mechanism of action of plant-derived anticancer drugs. Extract of the medicinal plant, Bacopa monnieri, inhibits tumor cell proliferation and accumulation of malignant ascites fluid. The crude sample when subjected to Soxhlet extraction yielded different solvent extracts of which the aqueous extract showed biological activity of apoptosis in Ehrlich ascites tumor cell lines (EAT). Bacopa monnieri water extract (BMWE) treatment of EAT cells produced apoptotic morphological characteristics and in-vivo DNA fragmentation, which is due to the activity of an endogenous endonuclease. The endonuclease responsible for DNA fragmentation acts downstream of caspase-3 activity and is also referred to as caspase-activated DNase (CAD). The CAD constitutively expressed in the cell cytoplasm is translocated into the nucleus upon BMWE treatment, as verified by Western blotting, leading to DNA fragmentation and to programmed cell death. The expression of the pro-apoptotic gene Bax was increased and the expression of the anti-apoptotic gene Bcl-2 was decreased by BMWE treatment. Considering the above results, BMWE was able induce apoptosis in EAT cells via Bax-related caspase-3 activation. This may provide experimental data for the further clinical use of BMWE in cancer.

  18. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    PubMed

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  19. 17beta-estradiol attenuates programmed cell death in cortical pericontusional zone following traumatic brain injury via upregulation of ERalpha and inhibition of caspase-3 activation.

    PubMed

    Li, Li-Zhuo; Bao, Yi-Jun; Zhao, Min

    2011-01-01

    Pericontusional zone (PCZ) of traumatic cerebral contusion is a target of pharmacological intervention. It is well studied that 17beta-estradiol has a protective role in ischemic brain injury, but its role in brain protection of traumatic brain damage deserves further investigation, especially in pericontusional zone. Here we show that 17beta-estradiol enhances the protein expression and mRNA induction of estrogen alpha receptor (ERalpha) and prevents from programmed cell death in cortical pericontusional zone. ERalpha specific antagonist blocks this protective effect of 17beta-estradiol. Caspase-3 activation occurs in cortical pericontusional zone of the oil-treated injured rat brain and its activation is inhibited by 17beta-estradiol treatment. Additionally, ERalpha specific antagonist reverses this inhibition. Pan-caspase inhibitor also protect cortical pericontusional zone from programmed cell death. Our present study indicates 17beta-estradiol protects from programmed cell death in cortical pericontusional zone via enhancement of ERalpha and decrease of caspase-3 activation.

  20. Linoleic acid derivative DCP-LA protects neurons from oxidative stress-induced apoptosis by inhibiting caspase-3/-9 activation.

    PubMed

    Yaguchi, Takahiro; Fujikawa, Hirokazu; Nishizaki, Tomoyuki

    2010-05-01

    The present study aimed at understanding the effect of the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) on oxidative stress-induced neuronal death. Sodium nitroprusside (SNP; 1 mM) reduced viability of cultured rat cerebral cortical neurons to 50% of basal levels, but DCP-LA significantly prevented the SNP effect in a concentration (1-100 nM)-dependent manner. In addition, DCP-LA (100 nM) rescued neurons from SNP-induced degradation. SNP (1 mM) activated caspase-3 and -9 in cultured rat cerebral cortical neurons, but DCP-LA (100 nM) abolished the caspase activation. For a mouse model of middle cerebral artery occlusion, oral administration with DCP-LA (1 mg/kg) significantly diminished degraded area due to cerebral infarction. The results of the present study, thus, demonstrate that DCP-LA protects neurons at least in part from oxidative stress-induced apoptosis by inhibiting activation of caspase-3/-9.

  1. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression

    PubMed Central

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC. PMID:27642320

  2. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression.

    PubMed

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC.

  3. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression.

    PubMed

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC. PMID:27642320

  4. MicroRNA-124 and -137 cooperativity controls caspase-3 activity through BCL2L13 in hippocampal neural stem cells

    PubMed Central

    Schouten, Marijn; Fratantoni, Silvina A.; Hubens, Chantal J.; Piersma, Sander R.; Pham, Thang V.; Bielefeld, Pascal; Voskuyl, Rob A.; Lucassen, Paul J.; Jimenez, Connie R.; Fitzsimons, Carlos P.

    2015-01-01

    Adult neurogenesis continuously contributes new neurons to hippocampal circuits and the programmed death of a subset of immature cells provides a primary mechanism controlling this contribution. Epileptic seizures induce strong structural changes in the hippocampus, including the induction of adult neurogenesis, changes in gene expression and mitochondrial dysfunction, which may all contribute to epileptogenesis. However, a possible interplay between this factors remains largely unexplored. Here, we investigated gene expression changes in the hippocampal dentate gyrus shortly after prolonged seizures induced by kainic acid, focusing on mitochondrial functions. Using comparative proteomics, we identified networks of proteins differentially expressed shortly after seizure induction, including members of the BCL2 family and other mitochondrial proteins. Within these networks, we report for the first time that the atypical BCL2 protein BCL2L13 controls caspase-3 activity and cytochrome C release in neural stem/progenitor cells. Furthermore, we identify BCL2L13 as a novel target of the cooperative action of microRNA-124 and microRNA-137, both upregulated shortly after seizure induction. This cooperative microRNA-mediated fine-tuning of BCL2L13 expression controls casp3 activity, favoring non-apoptotic caspase-3 functions in NSPC exposed to KA and thereby may contribute to the early neurogenic response to epileptic seizures in the dentate gyrus. PMID:26207921

  5. Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro.

    PubMed

    Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu; Mikkelsen, Jørn D; Meyer, Anne S

    2011-12-01

    Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3.

  6. Brazilein from Caesalpinia sappan L. Antioxidant Inhibits Adipocyte Differentiation and Induces Apoptosis through Caspase-3 Activity and Anthelmintic Activities against Hymenolepis nana and Anisakis simplex

    PubMed Central

    Liang, Chia-Hua; Chan, Leong-Perng; Chou, Tzung-Han; Chiang, Feng-Yu; Yen, Chuan-Min; Chen, Pin-Ju; Ding, Hsiou-Yu

    2013-01-01

    Brazilein, a natural, biologically active compound from Caesalpinia sappan L., has been shown to exhibit anti-inflammatory and antioxidant properties and to inhibit the growth of several cancer cells. This study verifies the antioxidant and antitumor characteristics of brazilein in skin cancer cells and is the first time to elucidate the inhibition mechanism of adipocyte differentiation, cestocidal activities against Hymenolepis nana, and reduction of spontaneous movement in Anisakis simplex. Brazilein exhibits an antioxidant capacity as well as the ability to scavenge DPPH• and ABTS•+ free radicals and to inhibit lipid peroxidation. Brazilein inhibited intracellular lipid accumulation during adipocyte differentiation in 3T3-L1 cells and suppressed the induction of peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, suggesting that brazilein presents the antiobesity effects. The toxic effects of brazilein were evaluated in terms of cell viability, induction of apoptosis, and the activity of caspase-3 in BCC cells. The inhibition of the growth of skin cancer cells (A431, BCC, and SCC25) by brazilein is greater than that of human skin malignant melanoma (A375) cells, mouse leukemic monocyte macrophage (RAW 264.7 cells), and noncancerous cells (HaCaT and BNLCL2 cells). The anthelmintic activities of brazilein against Hymenolepis nana are better than those of Anisakis simplex. PMID:23554834

  7. The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

    SciTech Connect

    Garcia-Rates, Sara; Camarasa, Jordi

    2010-05-01

    Previous work by our group demonstrated that homomeric alpha7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca{sup 2+} increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific alpha7 agonist PNU 282987 with IC{sub 50} values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human alpha7 but not with alpha4beta2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and alpha-bungarotoxin but not by dihydro-beta-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on alpha7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca{sup 2+} release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca{sup 2+} levels and induced an increase in alpha-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and alpha7 nAChR. Sustained calcium entry and calpain activation could favor the

  8. Induction of apoptosis by penta-O-galloyl-beta-D-glucose through activation of caspase-3 in human leukemia HL-60 cells.

    PubMed

    Pan, M H; Lin, J H; Lin-Shiau, S Y; Lin, J K

    1999-09-24

    Penta-O-galloyl-beta-D-glucose is structurally related to (-)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta-O-galloyl-beta-D-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta-O-galloyl-beta-D-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta-O-galloyl-beta-D-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta-O-galloyl-beta-D-glucose-induced apoptosis. Treatment with penta-O-galloyl-beta-D-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) inhibited penta-O-galloyl-beta-D-glucose-induced DNA fragmentation. Furthermore, treatment with penta-O-galloyl-beta-D-glucose (50 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta-O-galloyl-beta-D-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta-O-galloyl-beta-D-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta-O-galloyl-beta-D-glucose may provide a pivotal mechanism for its cancer chemopreventive action.

  9. Apoptosis induced by paclitaxel via Bcl-2, Bax and caspases 3 and 9 activation in NB4 human leukaemia cells is not modulated by ERK inhibition.

    PubMed

    Morales-Cano, Daniel; Calviño, Eva; Rubio, Virginia; Herráez, Angel; Sancho, Pilar; Tejedor, M Cristina; Diez, José C

    2013-11-01

    We have studied the role of pivotal bio-molecules involved in signalling of cytotoxic effects induced by paclitaxel (Ptx) on acute promyelocytic human leukaemia NB4 cells. A time-dependent increase in cell death and DNA cleavage was observed after 30μM Ptx treatment. Cell death induction by Ptx proceeds mainly as programmed cell death as shown by annexin V-FITC, reaching up to 30% of apoptotic cells after 24h. Significant reductions of p53, changes in Bax and Bcl-2 and activation of caspases 3 and 9 were observed as the treatment was applied for long times. Ptx treatments produced NFkB depletion with expression levels abolished at 19h what could be involved in reduction of survival signals. Phosphorylation of intracellular kinases showed that pERK1/2 decreased significantly at 19h of Ptx treatment. When these cells were preincubated for 90min with 20μM PD98059, 2'-amino-3'-methoxyflavone, an inhibitor of ERK phosphorylation, a slight reduction of cell viability was observed in comparison to that produced by Ptx alone. Pretreatment with PD98059 neither activated caspases nor significantly increased the apoptotic effect of Ptx. Taken together, our data reveal that the inhibition of ERK phosphorylation does not seem to be an essential pathway for bursting an increased induction of apoptosis by Ptx. Decrease of p53 and Bcl-2, fragmentation of DNA, increase of Bax and, finally, activation of caspases 3 and 9 in NB4 leukaemia cells make the apoptotic process induced by Ptx irreversible. Application of Ptx in leukaemia cells shows therefore a promising potential with particular effects on different leukaemia cell types.

  10. Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3.

    PubMed

    Miller, Terry J; Schneider, Randal J; Miller, James A; Martin, Brad P; Al-Ubaidi, Muayyad R; Agarwal, Neeraj; Dethloff, Lloyd A; Philbert, Martin A

    2006-01-01

    The nitroimidazole radiosensitizer CI-1010 ((R)-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide) causes selective, irreversible, retinal photoreceptor apoptosis in vivo. The mouse 661 W photoreceptor cell line was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in 661 W cells, as determined by ultrastructural analysis, agarose electrophoresis and analysis of TUNEL-positive nuclei. CI-1010 caused a loss of viability in 661 W cells, as measured by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A clear link was established between the onset of apoptosis and activity of caspase-3 and caspase-8, prior to poly[ADP-ribose]polymerase (PARP) cleavage. Pretreatment with caspase inhibitors, ZVAD.fmk or DEVD-CHO, prevented morphological changes in most CI-1010-treated cells. Evaluation of mitochondrial inner membrane potential (Deltapsi(m)) in live 661 W cells using the fluorescent dye, tetramethylrhodamine methyl ester revealed retention of (Deltapsi(m)) until after caspase activation. Absence of cytochrome c in the cytoplasm in treated cells further supports the hypothesis of a mitochondrial-independent mechanism of cell death. Significant increase in DNA crosslinks observed in 661 W cells correlates with induction and phosphorylation of p53 at multiple serine sites. Cell cycle analysis of 661 W cells reveals a G(2) arrest in response to CI-1010-induced DNA damage and neuronal cell death. Increased protein expression of Bax, Fas, and FasL, concomitant to the loss of Bcl-xL in treated 661 W cells may be modulated by p53. This study evaluates in vitro mechanisms of CI-1010-induced cell death in photoreceptors and provides evidence in support of a p53-linked activation of caspase-3 in response to DNA damage caused by CI-1010.

  11. Brain-derived neurotrophic factor prevents changes in Bcl-2 family members and caspase-3 activation induced by excitotoxicity in the striatum.

    PubMed

    Pérez-Navarro, Esther; Gavaldà, Núria; Gratacòs, Elena; Alberch, Jordi

    2005-02-01

    Brain-derived neurotrophic factor (BDNF) prevents the loss of striatal neurons caused by excitotoxicity. We examined whether these neuroprotective effects are mediated by changes in the regulation of Bcl-2 family members. We first analyzed the involvement of the phosphatidylinositol 3-kinase/Akt pathway in this regulation, showing a reduction in phosphorylated Akt (p-Akt) levels after both quinolinate (QUIN, an NMDA receptor agonist) and kainate (KA, a non-NMDA receptor agonist) intrastriatal injection. Our results also show that Bcl-2, Bcl-x(L) and Bax protein levels and heterodimerization are selectively regulated by NMDA and non-NMDA receptor stimulation. Striatal cell death induced by QUIN is mediated by an increase in Bax and a decrease in Bcl-2 protein levels, leading to reduced levels of Bax:Bcl-2 heterodimers. In contrast, changes in Bax protein levels are not required for KA-induced apoptotic cell death, but decreased levels of both Bax:Bcl-2 and Bax:Bcl-x(L) heterodimer levels are necessary. Furthermore, QUIN and KA injection activated caspase-3. Intrastriatal grafting of a BDNF-secreting cell line counter-regulated p-AKT, Bcl-2, Bcl-x(L) and Bax protein levels, prevented changes in the heterodimerization between Bax and pro-survival proteins, and blocked caspase-3 activation induced by excitotoxicity. These results provide a possible mechanism to explain the anti-apoptotic effect of BDNF against to excitotoxicity in the striatum through the regulation of Bcl-2 family members, which is probably mediated by Akt activation.

  12. Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

    PubMed

    Dar, Asif A; Pradhan, Trupti N; Kulkarni, Dakshayni P; Shah, Sagar U; Rao, Kanury V; Chaukar, Devendra A; D'Cruz, Anil K; Chiplunkar, Shubhada V

    2016-02-01

    Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions. PMID:26595239

  13. Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

    PubMed

    Dar, Asif A; Pradhan, Trupti N; Kulkarni, Dakshayni P; Shah, Sagar U; Rao, Kanury V; Chaukar, Devendra A; D'Cruz, Anil K; Chiplunkar, Shubhada V

    2016-02-01

    Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions.

  14. Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.

    PubMed

    Mori, M; Terui, Y; Tanaka, M; Tomizuka, H; Mishima, Y; Ikeda, M; Kasahara, T; Uwai, M; Ueda, M; Inoue, R; Itoh, T; Yamada, M; Hayasawa, H; Furukawa, Y; Ishizaka, Y; Ozawa, K; Hatake, K

    2001-06-01

    We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

  15. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation.

    PubMed

    Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer.

  16. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells

    PubMed Central

    Baharara, Javad; Ramezani, Tayebe; Divsalar, Adeleh; Mousavi, Marzieh; Seyedarabi, Arefeh

    2016-01-01

    Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose-dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. PMID:27141266

  17. PET imaging of in vivo caspase-3/7 activity following myocardial ischemia-reperfusion injury with the radiolabeled isatin sulfonamide analogue [18F]WC-4-116

    PubMed Central

    Thukkani, Arun K; Shoghi, Kooresh I; Zhou, Dong; Xu, Jinbin; Chu, Wenhua; Novak, Eric; Chen, Delphine L; Gropler, Robert J; Mach, Robert H

    2016-01-01

    The utility of [18F]WC-4-116, a PET tracer for imaging caspase-3 activation, was evaluated in an animal model of myocardial apoptosis. [18F]WC-4-116 was injected into rats at 3 hours after a 30 min period of ischemia induced by temporary occlusion of the left anterior descending coronary artery in Sprague-Dawley rats. [18F]WC-4-116 uptake was quantified by 1) autoradiography, 2) microPET imaging studies, and 3) post-PET biodistribution studies. MicroPET imaging also assessed uptake of the non-caspase-3-targeted tracer [18F]ICMT-18 at 3 hours postischemia. Enzyme assays and Western blotting assessed caspase-3 activation in both at-risk and not-at-risk regions. Caspase-3 enzyme activity increased in the at-risk but not in the not-at-risk myocardium. Quantitative autoradiographic analysis of [18F]WC-4-116 demonstrated nearly 2-fold higher uptake in the ischemia-reperfusion (IR) versus sham animals. [18F]WC-4-116 microPET imaging studies demonstrated that the IR animals was similarly elevated in relation to sham. [18F]ICMT-18 uptake did not increase in at-risk myocardium despite evidence of caspase-3 activation. Biodistribution studies with [18F]WC-4-116 confirmed the microPET findings. These data indicate that the caspase-3-PET tracer [18F]WC-4-116 can noninvasively image in vivo caspase activity during myocardial apoptosis and may be useful for clinical imaging in humans. PMID:27186438

  18. Poly β-cyclodextrin/TPdye nanomicelle-based two-photon nanoprobe for caspase-3 activation imaging in live cells and tissues.

    PubMed

    Yan, Huijuan; He, Leiliang; Zhao, Wenjie; Li, Jishan; Xiao, Yue; Yang, Ronghua; Tan, Weihong

    2014-11-18

    Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. β-cyclodextrin polymer (βCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/βCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or

  19. Bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex a new apoptotic agent through Flk-1 down regulation, caspase-3 activation and oligonucleosomes DNA fragmentation.

    PubMed

    Azab, Hassan A; Hussein, Belal H M; El-Azab, Mona F; Gomaa, Mohamed; El-Falouji, Abdullah I

    2013-01-01

    New bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex was synthesized and characterized. In vivo anti-angiogenic activities of bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex against Ehrlich ascites carcinoma (EAC) cells are described. The newly synthesized complex resulted in inhibition of proliferation of EAC cells and ascites formation. The anti-tumor effect was found to be through anti-angiogenic activity as evident by the reduction of microvessel density in EAC solid tumors. The anti-angiogenic effect is mediated through down-regulation of VEGF receptor type-2 (Flk-1). The complex was also found to significantly increase the level of caspase-3 in laboratory animals compared to the acridine ligand and to the control group. This was also consistent with the DNA fragmentation detected by capillary electrophoresis that proved the apoptotic effect of the new complex. Our complex exhibited anti-angiogenic and apoptotic activity in vivo, a thing that makes it a potential effective chemotherapeutic agent. The interaction of calf thymus DNA (ct-DNA) with bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex has been investigated using fluorescence technique. A competitive experiment of the europium(III)-acridine complex with ethidium bromide (EB) to bind DNA revealed that interaction between the europium(III)-acridine and DNA was via intercalation. The interaction of the synthesized complex with tyrosine kinases was also studied using molecular docking simulation to further substantiate its mode of action.

  20. Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway.

    PubMed

    Ying, Tsung-Ho; Yang, Shun-Fa; Tsai, Su-Ju; Hsieh, Shu-Ching; Huang, Yi-Chang; Bau, Da-Tian; Hsieh, Yi-Hsien

    2012-02-01

    Fisetin is a naturally occurring flavonoid that has been reported to inhibit the proliferation and to induce apoptotic cell death in several tumor cells. However, the apoptosis-inducing effect of fisetin on tumor cell lines was investigated besides HeLa cells. In this study, we found that fisetin induced apoptosis of HeLa cells in a dose- and time-dependent manner, as evidenced by nuclear staining of 4'-6-Diamidino-2-phenylindole (DAPI), flow cytometry assay, and Annexin-V/PI double-labeling. In addition, fisetin triggered the activations of caspases-3 and -8 and the cleavages of poly (ADP-ribose) polymerase, resulting in apoptosis induction. Moreover, treatment of HeLa cells with fisetin induced a sustained activation of the phosphorylation of ERK1/2, and inhibition of ERK1/2 by PD98059 (MEK1/2 inhibitor) or transfection with the mutant ERK1/2 expression vector significantly abolished the fisetin-induced apoptosis through the activation of caspase-8/-3 pathway. The in vivo xenograft mice experiments revealed that fisetin significantly reduced tumor growth in mice with HeLa tumor xenografts. In conclusion, our results indicated that fisetin exhibited anti-cancer effect and induced apoptosis in HeLa cell lines both in vitro and in vivo.

  1. Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line.

    PubMed

    Anand, T; Pandareesh, M D; Bhat, Pratiksha V; Venkataramana, M

    2014-10-01

    Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

  2. Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells.

    PubMed

    Kitazumi, Ikuko; Maseki, Yoko; Nomura, Yoshiko; Shimanuki, Akiko; Sugita, Yumi; Tsukahara, Masayoshi

    2010-01-01

    DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.

  3. Selective blockade of CaMKII-alpha inhibits NMDA-induced caspase-3-dependent cell death but does not arrest PARP-1 activation or loss of plasma membrane selectivity in rat retinal neurons.

    PubMed

    Goebel, Dennis J

    2009-02-23

    Calcium/calmodulin-dependent protein kinase II-alpha (CaMKII-alpha) has been implicated in a number of receptor mediated events in neurons. Pharmacological blockade of CaMKII-alpha has been shown to prevent phosphorylation of NMDA-R2A and R2B receptor subunits, suggesting that this enzyme may be linked to receptor trafficking of glutamate receptors and serve as a regulatory protein for neuronal cell death. In the retina, inhibition of CaMKII-alpha has been reported to be neuroprotective against NMDA-induced cell death by preventing the activation of the caspase-3 dependent pathway. However, the effects of CaMKII-alpha blockade on the caspase-3 independent, PARP-1 dependent and the non-programmed cell death pathways have not previously been investigated. In the present study, blockade of CaMKII-alpha with the highly specific antagonist myristoylated autocamtide-2-related inhibitory peptide (AIP) was used in a rat in vivo model of retinal toxicity to compare the effects of on NMDA-induced caspase-3-dependent, PARP-1 dependent and the non-programmed (necrosis) cell death pathways. Results confirmed that AIP fully attenuates caspase-3 activation for at least 8 h following NMDA insult and also significantly improves retinal ganglion cell survival. However, this blockade had little effect on reducing the loss of plasma membrane selectivity (LPMS, e.g. necrosis) in cells located in the ganglion cell and inner nuclear layers and did not alter NMDA-induced PARP-1 hyperactivation, or prevent TUNEL labeling following a moderate NMDA-insult. These findings support a specific role for CaMKII-alpha in mediating the caspase-3 dependent cell death pathway and provide evidence that it is not directly linked to the signaling of either the PARP-1 dependent or the non-programmed cell death pathways.

  4. RNase activity of sialic acid-binding lectin from bullfrog eggs drives antitumor effect via the activation of p38 MAPK to caspase-3/7 signaling pathway in human breast cancer cells

    PubMed Central

    Kariya, Yukiko; Tatsuta, Takeo; Sugawara, Shigeki; Kariya, Yoshinobu; Nitta, Kazuo; Hosono, Masahiro

    2016-01-01

    Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribo-nuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDA-MB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDA-MB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase-3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase-3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDA-MB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase-3/7. PMID:27513956

  5. P2X7 receptor blockade protects against cisplatin-induced nephrotoxicity in mice by decreasing the activities of inflammasome components, oxidative stress and caspase-3

    SciTech Connect

    Zhang, Yuanyuan; Yuan, Fahuan; Cao, Xuejiao; Zhai, Zhifang; Gang Huang; Du, Xiang; Wang, Yiqin; Zhang, Jingbo; Huang, Yunjian; Zhao, Jinghong; Hou, Weiping

    2014-11-15

    Nephrotoxicity is a common complication of cisplatin chemotherapy and thus limits the use of cisplatin in clinic. The purinergic 2X7 receptor (P2X7R) plays important roles in inflammation and apoptosis in some inflammatory diseases; however, its roles in cisplatin-induced nephrotoxicity remain unclear. In this study, we first assessed the expression of P2X7R in cisplatin-induced nephrotoxicity in C57BL/6 mice, and then we investigated the changes of renal function, histological injury, inflammatory response, and apoptosis in renal tissues after P2X7R blockade in vivo using an antagonist A-438079. Moreover, we measured the changes of nod-like receptor family, pyrin domain containing proteins (NLRP3) inflammasome components, oxidative stress, and proapoptotic genes in renal tissues in cisplatin-induced nephrotoxicity after treatment with A-438079. We found that the expression of P2X7R was significantly upregulated in the renal tubular epithelial cells in cisplatin-induced nephrotoxicity compared with that of the normal control group. Furthermore, pretreatment with A-438079 markedly attenuated the cisplatin-induced renal injury while lightening the histological damage, inflammatory response and apoptosis in renal tissue, and improved the renal function. These effects were associated with the significantly reduced levels of NLRP3 inflammasome components, oxidative stress, p53 and caspase-3 in renal tissues in cisplatin-induced nephrotoxicity. In conclusions, our studies suggest that the upregulated activity of P2X7R might play important roles in the development of cisplatin-induced nephrotoxicity, and P2X7R blockade might become an effective therapeutic strategy for this disease. - Highlights: • The P2X7R expression was markedly upregulated in cisplatin-induced nephrotoxicity. • P2X7R blockade significantly attenuated the cisplatin-induced renal injury. • P2X7R blockade reduced activities of NLRP3 inflammasome components in renal tissue. • P2X7R blockade

  6. Increased anticancer activity of the thymidylate synthase inhibitor BGC9331 combined with the topoisomerase I inhibitor SN-38 in human colorectal and breast cancer cells: induction of apoptosis and ROCK cleavage through caspase-3-dependent and -independent mechanisms.

    PubMed

    Coudray, Anne-Marie; Louvet, Christophe; Kornprobst, Michel; Raymond, Eric; André, Thierry; Tournigand, Christophe; Faivre, Sandrine; De Gramont, Aimery; Larsen, Annette K; Gespach, Christian

    2005-08-01

    The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolytic activation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

  7. Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

    PubMed

    Meesarapee, Benjawan; Thampithak, Anusorn; Jaisin, Yamaratee; Sanvarinda, Pimtip; Suksamrarn, Apichart; Tuchinda, Patoomratana; Morales, Noppawan Phumala; Sanvarinda, Yupin

    2014-04-01

    6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 μM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration.

  8. Phyllanthus amarus inhibits cell growth and induces apoptosis in Dalton's lymphoma ascites cells through activation of caspase-3 and downregulation of Bcl-2.

    PubMed

    Harikumar, Kuzhuvelil B; Kuttan, Girija; Kuttan, Ramadasan

    2009-06-01

    The authors found in an earlier study that Phyllanthus amarus extract could significantly inhibit the solid and ascites tumor development in mice induced by Dalton's lymphoma ascites (DLA) cells. In the present study, the apoptotic effects of P. amarus against DLA cells in culture was evaluated. P. amarus produced significant reduction in cell viability as determined by the MTT assay. It also induces the formation of apoptotic bodies with characteristic features like plasma membrane invagination, elongation, fragmentation, and chromatin condensation. P. amarus at concentrations of 100 and 200 microg/mL is shown to induce DNA fragmentation. Gene expression analysis reveals that P. amarus induces the expression of caspase-3 and inhibits the expression of Bcl-2, which is an antiapoptotic protein. So the present study provides some insights into the possible mechanism by which P. amarus brings about apoptosis and growth inhibition in DLA cells. PMID:19223368

  9. Allicin induces apoptosis of the MGC-803 human gastric carcinoma cell line through the p38 mitogen-activated protein kinase/caspase-3 signaling pathway.

    PubMed

    Zhang, Xuecheng; Zhu, Yong; Duan, Wei; Feng, Chen; He, Xuan

    2015-04-01

    Gastric cancer is one of the most common forms of malignant tumor, and the development of anti‑gastric cancer drugs with minimal toxicity is of clinical importance. Allicin is extracted from Allium sativum (garlic). Recent research, including clinical experiments, has shown that garlic has anticancer and tumor suppressive effects. The present study aimed to investigate the effects of allicin on the MGC‑803 human gastric carcinoma cell line, and to further explore the possible mechanisms of its tumor suppressor effects. The effects of allicin on the MGC‑803 cells were initially examined using an 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. Hoechst staining was also used, in order to demonstrate the impact of allicin on MGC‑803 cell apoptosis. In addition, western blot analysis was performed to determine the abnormal expression levels of apoptosis‑associated proteins, following the treatment of MGC‑803 cells with allicin. Western blotting was also used to investigate the specific mechanisms underlying allicin‑induced apoptosis of MGC‑803 cells. The rate of MGC‑803 apoptosis was significantly increased, when the concentration and treatment time of allicin were increased. Hoechst staining detected an enhanced rate of apoptosis, and enhanced expression levels of cleaved caspase 3 were determined by western blotting. Notably, the protein expression levels of p38 were increased when the MGC‑803 cells were treated with allicin. The results of the present study suggest that allicin may inhibit the proliferation and induce the apoptosis of MGC‑803 human gastric carcinoma cells, and this may partially be achieved through the enhanced expression of p38 and cleaved caspase 3. PMID:25523417

  10. α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor activation protects against phencyclidine-induced caspase-3 activity by activating voltage-gated calcium channels.

    PubMed

    Timpe, Jennifer M; Wang, Cheng Z; Kim, Jisoo; Johnson, Kenneth M

    2014-12-01

    Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine-sensitive, schizophrenia-like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) inhibited PCP-induced apoptosis in organotypic neonatal rat brain slices in a concentration-dependent and 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage-dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA-mediated protection against PCP. Furthermore, the L-type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L-type VDCCs are required for AMPA-mediated protection against PCP-induced neurotoxicity. Whereas the P/Q-type inhibitor ω-agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N-type VDCC inhibitor ω-conotoxin (2 µM) had no effect. Decreased AMPA-mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain-derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L-type, and to a lesser extent P/Q-type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. PMID:24995437

  11. Abdominal surgery activates nesfatin-1 immunoreactive brain nuclei in rats.

    PubMed

    Stengel, Andreas; Goebel, Miriam; Wang, Lixin; Taché, Yvette

    2010-02-01

    Abdominal surgery-induced postoperative gastric ileus is well established to induce Fos expression in specific brain nuclei in rats within 2-h after surgery. However, the phenotype of activated neurons has not been thoroughly characterized. Nesfatin-1 was recently discovered in the rat hypothalamus as a new anorexigenic peptide that also inhibits gastric emptying and is widely distributed in rat brain autonomic nuclei suggesting an involvement in stress responses. Therefore, we investigated whether abdominal surgery activates nesfatin-1-immunoreactive (ir) neurons in the rat brain. Two hours after abdominal surgery with cecal palpation under short isoflurane anesthesia or anesthesia alone, rats were transcardially perfused and brains processed for double immunohistochemical labeling of Fos and nesfatin-1. Abdominal surgery, compared to anesthesia alone, induced Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), Edinger-Westphal nucleus (EW), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM). Double Fos/nesfatin-1 labeling showed that of the activated cells, 99% were nesfatin-1-immunoreactive in the SON, 91% in the LC, 82% in the rRPa, 74% in the EW and VLM, 71% in the anterior parvicellular PVN, 47% in the lateral magnocellular PVN, 41% in the medial magnocellular PVN, 14% in the NTS and 9% in the medial parvicellular PVN. These data established nesfatin-1 immunoreactive neurons in specific nuclei of the hypothalamus and brainstem as part of the neuronal response to abdominal surgery and suggest a possible implication of nesfatin-1 in the alterations of food intake and gastric transit associated with such a stressor. PMID:19944727

  12. Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis

    PubMed Central

    Ge, Y; Cai, Y-M; Bonneau, L; Rotari, V; Danon, A; McKenzie, E A; McLellan, H; Mach, L; Gallois, P

    2016-01-01

    Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation. PMID:27058316

  13. Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis.

    PubMed

    Ge, Y; Cai, Y-M; Bonneau, L; Rotari, V; Danon, A; McKenzie, E A; McLellan, H; Mach, L; Gallois, P

    2016-09-01

    Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation. PMID:27058316

  14. Effect of polysaccharides extract of rhizoma atractylodis macrocephalae on thymus, spleen and cardiac indexes, caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein and mRNA expression levels in aged rats.

    PubMed

    Guo, Ling; Sun, Yong Le; Wang, Ai Hong; Xu, Chong En; Zhang, Meng Yuan

    2012-10-01

    This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

  15. Blocking caspase-3-dependent pathway preserves hair cells from salicylate-induced apoptosis in the guinea pig cochlea.

    PubMed

    Feng, Hao; Yin, Shi-Hua; Tang, An-Zhou

    2011-07-01

    In the present study, we aim to explore whether the caspase-3-dependent pathway is involved in the apoptotic cell death that occurs in the hair cells (HCs) of guinea pig cochlea following a salicylate treatment. Guinea pigs received sodium salicylate (Na-SA), at a dose of 200 mg·kg(-1)·d(-1) i.p., as a vehicle for 5 consecutive days. In some experiments, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-FMK), a specific apoptosis inhibitor, was directly applied into the cochlea via the round window niche (RWN) prior to salicylate treatment for determination of caspase-3 activation. Alterations in auditory function were evaluated with auditory brainstem responses (ABR) thresholds. Caspase-3 activity was determined by measuring the proteolytic cleavage product of caspase-3 (N-terminated peptide substrate). DNA fragmentation within the nuclei was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Ultrastructure variation in the target cell was assessed by electron microscopy (EM). Salicylate treatment initiated an obvious elevation in ABR thresholds with a maximum average shift of 60 dB sound pressure level (SPL), and caused significant apoptosis in both inner (IHCs) and outer (OHCs) hair cells resulted from an evident increasing in immunoreactivity to caspase-3 protease. Transmission electron microscopy (TEM) displayed chromatin condensation and nucleus margination accompanied by cell body shrinkage in the OHCs, but not in the IHCs. Scanning electron microscopy (SEM) showed breakdown, fusion, and loss in the stereociliary bundles at the apex of OHCs rather than IHCs. zDEVD-FMK pretreatment prior to salicylate injection substantially attenuated an expression of the apoptotic protease and protected HCs against apoptotic death, followed by a moderate relief in the thresholds of ABR, an alleviation in the submicroscopic structure was also identified. In particular, disorientation and insertion in the

  16. A novel nano-copper-bearing stainless steel with reduced Cu2+ release only inducing transient foreign body reaction via affecting the activity of NF-κB and Caspase 3

    PubMed Central

    Wang, Lei; Ren, Ling; Tang, Tingting; Dai, Kerong; Yang, Ke; Hao, Yongqiang

    2015-01-01

    Foreign body reaction induced by biomaterials is a serious problem in clinical applications. Although 317L-Cu stainless steel (317L-Cu SS) is a new type of implant material with antibacterial ability and osteogenic property, the foreign body reaction level still needs to be assessed due to its Cu2+ releasing property. For this purpose, two macrophage cell lines were selected to detect cellular proliferation, apoptosis, mobility, and the secretions of inflammatory cytokines with the influence of 317L-Cu SS. Our results indicated that 317L-Cu SS had no obvious effect on the proliferation and apoptosis of macrophages; however, it significantly increased cellular migration and TNF-α secretion. Then, C57 mice were used to assess foreign body reaction induced by 317L-Cu SS. We observed significantly enhanced recruitment of inflammatory cells (primarily macrophages) with increased TNF-α secretion and apoptosis level in tissues around the materials in the early stage of implantation. With tissue healing, both inflammation and apoptosis significantly decreased. Further, we discovered that NF-κB pathway and Caspase 3 played important roles in 317L-Cu SS induced inflammation and apoptosis. We concluded that 317L-Cu SS could briefly promote the inflammation and apoptosis of surrounding tissues by regulating the activity of NF-κB pathway and Caspase 3. All these discoveries demonstrated that 317L-Cu SS has a great potential for clinical application. PMID:26604748

  17. Synergistic effect of fisetin combined with sorafenib in human cervical cancer HeLa cells through activation of death receptor-5 mediated caspase-8/caspase-3 and the mitochondria-dependent apoptotic pathway.

    PubMed

    Lin, Ming-Te; Lin, Chia-Liang; Lin, Tzu-Yu; Cheng, Chun-Wen; Yang, Shun-Fa; Lin, Chu-Liang; Wu, Chih-Chien; Hsieh, Yi-Hsien; Tsai, Jen-Pi

    2016-05-01

    Combining antitumor agents with bioactive compounds is a potential strategy for improving the effect of chemotherapy on cancer cells. The goal of this study was to elucidate the antitumor effect of the flavonoid, fisetin, combined with the multikinase inhibitor, sorafenib, against human cervical cancer cells in vitro and in vivo. The combination of fisetin and sorafenib synergistically induced apoptosis in HeLa cells, which is accompanied by a marked increase in loss of mitochondrial membrane potential. Apoptosis induction was achieved by caspase-3 and caspase-8 activation which increased the ratio of Bax/Bcl-2 and caused the subsequent cleavage of PARP level while disrupting the mitochondrial membrane potential in HeLa cells. Decreased Bax/Bcl-2 ratio level and mitochondrial membrane potential were also observed in siDR5-treated HeLa cells. In addition, in vivo studies revealed that the combined fisetin and sorafenib treatment was clearly superior to sorafenib treatment alone using a HeLa xenograft model. Our study showed that the combination of fisetin and sorafenib exerted better synergistic effects in vitro and in vivo than either agent used alone against human cervical cancer, and this synergism was based on apoptotic potential through a mitochondrial- and DR5-dependent caspase-8/caspase-3 signaling pathway. This combined fisetin and sorafenib treatment represents a novel therapeutic strategy for further clinical developments in advanced cervical cancer.

  18. Deficiency of caspase 3 in tumor xenograft impairs therapeutic effect of measles virus Edmoston strain.

    PubMed

    Wang, Biao; Yan, Xu; Guo, Qingguo; Li, Yan; Zhang, Haiyan; Xie, Ji Sheng; Meng, Xin

    2015-06-30

    The oncolytic measles virus Edmonston (MV-Edm) strain shows considerable oncolytic activity against a variety of human tumors. In this study, we report MV-Edm is able to trigger apoptosis pathways in infected tumor cells and elucidate the roles of cellular apoptosis in the whole oncolytic process. We also show that activated caspase 3, a key executioner of apoptosis, plays key roles in the oncolytic virotherapy. Activated caspase 3 can accelerate viral replication in cervical cancer cells and enhance the killing effects of the virus. Deficiency of caspase 3 either in tumor cells or in tumor xenograft significantly desensitized tumor to oncolysis with MV-Edm. In the infected cells, caspase 3 regulates interferon α release, which can inhibit viral replication in neighboring tumor cells. We propose that caspase-3 activation enhances the oncolytic effects of MV-Edm, thus inhibiting tumor growth in mice.

  19. Caspase-3, Shears for Synapse Pruning

    PubMed Central

    Shen, Chengyong; Xiong, Wen C.; Mei, Lin

    2014-01-01

    Motor neurons regulate neuromuscular junction formation by using agrin to stimulate acetylcholine receptor clustering and using acetylcholine to disperse unnecessary receptor clusters on muscle fibers. Wang et al. now report in this issue of Developmental Cella critical role for caspase-3 in intracellular mechanisms of acetylcholine-induced dispersal. PMID:24697895

  20. Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

    NASA Technical Reports Server (NTRS)

    Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

    2003-01-01

    In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

  1. Combination of the histone deacetylase inhibitor depsipeptide and 5-fluorouracil upregulates major histocompatibility complex class II and p21 genes and activates caspase-3/7 in human colon cancer HCT-116 cells

    PubMed Central

    Okada, Kouji; Hakata, Shuko; Terashima, Jun; Gamou, Toshie; Habano, Wataru; Ozawa, Shogo

    2016-01-01

    Epigenetic anticancer drugs such as histone deacetylase (HDAC) inhibitors have been combined with existing anticancer drugs for synergistic or additive effects. In the present study, we found that a very low concentration of depsipeptide, an HDAC inhibitor, potentiated the antitumor activity of 5-fluorouracil (5-FU) in a human colon cancer cell model using HCT-116, HT29, and SW48 cells via the inhibition of colony formation ability or cellular viability. Exposure to a combination of 5-FU (1.75 µM) and 1 nM depsipeptide for 24 and 48 h resulted in a 3- to 4-fold increase in activated caspase-3/7, while 5-FU alone failed to activate caspase-3/7. Microarray and subsequent gene ontology analyses revealed that compared to 5-FU or depsipeptide alone, the combination treatment of 5-FU and depsipeptide upregulated genes related to cell death and the apoptotic process consistent with the inhibition of colony formation and caspase-3/7 activation. These analyses indicated marked upregulation of antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class (GO:0002504) and MHC protein complex (GO:0042611). Compared with vehicle controls, the cells treated with the combination of 5-FU and depsipeptide showed marked induction (3- to 8.5-fold) of expression of MHC class II genes, but not of MHC class I genes. Furthermore, our global analysis of gene expression, which was focused on genes involved in the molecular regulation of MHC class II genes, showed enhancement of pro-apoptotic PCAF and CIITA after the combination of 5-FU and depsipeptide. These results may indicate a closer relationship between elevation of MHC class II expression and cellular apoptosis induced by the combination of depsipeptide and 5-FU. To the best of our knowledge, this is the first study to report that the combination of 5-FU and depsipeptide induces human colon cancer cell apoptosis in a concerted manner with the induction of MHC class II gene

  2. Combination of the histone deacetylase inhibitor depsipeptide and 5-fluorouracil upregulates major histocompatibility complex class II and p21 genes and activates caspase-3/7 in human colon cancer HCT-116 cells.

    PubMed

    Okada, Kouji; Hakata, Shuko; Terashima, Jun; Gamou, Toshie; Habano, Wataru; Ozawa, Shogo

    2016-10-01

    Epigenetic anticancer drugs such as histone deacetylase (HDAC) inhibitors have been combined with existing anticancer drugs for synergistic or additive effects. In the present study, we found that a very low concentration of depsipeptide, an HDAC inhibitor, potentiated the antitumor activity of 5-fluorouracil (5-FU) in a human colon cancer cell model using HCT-116, HT29, and SW48 cells via the inhibition of colony formation ability or cellular viability. Exposure to a combination of 5-FU (1.75 µM) and 1 nM depsipeptide for 24 and 48 h resulted in a 3- to 4-fold increase in activated caspase-3/7, while 5-FU alone failed to activate caspase-3/7. Microarray and subsequent gene ontology analyses revealed that compared to 5-FU or depsipeptide alone, the combination treatment of 5-FU and depsipeptide upregulated genes related to cell death and the apoptotic process consistent with the inhibition of colony formation and caspase-3/7 activation. These analyses indicated marked upregulation of antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class (GO:0002504) and MHC protein complex (GO:0042611). Compared with vehicle controls, the cells treated with the combination of 5-FU and depsipeptide showed marked induction (3- to 8.5-fold) of expression of MHC class II genes, but not of MHC class I genes. Furthermore, our global analysis of gene expression, which was focused on genes involved in the molecular regulation of MHC class II genes, showed enhancement of pro-apoptotic PCAF and CIITA after the combination of 5-FU and depsipeptide. These results may indicate a closer relationship between elevation of MHC class II expression and cellular apoptosis induced by the combination of depsipeptide and 5-FU. To the best of our knowledge, this is the first study to report that the combination of 5-FU and depsipeptide induces human colon cancer cell apoptosis in a concerted manner with the induction of MHC

  3. Cytosolic TDP-43 expression following axotomy is associated with caspase 3 activation in NFL-/- mice: support for a role for TDP-43 in the physiological response to neuronal injury.

    PubMed

    Moisse, Katie; Mepham, Jennifer; Volkening, Kathryn; Welch, Ian; Hill, Tracy; Strong, Michael J

    2009-11-01

    TAR DNA binding protein (TDP-43) mislocalization has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have recently reported that TDP-43 and PGRN expression is altered in response to axotomy in C57BL6 mice and that normal expression is restored following recovery. We have performed axotomies in two different presymptomatic models of motor neuron degeneration, low molecular weight neurofilament knockout (NFL(-/-)) mice and mutant SOD1(G93A) transgenic (mtSOD1(G93A)) mice aged 6 weeks, and observed TDP-43 and PGRN expression patterns in axotomized spinal motor neurons over 28 days. In contrast to both C57BL6 mice and mtSOD1(G93A) mice, behavioural deficits in NFL(-/-) mice were sustained. We did not observe differences in TDP-43 or PGRN expression between C57BL6 mice and mtSOD1(G93A) mice throughout the observation period. However, compared to C57BL6 mice and mtSOD1(G93A) mice, NFL(-/-) mice exhibited late upregulation of cytosolic TDP-43 expression and persistent downregulation of neuronal PGRN expression accompanied by caspase 3 activation on post-injury day 28. By post-injury day 42, no cytosolic TDP-43-positive neurons remained in NFL(-/-) mice, suggesting that they had undergone apoptotic cell death. These findings suggest that whereas TDP-43 expression is normally upregulated transiently following axotomy, in the absence of NFL this response is delayed and associated with caspase 3 activation and neuronal death. These results further support that TDP-43 is involved in neurofilament mRNA metabolism and transport, and provide insight into the pathogenesis of motor neuron death in ALS in which NFL mRNA levels are selectively suppressed.

  4. MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3

    PubMed Central

    Zhang, Nan; Zhong, Jie; Han, Song; Li, Yun; Yin, Yanling; Li, Junfa

    2016-01-01

    miRNAs have been linked to many human diseases, including ischemic stroke, and are being pursued as clinical diagnostics and therapeutic targets. Among the aberrantly expressed miRNAs in our previous report using large-scale microarray screening, the downregulation of miR-378 in the peri-infarct region of middle cerebral artery occluded (MCAO) mice can be reversed by hypoxic preconditioning (HPC). In this study, the role of miR-378 in the ischemic injury was further explored. We found that miR-378 levels significantly decreased in N2A cells following oxygen-glucose deprivation (OGD) treatment. Overexpression of miR-378 significantly enhanced cell viability, decreased TUNEL-positive cells and the immunoreactivity of cleaved-caspase-3. Conversely, downregulation of miR-378 aggravated OGD-induced apoptosis and ischemic injury. By using bioinformatic algorithms, we discovered that miR-378 may directly bind to the predicted 3′-untranslated region (UTR) of Caspase-3 gene. The protein level of caspase-3 increased significantly upon OGD treatment, and can be downregulated by pri-miR-378 transfection. The luciferase reporter assay confirmed the binding of miR-378 to the 3′-UTR of Caspase-3 mRNA and repressed its translation. In addition, miR-378 agomir decreased cleaved-caspase-3 ratio, reduced infarct volume and neural cell death induced by MCAO. Furthermore, caspase-3 knockdown could reverse anti-miR-378 mediated neuronal injury. Taken together, our data demonstrated that miR-378 attenuated ischemic injury by negatively regulating the apoptosis executioner, caspase-3, providing a potential therapeutic target for ischemic stroke. PMID:27598143

  5. MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3.

    PubMed

    Zhang, Nan; Zhong, Jie; Han, Song; Li, Yun; Yin, Yanling; Li, Junfa

    2016-01-01

    miRNAs have been linked to many human diseases, including ischemic stroke, and are being pursued as clinical diagnostics and therapeutic targets. Among the aberrantly expressed miRNAs in our previous report using large-scale microarray screening, the downregulation of miR-378 in the peri-infarct region of middle cerebral artery occluded (MCAO) mice can be reversed by hypoxic preconditioning (HPC). In this study, the role of miR-378 in the ischemic injury was further explored. We found that miR-378 levels significantly decreased in N2A cells following oxygen-glucose deprivation (OGD) treatment. Overexpression of miR-378 significantly enhanced cell viability, decreased TUNEL-positive cells and the immunoreactivity of cleaved-caspase-3. Conversely, downregulation of miR-378 aggravated OGD-induced apoptosis and ischemic injury. By using bioinformatic algorithms, we discovered that miR-378 may directly bind to the predicted 3'-untranslated region (UTR) of Caspase-3 gene. The protein level of caspase-3 increased significantly upon OGD treatment, and can be downregulated by pri-miR-378 transfection. The luciferase reporter assay confirmed the binding of miR-378 to the 3'-UTR of Caspase-3 mRNA and repressed its translation. In addition, miR-378 agomir decreased cleaved-caspase-3 ratio, reduced infarct volume and neural cell death induced by MCAO. Furthermore, caspase-3 knockdown could reverse anti-miR-378 mediated neuronal injury. Taken together, our data demonstrated that miR-378 attenuated ischemic injury by negatively regulating the apoptosis executioner, caspase-3, providing a potential therapeutic target for ischemic stroke. PMID:27598143

  6. Purification of nasulysin-1: A new toxin from Porthidium nasutum snake venom that specifically induces apoptosis in leukemia cell model through caspase-3 and apoptosis-inducing factor activation.

    PubMed

    Bonilla-Porras, Angelica Rocio; Vargas, Leidy Johana; Jimenez-Del-Rio, Marlene; Nuñez, Vitelbina; Velez-Pardo, Carlos

    2016-09-15

    Nasulysin-1, a new zinc-metalloproteinase from the snake venom of the hognose pit viper Porthidium nasutum, was purified to homogeneity using molecular exclusion chromatography and high performance liquid chromatography on a reverse phase column. The molecular mass of the purified enzyme was 25,900 kDa and pI 4.1, as determined by 1D and 2D polyacrylamide gel electrophoresis. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the N-terminal amino acid sequence (1FSPRYIELVVVADHGMFKKYNSNLNTIR28; 1TASLANLEVWSK12; 1DLLPR6) of the purified nasulysin-1, shows close structural homology with other snake venom metalloproteinases isolated from different snake venoms. The purified nasulysin-1 showed specific apoptosis-inducing activity in Jurkat and K562 cells, a T-cell acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (AML) cell model, respectively, without affecting the viability of human lymphocyte cells. After 48 h treatment, nasulysin-1 (20 μg/mL) induced loss of the mitochondrial membrane potential (ΔΨm), activated the apoptosis-inducing factor (AIF), activated the protease caspase-3, and induced chromatin condensation and DNA fragmentation, all hallmarks of apoptosis. These results strongly suggest that nasulysin-1 selectively induces apoptosis to eliminate leukemia cells. Thus, these data warrant further investigation into the use of the metalloproteinase protein, nasulysin-1 as a potential therapeutic agent for treating leukemia. PMID:27530665

  7. Effects of inhibitors on the synergistic interaction between calpain and caspase-3 during post-mortem aging of chicken meat.

    PubMed

    Chen, Lin; Feng, Xian Chao; Zhang, Wan Gang; Xu, Xing Lian; Zhou, Guang Hong

    2012-08-29

    Calpain has been considered to be the most important protease involved in tenderization during the conversion of muscle into meat. However, recent evidence suggests the possible involvement of the key apoptosis protease, caspase, on post-mortem tenderization. This study used inhibitors of calpain and caspase-3 to treat chicken muscle immediately after slaughter and followed the changes in caspase-3 and calpain activities together with their expression during 5 days of aging. Addition of calpain inhibitors to the system resulted in significantly higher caspase-3 activities (p < 0.01) during storage. Western blot analysis of pro-caspase-3 and α-spectrin cleavage of the 120 kDa peptide (SBDP 120) showed that the addition of calpain inhibitors resulted in the formation of higher amounts of the active form of caspase-3 compared with the control (p < 0.01). Inclusion of inhibitors of caspase-3 led to lower calpain activities (p < 0.01) and dramatically reduced the expression of calpain-1 and calpain-2 (p < 0.01). Concomitantly, this inhibition resulted in greater calpastatin expression compared with the control (p < 0.01). The findings of this investigation show that calpain prevented the activation of caspase-3, whereas caspase-3 appeared to enhance the calpain activity during post-mortem aging through inhibition of calpastatin. It is therefore suggested that there is a relationship between caspase-3 and calpain which contributes to the tenderizing process during the conversion of muscle tissue into meat.

  8. Caspase-3 expression in normal oral epithelium, oral submucous fibrosis and oral squamous cell carcinoma

    PubMed Central

    Veeravarmal, Veeran; Austin, Ravi David; Siddavaram, Nagini; Thiruneelakandan, Sambanthan; Nassar, Mohamed Hanifa Mohamed

    2016-01-01

    Context: The epithelium atrophy, as the oral submucous fibrosis (OSMF) progresses, is believed to be an after effect of stromal fibrosis, hyalinization, decrease in vascularity and cellularity and is considered as “ischemic atrophy.” Due to hypoxia, caspase-3 get activation and subsequent decrease in viable cell count can occur. Aims and Objectives: To determine caspase-3 expression in various grades of OSMF and oral squamous cell carcinoma (OSCC) to find out whether upregulation of apoptosis is responsible for the epithelial changes in OSMF. Subjects and Methods: The control tissue (15 samples from normal oral mucosa) and study group comprising 97 cases of OSMF of different grades and OSCC associated with OSMF were stained with caspase-3 antibody, and the percentage of positive cells was calculated using ImageJ software. Statistical Analysis: The results obtained were statistically analyzed using ANOVA and Tukey's honest significance difference test and Mann–Whitney U-test. Results: There was a nuclear expression of caspase-3 in basal and parabasal layers of normal epithelium. There was cytoplasmic expression of caspase-3 in OSMF without dysplasia, total absence of caspase-3 expression in dysplastic epithelium and in majority cases of OSCC. The caspase-3 percentage was increased in OSMF (0%–53%) when compared with OSCC (0%–8%). The statistical comparison of caspase-3 among normal, OSMF and OSCC patients revealed significant correlation (P < 0.00010). The comparison within different grades of OSMF and between dysplastic and nondysplastic epithelium OSMF also showed significance (P < 0.019). Conclusions: The decreased expression of caspase-3 in disease progression reflects its role in the malignant transformation. PMID:27721610

  9. Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus–ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor

    PubMed Central

    Mocan, Lucian; Matea, Cristian; Tabaran, Flaviu A; Mosteanu, Ofelia; Pop, Teodora; Mocan, Teodora; Iancu, Cornel

    2015-01-01

    We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 μg/mL) to 48.2% (for 50 μg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 μg/mL) to 52.3% (50 μg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus–endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating. PMID:26346915

  10. Delayed growth of glioma by a polysaccharide from Aster tataricus involve upregulation of Bax/Bcl-2 ratio, activation of caspase-3/8/9, and downregulation of the Akt.

    PubMed

    Du, Lei; Mei, Hai-Feng; Yin, Xue; Xing, Yi-Qiao

    2014-03-01

    In this study, a homogeneous polysaccharide (ATP-II), with a molecular weight of 3.4 × 10(4) Da, was successfully purified from Aster tataricus by DEAE-Sepharose CL-6B ion exchange and Sepharose CL-6B gel filtration chromatography. Monosaccharide component analysis indicated that ATP-II was composed of glucose, galactose, mannose, rhamnose, and arabinose in molar ratios of 2.1:5.2:2.1:1.0:1.2. We evaluated the anticancer efficacy and associated mechanisms of ATP-II on glioma C6 cells in vitro and in vivo. The results showed that treatment of C6 cells with ATP-II inhibited cell proliferation and this biological response came from induction of DAN damage and consequent inducing apoptosis. Likewise, oral ATP-II administration resulted in consistent regression of glioma tumors and induced apoptosis of transplanted tumor tissues by increasing the ratio of Bax/Bcl-2 and activation of caspase-3, caspase-8, and caspase-9 cascade. Importantly, the efficient downregulation of Akt, which is successfully detected in tumor tissues, is a unique contribution to retard the tumor growth by ATP-II. These data suggest that ATP-II may be a potential candidate for glioma treatment.

  11. Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor.

    PubMed

    Mocan, Lucian; Matea, Cristian; Tabaran, Flaviu A; Mosteanu, Ofelia; Pop, Teodora; Mocan, Teodora; Iancu, Cornel

    2015-01-01

    We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 μg/mL) to 48.2% (for 50 μg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 μg/mL) to 52.3% (50 μg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus-endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating.

  12. High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

    PubMed

    Okuyama, Ryuhei; Nguyen, Bach-Cuc; Talora, Claudio; Ogawa, Eisaku; Tommasi di Vignano, Alice; Lioumi, Maria; Chiorino, Giovanna; Tagami, Hachiro; Woo, Minna; Dotto, G Paolo

    2004-04-01

    Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

  13. Phorbol esters from Jatropha meal triggered apoptosis, activated PKC-δ, caspase-3 proteins and down-regulated the proto-oncogenes in MCF-7 and HeLa cancer cell lines.

    PubMed

    Oskoueian, Ehsan; Abdullah, Norhani; Ahmad, Syahida

    2012-09-10

    Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs). The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7) and cervical (HeLa) cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC₅₀ of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA) as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC₅₀ concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun). These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.

  14. Caspase 3 in dying tumor cells mediates post-irradiation angiogenesis

    PubMed Central

    Zhang, Zhengxiang; Yu, Yang; Cheng, Jin; Gong, Yanping; Li, Chuan-Yuan; Huang, Qian

    2015-01-01

    Cytotoxic radiotherapy unfavorably induces tumor cells to generate various proangiogenic substances, promoting post-irradiation angiogenesis (PIA), which is one of major causes of radiotherapy failure. Though several studies have reported some mechanisms behind PIA, they have not yet described the beginning proangiogenic motivator buried in the irradiated microenvironment. In this work, we revealed that dying tumor cells induced by irradiation prompted PIA via a caspase 3 dependent mechanism. Proteolytic inactivation of caspase 3 in dying tumor cells by transducing a dominant-negative version weakened proangiogenic effects in vitro and in vivo. In addition, inhibition of caspase 3 activity suppressed tumor angiogenesis and tumorigenesis in xenograft mouse model. Importantly, we identified vascular endothelial growth factor (VEGF)-A as a downstream proangiogenic factor regulated by caspase 3 possibly through Akt signaling. Collectively, these findings indicated that besides acting as a key executioner in apoptosis, caspase 3 in dying tumor cells may play a central role in driving proangiogenic response after irradiation. Thus, radiotherapy in combination with caspase 3 inhibitors may be a novel promising therapeutic strategy to reduce tumor recurrence due to restrained PIA. PMID:26431328

  15. Suppressive effects of long-term exposure to P-nitrophenol on gonadal development, hormonal profile with disruption of tissue integrity, and activation of caspase-3 in male Japanese quail (Coturnix japonica).

    PubMed

    Ahmed, Eman; Nagaoka, Kentaro; Fayez, Mostafa; Abdel-Daim, Mohamed M; Samir, Haney; Watanabe, Gen

    2015-07-01

    P-Nitrophenol (PNP) is considered to be one of nitrophenol derivatives of diesel exhaust particles. PNP is a major metabolite of some organophosphorus compounds. PNP is a persistent organic pollutant as well as one of endocrine-disrupting compounds. Consequently, bioaccumulation of PNP potentiates toxicity. The objectives of the current study were to assess in vivo adverse effects of long-term low doses of PNP exposure on reproductive system during development stage. Twenty-eight-day-old male Japanese quails were orally administered different doses of PNP (0, 0.01, 0.1, 1 mg/kg body weight) daily for 2.5 months. Testicular histopathology, hormones, caspase-3 (CASP3), and claudin-1 (CLDN1) tight junction protein, as well as plasma hormones were analyzed. The results revealed that long-term PNP exposure caused testicular histopathological changes such as vacuolation of spermatogenic cell and spermatocyte with significant testicular and cloacal gland atrophy. PNP activated CASP3 enzyme that is an apoptosis-related cysteine peptidase. Besides, it disrupted the expression of CLDN1. Furthermore, a substantial decrease in plasma concentrations of luteinizing hormone (LH) and testosterone was observed after 2 and 2.5 months in the PNP-treated groups. Meanwhile, the pituitary LH did not significantly change. Site of action of PNP may be peripheral on testicular development and/or centrally on the hypothalamic-pituitary-gonadal axis through reduction of pulsatile secretion of gonadotrophin-releasing hormone. Consequently, it may reduce the sensitivity of the anterior pituitary gland to secrete LH. In conclusion, PNP induced profound endocrine disruption in the form of hormonal imbalance, induction of CASP3, and disruption of CLDN1 expression in the testis. Hence, it may hinder the reproductive processes.

  16. Dynamic role of postsynaptic caspase-3 and BIRC4 in zebra finch song response habituation

    PubMed Central

    Huesmann, Graham R.; Clayton, David F.

    2007-01-01

    Summary Activation of the protease caspase-3 is commonly thought to cause apoptotic cell death. Here we show that caspase-3 activity is regulated at postsynaptic sites in brain following stimuli associated with memory (neural activation and subsequent response habituation) instead of cell death. In the zebra finch auditory forebrain, the concentration of caspase-3 active sites increases briefly within minutes after exposure to tape-recorded birdsong. With confocal and immunoelectron microscopy, we localize the activated enzyme to dendritic spines. The activated caspase-3 protein is present even in unstimulated brain but bound to an endogenous inhibitor, BIRC4 (xIAP), suggesting a mechanism for rapid release and sequestering at specific synaptic sites. Caspase-3 activity is necessary to consolidate a persistent physiological trace of the song stimulus, as demonstrated using pharmacological interference and the zenk gene habituation assay. Thus the brain appears to have adapted a core component of cell death machinery to serve a unique role in learning and memory. PMID:17178408

  17. Zinc pyrithione inhibits caspase-3 activity, promotes ErbB1-ErbB2 heterodimerization and suppresses ErbB2 downregulation in cardiomyocytes subjected to ischemia/reperfusion.

    PubMed

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

    2015-12-01

    Heart tissue becomes zinc-depleted and the capacity to mobilize labile zinc is diminished, indicating zinc dyshomeostasis during ischemia/reperfusion (I/R). Apparently, zinc pyrithione restores the basal zinc levels during I/R and prevents apoptosis by activating phosphatidyl inositol-3-kinase/Akt and targeting mitochondrial permeability transition. Receptor tyrosine kinases of the ErbB family (ErbB1 to ErbB4) are cell surface proteins that can regulate cell growth, proliferation and survival. Previous studies have shown that zinc pyrithione-induced activation of PI3kinase/Akt requires ErbB2 expression. On the other hand, while I/R decreases ErbB2 levels causing cardiomyocyte dysfunction and cell death, zinc pyrithione restores ErbB2 levels and maintains cardiomyocyte function. H9c2 cells expressed all the four ErbBs, although the expression of ErbB1 and ErbB2 were higher compared to ErbB3 and ErbB4. Hypoxia/Reoxygenation (H/R) had opposing effects on the mRNA expression of ErbB1 and ErbB2. ErbB2 mRNA levels were enhanced, but corresponding ErbB2 protein levels decreased after reoxygenation. H/R induced the degradation of ErbB2 in caspase-3 dependent manner, with the formation of a 25kDa fragment. This fragment could be detected after H/R only upon treatment of the cells with a proteasomal inhibitor, ALLN, suggesting that caspase-mediated cleavage of 185kDa ErbB2 results in C-terminal cleavage and formation of 25kDa fragment, which is further degraded by proteasome. Heterodimerization and phosphorylation of ErbB2/ErbB1 which decreased upon reoxygenation, was promoted by zinc pyrithione. Zinc pyrithione effectively suppressed the caspase activation, decreased the proteolytic cleavage of ErbB2, enhanced the phosphorylation and activation of ErbB1-ErbB2 complexes and improved the cell survival after hypoxia/reoxygenation. PMID:26436560

  18. CO{sub 2} impairs peroxynitrite-mediated inhibition of human caspase-3

    SciTech Connect

    Ascenzi, Paolo . E-mail: ascenzi@uniroma3.it; Marino, Maria; Menegatti, Enea

    2006-10-13

    Peroxynitrite (ONOO{sup -}) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide ({sup ?}NO) with superoxide (O2?-). The peroxynitrite reactivity is modulated by carbon dioxide (CO{sub 2}) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO{sub 2} on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the S{gamma} atom of the Cys catalytic residue. In the absence of CO{sub 2}, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO{sub 2} (=1.3x10{sup -3}M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the k{sub cat} value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering K{sub m}. Both in the absence and presence of CO{sub 2}, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent First evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO{sub 2}, supporting the role of CO{sub 2} in fine tuning of cell processes (e.g., apoptosis)

  19. Caspase-3 deficiency results in disrupted synaptic homeostasis and impaired attention control.

    PubMed

    Lo, Shih-Ching; Wang, Yuanyuan; Weber, Martin; Larson, Jessica L; Scearce-Levie, Kimberly; Sheng, Morgan

    2015-02-01

    The ability to attend to relevant stimuli and to adapt dynamically as demands change is a core aspect of cognition, and one that is impaired in several neuropsychiatric diseases, including attention deficit/hyperactivity disorder. However, the cellular and molecular mechanisms underlying such cognitive adaptability are poorly understood. We found that deletion of the caspase-3 gene, encoding an apoptosis protease with newly discovered roles in neural plasticity, disrupts attention in mice while preserving multiple learning and memory capabilities. Attention-related deficits include distractibility, impulsivity, behavioral rigidity, and reduced habituation to novel stimuli. Excess exploratory activity in Casp3(-/-) mice was correlated with enhanced novelty-induced activity in the dentate gyrus, which may be related to our findings that caspase-3 is required for homeostatic synaptic plasticity in vitro and homeostatic expression of AMPA receptors in vivo in response to chronic or repeated stimuli. These results suggest an important role for caspase-3 in synaptic suppression of irrelevant stimuli.

  20. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide

    PubMed Central

    Roy, Sophie; Bayly, Christopher I.; Gareau, Yves; Houtzager, Vicky M.; Kargman, Stacia; Keen, Sabina L. C.; Rowland, Kathleen; Seiden, Isolde M.; Thornberry, Nancy A.; Nicholson, Donald W.

    2001-01-01

    Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp “safety-catch” regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this “safety catch” results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation. PMID:11353841

  1. Caspase 3-Mediated Inactivation of Rac GTPases Promotes Drug-Induced Apoptosis in Human Lymphoma Cells

    PubMed Central

    Zhang, Baolin; Zhang, Yaqin; Shacter, Emily

    2003-01-01

    The Rac members of the Rho family GTPases control signaling pathways that regulate diverse cellular activities, including cytoskeletal organization, gene transcription, and cell transformation. Rac is implicated in apoptosis, but little is known about the mechanism by which it responds to apoptotic stimuli. Here we demonstrate that endogenous Rac GTPases are caspase 3 substrates that are cleaved in human lymphoma cells during drug-induced apoptosis. Cleavage of Rac1 occurs at two unconventional caspase 3 sites, VVGD11/G and VMVD47/G, and results in inactivation of the GTPase and effector functions of the protein (binding to the p21-activated protein kinase PAK1). Expression of caspase 3-resistant Rac1 mutants in the cells suppresses drug-induced apoptosis. Thus, proteolytic inactivation of Rac GTPases represents a novel, irreversible mechanism of Rac downregulation that allows maximal cell death following drug treatment. PMID:12897143

  2. Microsphere-based intracellular sensing of caspase-3/7 in apoptotic living cells.

    PubMed

    Cárdenas-Maestre, Juan Manuel; Pérez-López, Ana M; Bradley, Mark; Sánchez-Martín, Rosario M

    2014-07-01

    A novel multifunctional probe to monitor intracellular enzymatic activity in living cells is successfully developed. Their use as accurate intracellular sensors by conjugation of an internal control (that gives an extra feature to both evaluate cellular-uptake efficiency and track probes over time) is reported. In particular, a specific application of these multifunctional microspheres as sensors of caspase-3/7 to monitor apoptosis by flow cytometry is described. The preparation of these devices together with a kinetic study towards caspase-3 and caspase-7 and their evaluation as flow cytometry probe in apoptotic living cells are reported.

  3. Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    PubMed Central

    Wang, Shan; He, Meifang; Li, Linmei; Liang, Zhihua; Zou, Zehong

    2016-01-01

    Purpose Cell-in-cell structures are created by one living cell entering another homotypic or heterotypic living cell, which usually leads to the death of the internalized cell, specifically through caspase-dependent cell death (emperitosis) or lysosome-dependent cell death (entosis). Although entosis has attracted great attention, its occurrence is controversial, because one cell line used in its study (MCF-7) is deficient in caspase-3. Methods We investigated this issue using MCF-7 and A431 cell lines, which often display cell-in-cell invasion, and have different levels of caspase-3 expression. Cell-in-cell death morphology, microstructures, and signaling pathways were compared in the two cell lines. Results Our results confirmed that MCF-7 cells are caspase-3 deficient with a partial deletion in the CASP-3 gene. These cells underwent cell death that lacked typical apoptotic properties after staurosporine treatment, whereas caspase-3-sufficient A431 cells displayed typical apoptosis. The presence of caspase-3 was related neither to the lysosome-dependent nor to the caspase-dependent cell-in-cell death pathway. However, the existence of caspase-3 was associated with a switch from lysosome-dependent cell-in-cell death to the apoptotic cell-in-cell death pathway during entosis. Moreover, cellular hypoxia, mitochondrial swelling, release of cytochrome C, and autophagy were observed in internalized cells during entosis. Conclusion The occurrence of caspase-independent entosis is not a cell-specific process. In addition, entosis actually represents a cellular self-repair system, functioning through autophagy, to degrade damaged mitochondria resulting from cellular hypoxia in cell-in-cell structures. However, sustained autophagy-associated signal activation, without reduction in cellular hypoxia, eventually leads to lysosome-dependent intracellular cell death. PMID:27721872

  4. Khz (fusion product of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis in human colon carcinoma HCT116 cells, accompanied by an increase in reactive oxygen species, activation of caspase 3, and increased intracellular Ca²⁺.

    PubMed

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2015-03-01

    Khz (a fusion mycelium of Ganoderma lucidum and Polyporus umbellatus mycelia) is isolated from ganoderic acid and P. umbellatus and it exerts antiproliferative effects against malignant cells. However, no previous study has reported the inhibitory effects of Khz on the growth of human colon cancer cells. In the present study, we found that Khz suppressed cell division and induced apoptosis in HCT116 cells. Khz cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Khz reduced cell viability and mitochondrial membrane potential levels and it also induced disruption of the mitochondrial membrane potential and increased calcium concentration and reactive oxygen species generation. Khz increased caspase 3, PARP, caspase 7, and caspase 9 levels, but reduced Bcl-2 protein levels. Flow cytometry showed that the percentage of HCT116 cells in the sub-G1 phase of the cell cycle increased in response to Khz treatment.

  5. CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat.

    PubMed

    Krupinski, J; Ferrer, I; Barrachina, M; Secades, J J; Mercadal, J; Lozano, R

    2002-05-01

    Citicoline has been demonstrated to be beneficial in several models of cerebral ischaemia. We tested the hypothesis that citicoline may provide apoptotic pathways following focal cerebral ischaemia. Focal cerebral ischaemia was produced by distal, permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. The animals were randomised into four groups: (B+A) Citicoline 500 mg/kg IP 24 and 1 h before MCAO, and 23 h after MCAO; (A) citicoline 500 mg/kg IP, within 30 min after MCAO, and 23 h after MCAO; (C) vehicle IP; and (D) sham-operated. The animals were sacrificed at 12 h (n=8 per group) and 24 h (n=8 per group) after MCAO. Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6 and -8, and in paraffin-embedded sections processed for cleaved caspase-3 (17 kDa) immunohistochemistry. Finally, some sections were stained with the method of in situ end-labelling of nuclear DNA fragmentation. For gel electrophoresis and Western blotting, antibodies to poly (ADP-ribose) polymerase (PARP) products of 89 kDa were used to reveal specific cleavage substrates of caspases. MCAO induced the expression of all procaspases and the expression of PARP products of 89 kDa, as well as cells with nuclear DNA fragmentation, at 12 and 24 h, in the infarcted core and penumbra. Citicoline reduced the expression of all procaspases at 12 and 24 h after MCAO, as well as the expression of cleaved caspase-3 in cells in the penumbra area. This was accompanied by a reduction in the number of cells bearing nuclear DNA fragments. The expression of caspase-cleaved products of PARP (PARP 89 kDa) was reduced in citicoline-treated ischaemic rats. These results show that citicoline inhibits the expression of proteins involved in apoptosis following MCAO.

  6. Caspase-3–Dependent Organ Apoptosis Early After Burn Injury

    PubMed Central

    Fukuzuka, Kunitaro; Rosenberg, Jason J.; Gaines, Gregory C.; Edwards, Carl K.; Clare-Salzler, Michael; MacKay, Sally L. D.; Moldawer, Lyle L.; Copeland, Edward M.; Mozingo, David W.

    1999-01-01

    Objective To examine the role played by endotoxin, tumor necrosis factor-alpha (TNF-α), and caspase-3 in the increased apoptosis seen in solid organs in the early period after a burn injury. Summary Background Data Burn injury is often associated with immune suppression. Bacterial translocation and systemic endotoxemia have been reported after a burn injury, and caspase-3 activation due to TNF-α and Fas ligand (FasL) are presumed to initiate apoptosis. We hypothesized that endotoxin-induced TNF-α expression and caspase-3 activation could be the stimulus for the apoptosis after burn injury. Methods A 20% full-thickness scald burn was used in C57BL/6 mice. Three hours after burn injury, tissue samples were obtained from the thymus, lung, liver, and spleen. Lipopolysaccharide-nonresponsive (C3H/HeJ) and TNFα null B6x129tnf−/− mice were also used. To detect apoptosis, hematoxylin and eosin stain, in situTUNEL, DNA extraction, and gel electrophoresis were all performed. Caspase-3 activity and TNF-α and FasL mRNA were also measured. Results Increased apoptosis and caspase-3 activity were observed in the thymus and spleen 3 hours after burn injury but were not seen in liver or lung. In the thymus and spleen, increased expression of FasL mRNA was also observed, whereas increased TNF-α mRNA was not. Increased apoptosis in thymus and spleen were also observed in C3H/HeJ and B6x129tnf−/− mice after burn injury. An inhibitor of the caspase-3 (Z-VAD-fmk) reduced apoptosis in both thymus and spleen. Conclusions In the early period after a burn injury, increased apoptosis is observed primarily in the lymphoid organs and is independent of endotoxin or TNF-α. The increased caspase-3 activity in thymus and spleen contributes to apoptosis in these organs. PMID:10363899

  7. Neural cell apoptosis induced by microwave exposure through mitochondria-dependent caspase-3 pathway.

    PubMed

    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 2.856 GHz for 5 min and 15 min, 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.

  8. 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

  9. TPEN Induces Apoptosis Independently of Zinc Chelator Activity in a Model of Acute Lymphoblastic Leukemia and Ex Vivo Acute Leukemia Cells through Oxidative Stress and Mitochondria Caspase-3- and AIF-Dependent Pathways

    PubMed Central

    Mendivil-Perez, Miguel; Velez-Pardo, Carlos; Jimenez-Del-Rio, Marlene

    2012-01-01

    Acute lymphoblastic leukemia is still an incurable disease with resistance to therapy developing in the majority of patients. We investigated the effect of TPEN, an intracellular zinc chelator, in Jurkat and in ex vivo acute lymphoblastic leukemia (ALL) cells resistant to chemotherapy. Changes of nuclei morphology, reactive oxygen species generation, presence of hypodiploid cells, phosphatidylserine translocation, mitochondrial membrane depolarization, immunohistochemical identification of cell death signalling molecules, and pharmacological inhibition were assayed to detect the apoptotic cell death pathways. We found that TPEN induces apoptosis in both types of cells by a molecular oxidative stress pathway involving O2•− > H2O2 ≫ NF-κB (JNK/c-Jun) >p53> loss ΔΨm> caspase-3, AIF > chromatin condensation/DNA fragmentation. Interestingly, TPEN induced apoptosis independently of glucose; leukemic cells are therefore devoid of survival capacity by metabolic resistance to treatment. Most importantly, TPEN cytotoxic effect can eventually be regulated by the antioxidant N-acetyl-cysteine and zinc ions. Our data suggest that TPEN can be used as a potential therapeutic prooxidant agent against refractory leukemia. These data contribute to understanding the importance of oxidative stress in the treatment of ALL. PMID:23320127

  10. FITC-quencher based caspase 3-activatable nanoprobes for effectively sensing caspase 3 in vitro and in cells

    NASA Astrophysics Data System (ADS)

    Tang, Anming; Mei, Bin; Wang, Weijuan; Hu, Wanglai; Li, Fang; Zhou, Jun; Yang, Qing; Cui, Hua; Wu, Mian; Liang, Gaolin

    2013-09-01

    By employing fluorescence resonance energy transfer (FRET) quenching, we rationally designed two new FITC-quencher based nanoprobes for effectively sensing caspase 3 (Casp3) in vitro and in cells. Our nanoprobes hold promise for assessing the chemotherapeutic effect of cancer treatment.By employing fluorescence resonance energy transfer (FRET) quenching, we rationally designed two new FITC-quencher based nanoprobes for effectively sensing caspase 3 (Casp3) in vitro and in cells. Our nanoprobes hold promise for assessing the chemotherapeutic effect of cancer treatment. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03339b

  11. Activated caspase-9 immunoreactivity in glial and neuronal cytoplasmic inclusions in multiple system atrophy.

    PubMed

    Kawamoto, Yasuhiro; Ayaki, Takashi; Urushitani, Makoto; Ito, Hidefumi; Takahashi, Ryosuke

    2016-08-15

    The mitochondria play an important role in apoptotic cell death, and the released cytochrome c from the mitochondria promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9 and the promotion of the apoptotic cascade. To investigate the role of mitochondria-dependent apoptotic cell death in patients with multiple system atrophy (MSA), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects and 10 patients with MSA. We then performed double-labeling immunohistochemistry for activated caspase-9 and α-synuclein in some sections from 10 patients with MSA. In the brains with MSA, glial cytoplasmic inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) were intensely immunoreactive for cytochrome c, Apaf-1 and caspase-9. Activated caspase-9 immunoreactivities were also confirmed to be densely localized to both GCIs and NCIs using two types of anti-cleaved caspase-9 antibodies. The semiquantitative analyses using the upper pontine sections double-immunostained with cleaved caspase-9 and α-synuclein demonstrated that approximately 80% of GCIs and NCIs were immunopositive for cleaved caspase-9. Our results suggest that the formation of the apoptosome accompanied by the activation of caspase-9 may occur in brains affected by MSA, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of MSA. PMID:27345387

  12. Caspase-3-independent pathways proceeding in bystander effect of HSV-tk/GCV system

    NASA Astrophysics Data System (ADS)

    Lin, Juqiang; Ma, Yan; Zeng, Shaoqun; Zhang, Zhihong

    2008-02-01

    HSV-tk/GCV system, which is the virus-directed enzyme/prodrug therapy of herpes simplex virus (HSV) thymidine kinase (tk) gene / the anti-viral reagent ganciclovir (GCV), is one of the promising approaches in the rapidly growing area of gene therapy. As gene therapy of cancer such as suicide gene therapy has entered the clinic, another therapy effect which is called 'bystander effect' was reported. Bystander effect can lead to killing of non-transduced tumor cells in the immediate vicinity of GCV-treated HSV-TK-positive cells. Now the magnitude of 'bystander effect' is an essential factor for this anti-tumor approach in vivo. However, the mechanism which HSV-tk/ACV brings "bystander effect" is poorly understood. In this study, we monitor the activation of caspase-3 in HSV-tk/GCV system by a FRET probe CD3, a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. Through application of CD3 we have visualized the activation of caspase-3 in tk gene positive human adenoid cystic carcinoma (ACC-M) cells but not in bystander effect of HSV-tk/GCV system induced by GCV. This finding provides needed information for understanding the mechanisms by which suicide gene approaches actually kill cancer cells, and may prove to be helpful for the clinical treatment of cancers.

  13. A polymorphism in JMJD2C alters the cleavage by caspase-3 and the prognosis of human breast cancer

    PubMed Central

    Liu, Guangyu; Shao, Zhiming

    2014-01-01

    JMJD2C is a candidate oncogene that encodes a histone lysine demethylase with the ability to demethylate the lysine 9 residue of histone H3 (H3K9). The expression levels of JMJD2C are associated with tumor development and clinical outcome. Here we identify JMJD2C as a new substrate for caspase-3. JMJD2C is cleaved by caspase-3 at DEVD396G motif and then loses its demethylase activity. Additionally, we uncover D396N polymorphism (rs2296067) in the cleavage site of JMJD2C and establish its influence on the resistant to the cleavage by caspase-3. Importantly, we determined that D396N polymorphism is significantly associated with the prognosis of human breast cancer. We further found that the basal levels of DSB (double strand DNA break) repair proteins γ-H2AX (gamma-H2AX) increased when cells were treated with tumor necrosis factor-α (TNF-α) which activates caspase-3 activity. We also show that knockdown of JMJD2C expression results in up-regulation of basal γ-H2AX. We propose that D396N polymorphism of JMJD2C affects the prognosis of human breast cancer via altering the cleavage by caspase-3 and the ability of DSB repair which may contribute to therapy resistance. PMID:24952432

  14. Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors

    SciTech Connect

    Wang, Zhigang; Watt, William; Brooks, Nathan A.; Harris, Melissa S.; Urban, Jan; Boatman, Douglas; McMillan, Michael; Kahn, Michael; Heinrikson, Robert L.; Finzel, Barry C.; Wittwer, Arthur J.; Blinn, James; Kamtekar, Satwik; Tomasselli, Alfredo G.

    2010-09-17

    Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel {beta}-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

  15. Caspase 3 cleavage of Pax7 inhibits self-renewal of satellite cells

    PubMed Central

    Dick, Sarah A.; Chang, Natasha C.; Dumont, Nicolas A.; Bell, Ryan A. V.; Putinski, Charis; Kawabe, Yoichi; Litchfield, David W.; Rudnicki, Michael A.; Megeney, Lynn A.

    2015-01-01

    Compensatory growth and regeneration of skeletal muscle is dependent on the resident stem cell population, satellite cells (SCs). Self-renewal and maintenance of the SC niche is coordinated by the paired-box transcription factor Pax7, and yet continued expression of this protein inhibits the myoblast differentiation program. As such, the reduction or removal of Pax7 may denote a key prerequisite for SCs to abandon self-renewal and acquire differentiation competence. Here, we identify caspase 3 cleavage inactivation of Pax7 as a crucial step for terminating the self-renewal process. Inhibition of caspase 3 results in elevated Pax7 protein and SC self-renewal, whereas caspase activation leads to Pax7 cleavage and initiation of the myogenic differentiation program. Moreover, in vivo inhibition of caspase 3 activity leads to a profound disruption in skeletal muscle regeneration with an accumulation of SCs within the niche. We have also noted that casein kinase 2 (CK2)-directed phosphorylation of Pax7 attenuates caspase-directed cleavage. Together, these results demonstrate that SC fate is dependent on opposing posttranslational modifications of the Pax7 protein. PMID:26372956

  16. An Unbalanced Rearrangement of Chromosomes 4:20 is Associated with Childhood Osteoporosis and Reduced Caspase-3 Levels.

    PubMed

    Kinning, Esther; McMillan, Martin; Shepherd, Sheila; Helfrich, Miep; Hof, Rob Vant; Adams, Christopher; Read, Heather; Wall, Daniel M; Ahmed, S Faisal

    2016-09-01

    The purpose of this study was to investigate the association of a chromosome 4:20 imbalance with osteoporosis in three related children. Bone biochemistry, bone turnover markers, and dual-energy X-ray absorptiometry (DXA) scanning were performed in all three cases and bone biopsy and histomorphometry in one. The chromosome imbalance was delineated by array comparative genomic hybridization (aCGH) and analyzed for candidate genes. A potential candidate gene within the deleted region is caspase-3, previously linked to low bone mineral density (BMD) in heterozygous mice thus caspase-3 activity was measured in cases and controls. Routine bone biochemistry and markers of bone turnover did not reveal any abnormality. DXA showed reduced total and lumbar spine bone mineral content. aCGH showed an 8 megabase (Mb) deletion of terminal chromosome 4q incorporating a region previously linked to low BMD and a 15 Mb duplication of terminal chromosome 20p. Bone biopsy showed a high bone turnover state, trabecularisation of cortical bone and numerous small osteoclasts coupled with normal bone formation. Basal serum caspase-3 activity was lower in cases compared with controls. We conclude that the early-onset osteoporosis with low basal levels of caspase-3 and abnormal osteoclasts is a feature of this chromosomal translocation. Further investigation of the role of the deleted and duplicated genes and especially caspase-3 is required. PMID:27617159

  17. Prognostic significance of survivin and caspase-3 immunohistochemical expression in patients with diffuse large B-cell lymphoma treated with rituximab and CHOP.

    PubMed

    Mitrović, Zdravko; Ilić, Ivana; Aurer, Igor; Kinda, Sandra Bašić; Radman, Ivo; Dotlić, Snježana; Ajduković, Radmila; Labar, Boris

    2011-06-01

    Survivin is an inhibitor of apoptosis whose expression may be associated with inferior outcome in patients with diffuse large B-cell lymphoma (DLBCL) treated without rituximab. Caspase-3 is the final caspase of the apoptotic cascade and its pattern of expression may also be related to patients' outcome. In this study we investigated immunohistochemical expression of survivin and caspase-3 (CPP32) in 57 patients with DLBCL treated with rituximab and CHOP (R-CHOP). According to previously published criteria, we separately analyzed correlation of different types of survivin expression with patients' outcome. Nuclear survivin was expressed in only 26% of cases, cytoplasmic survivin was expressed in 81% of cases while application of immunoreactivity scoring system yielded 58% of survivin positive cases. Caspase-3 was expressed in 77% of cases. There were no significant correlations between any type of survivin expression and response to treatment or survival of the patients. The expression of caspase-3 was also not associated with patients' outcome. We conclude that survivin and caspase-3 have no significant prognostic significance in patients with DLBCL treated with R-CHOP. PMID:20853074

  18. Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets.

    PubMed

    Brandhorst, Daniel; Brandhorst, Heide; Maataoui, Vidya; Maataoui, Adel; Johnson, Paul R V

    2013-06-01

    Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 μmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.

  19. Pharmacophore Modeling and Docking Studies on Some Nonpeptide-Based Caspase-3 Inhibitors

    PubMed Central

    Sharma, Simant; Basu, Arijit; Agrawal, R. K.

    2013-01-01

    Neurodegenerative disorders are major consequences of excessive apoptosis caused by a proteolytic enzyme known as caspase-3. Therefore, caspase-3 inhibition has become a validated therapeutic approach for neurodegenerative disorders. We performed pharmacophore modeling on some synthetic derivatives of caspase-3 inhibitors (pyrrolo[3,4-c]quinoline-1,3-diones) using PHASE 3.0. This resulted in the common pharmacophore hypothesis AAHRR.6 which might be responsible for the biological activity: two aromatic rings (R) mainly in the quinoline nucleus, one hydrophobic (H) group (CH3), and two acceptor (A) groups (–C=O). After identifying a valid hypothesis, we also developed an atom-based 3D-QSAR model applying the PLS algorithm. The developed model was statistically robust (q2 = 0.53; pred_r2 = 0.80). Additionally, we have performed molecular docking studies, cross-validated our results, and gained a deeper insight into its molecular recognition process. Our developed model may serve as a query tool for future virtual screening and drug designing for this particular target. PMID:24089669

  20. Multidrug resistance-associated protein 3 confers resistance to chemoradiotherapy for rectal cancer by regulating reactive oxygen species and caspase-3-dependent apoptotic pathway.

    PubMed

    Yu, Zhiqi; Zhang, Chang; Wang, Hao; Xing, Junjie; Gong, Haifeng; Yu, Enda; Zhang, Wei; Zhang, Xiaoqing; Cao, Guangwen; Fu, Chuangang

    2014-10-28

    This study aimed to clarify the role of multidrug resistance-associated protein 3 (MRP3) in resistance to neoadjuvant chemoradiotherapy and long-term prognosis of advanced rectal cancer. Immunohistochemistry was used to measure MRP3 expression in biopsy specimens of 144 stage II-III rectal cancer patients who received preoperative chemoradiotherapy. The effect of MRP3 expression on short-term pathological response and postoperative long-term prognosis were assessed using the Cox proportional hazards model. Short interfering RNAs targeting MRP3 were synthesized and used to transfect human colorectal carcinoma cell lines. The effect of MRP3 down-regulation on cell proliferation and apoptosis in response to 5-fluorouracil and/or irradiation were examined in vitro and in xenograft mouse models, respectively. The content of intracellular reactive oxygen species and the activity of caspase-3-dependent apoptotic pathway in response to irradiation were further evaluated. High expression (immunoreactive score > 6) of MRP3 significantly predicted poor pathological response to chemoradiotherapy (tumor regression grade ≤ 2 vs. ≥3, p = 0.002) in univariate analysis and unfavorable long-term prognosis (5-year overall survival: HR = 1.612, 95% CI, 1.094-2.375, p = 0.016; 5-year disease-free survival: HR = 1.513, 95% CI, 1.041-2.200, p = 0.030) in multivariate Cox analysis. MRP3 down-regulation significantly increased 5-fluorouracil or irradiation-induced cell apoptosis and attenuated tumor growth following irradiation in animal models. MRP3 inhibition significantly reduced intracellular reactive oxygen species exporting from cells following irradiation, and increased expression of cleaved poly ADP-ribose polymerase and caspase-3. Aberrant expression of MRP3 in rectal cancer confers chemo-radioresistance. MRP3 might be a predictive factor and an attractive target in treating advanced rectal cancer.

  1. Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats.

    PubMed

    Liu, Guangyi; Wang, Tao; Wang, Tinging; Song, Jinming; Zhou, Zhen

    2013-11-01

    the experimental group at 48 h following reperfusion. In conclusion, cerebral ischemia/reperfusion injury may cause neurological impairment and lead to a change of ethology, and neuron apoptosis may be associated with the activation of caspase-3 and Bax and the downregulation of Bcl-2. PMID:24649043

  2. Characterization of Social Behaviors in caspase-3 deficient mice

    PubMed Central

    Lo, Shih-Ching; Scearce-Levie, Kimberly; Sheng, Morgan

    2016-01-01

    Impaired social interaction is a defining feature of autism spectrum disorder, a neurodevelopmental disorder that shows a strong male preponderance in prevalence. Studies have identified neural circuits, neuromodulators and genetic factors involved in social behaviors, but mechanistic understanding of gender-specific social deficits is lacking. We report that deletion of the caspase-3 gene, encoding a protease with functions in apoptosis and neural plasticity, alters specific social behaviors in male mice, while leaving females unaffected. Casp3−/− mice showed normal behavioral responses to olfactory cues from food, neutral chemical and biological sources. Both Casp3−/− males and females displayed robust social exploration, sociability, recognition and preference for an enclosed novel mouse in the three-chamber test. However, Casp3−/− males showed significantly reduced social interaction behaviors when exposed to a freely moving novel mouse, including decreased interaction time and diminished mounting. Thus caspase-3 is essential for a subset of social behaviors, but despite similar hyper-locomotion in both sexes, only male Casp3−/− mice exhibited social interaction deficits, which is interesting given the male bias of autism. PMID:26783106

  3. TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

    PubMed

    Alvarez, Susana; Blanco, Almudena; Fresno, Manuel; Muñoz-Fernández, Ma Ángeles

    2011-01-27

    There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca(2+) independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

  4. Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

    NASA Astrophysics Data System (ADS)

    Wang, Huiying; Chen, Tongsheng; Sun, Lei

    2008-02-01

    Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

  5. Chronic glial activation, neurodegeneration, and APP immunoreactive deposits following acute administration of double-stranded RNA.

    PubMed

    Melton, Lisa M; Keith, Alexander B; Davis, Sue; Oakley, Arthur E; Edwardson, James A; Morris, Christopher M

    2003-10-01

    Several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, are associated with immunocompetent microglia, leading to the suggestion that chronic glial-mediated inflammation contributes to the neurodegeneration seen in these diseases. Little direct evidence supports this hypothesis, and no suitable rodent models exist that do not involve the use of blunt trauma or ischaemia, events that are infrequently encountered in the human disease state. In the present study, we report that administration of double-stranded RNA, a classical inducer of interferon-gamma (IFN-gamma), causes rapid and persistent activation of microglia and astrocytes, as well as induction of interleukin-1beta (IL-beta) and nitric oxide synthase. In close temporal succession to glial activation, there is neurodegeneration, with neuron loss involving apoptosis in selected brain regions including the septal nucleus, hippocampus, cortex and thalamus, along with hippocampal atrophy. This neuronal loss is accompanied by punctate deposits of material that are immunoreactive for amyloid precursor protein, beta-amyloid peptide (Abeta), and apolipoprotein E. The findings may have clinical relevance, since the administration of the nonsteroidal antiinflammatory agent (NSAID) ibuprofen markedly reduces the neurodegeneration observed in the absence of significant glial inhibition. These findings may be relevant to the pathogenesis of Alzheimer's disease in particular, and to other neurodegenerative diseases involving inflammation.

  6. A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3

    NASA Astrophysics Data System (ADS)

    Murthy, Aditya; Li, Yun; Peng, Ivan; Reichelt, Mike; Katakam, Anand Kumar; Noubade, Rajkumar; Roose-Girma, Merone; Devoss, Jason; Diehl, Lauri; Graham, Robert R.; van Lookeren Campagne, Menno

    2014-02-01

    Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

  7. Pituitary adenylatecyclase-activating polypeptide-immunoreactive nerve fibers in the rat epiglottis and pharynx.

    PubMed

    Kano, Mitsuhiro; Shimizu, Yoshinaka; Suzuki, Yujiro; Furukawa, Yusuke; Ishida, Hiroko; Oikawa, Miho; Kanetaka, Hiroyasu; Ichikawa, Hiroyuki; Suzuki, Toshihiko

    2011-12-20

    The distribution of pituitary adenylatecyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers was studied in the rat epiglottis and pharynx. PACAP-IR nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant on the laryngeal side of the epiglottis and in the dorsal and lateral border region between naso-oral and laryngeal parts of the pharynx. PACAP-IR nerve fibers were also detected in taste buds within the epiglottis and pharynx. In addition, many PACAP-IR nerve fibers were found around acinar cells and blood vessels. The double immunofluorescence method demonstrated that distribution of PACAP-IR nerve fibers was similar to that in CGRP-IR nerve fibers in the epithelium and taste bud. However, distributions of PACAP-IR and CGRP-IR nerve fibers innervating mucous glands and blood vessels were different. The retrograde tracing method also demonstrated that PACAP and CGRP were co-expressed by vagal and glossopharyngeal sensory neurons innervating the pharynx. These findings suggest that PACAP-IR nerve fibers in the epithelium and taste bud of the epiglottis and pharynx which originate from the vagal and glossopharyngeal sensory ganglia include nociceptors and chemoreceptors. The origin of PACAP-IR nerve fibers which innervate mucous glands and blood vessels may be the autonomic ganglion.

  8. Studies on porcine pancreatic elastase activity. II. Immunoreactive elastase level during acute hemorrhagic pancreatitis in pigs.

    PubMed

    Nakajima, Y; Matsuno, S; Noto, N; Saitoh, Y; Sato, T

    1980-06-01

    Acute hemorrhagic pancreatitis was produced in pig to study serum concentration of elastase and its physiological role. Pancreatitis was induced in two groups of young pigs by the injection of autologous bile. One group was injected with autologous bile (0.5 ml/kg) at high pressure, and the second group was injected as low pressure (100 cm H2O). Then femoral blood, portal blood and thoracic lymph were sampled at scheduled time intervals. The control level of immunoreactive elastase was around 90 ng/ml in each site, which significantly increased beginning 15 min after bile injection; the level of immunoreactive elastase was higher in the thoracic lymph duct than in the femoral and portal vein. The total and free elastase of both groups in pancreatic tissue were significantly decreased in pancreatitis, and an abundance of immunoreactive elastase was found in the ascites. The increasing pattern of immunoreactive elastase and amylase after bile injection was very similar. Therefore, the level of immunoreactive elastase was considered to be inadequate to determine the grade of severity of pancreatitis as well as the level of amylase which is already known.

  9. Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases.

    PubMed Central

    Jäättelä, M; Wissing, D; Kokholm, K; Kallunki, T; Egeblad, M

    1998-01-01

    The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions. PMID:9799222

  10. PCNA immunoreactivity revealing normal proliferative activity in the brain of adult Lampetra planeri (Bloch, 1784).

    PubMed

    Margotta, Vito; Caronti, Brunella; Colombari, Paolo Tito; Castiglia, Riccardo

    2007-01-01

    It is now well known that the Teleosts among Osteichthyes, Urodele and Anuran Amphibians, Lacertilian Reptiles possess encephalic natural proliferative activities even into adulthood, as demonstrated by a great number of researches performed both under normal and various experimental conditions. Few years ago we have undertaken in adult heterothermic vertebrates a reappraisal on spontaneous cerebral proliferative events involving some organisms (Podarcis sicula, Triturus carnifex, Rana esculenta, Carassius carassius) representative of these vertebrates and belonging to the same or phylogenetically similar species used by previous researchers in studies having the same object. In our investigations, these performances were revealed by a proliferative immunocytochemical marker, the Proliferating Cell Nuclear Antigen (PCNA). At this point of our study in the scenario emerging from findings a missing piece is represented by Petromyzontidae. To fill up this gap in the present investigation, using our usual test, we have paid attention to adult specimens of Lampetra planeri. The obtained immunostaining panorama has revealed the presence of a considerable number of spontaneous proliferative activities. These events might differ in quantity, in various encephalic districts. PCNA-labelled cells appeared scattered in the cranial portion of olfactory bulbs, while the PCNA expression has been observed steadily localized with a distinctly continous distribution in cells interposed among the ependymal epithelium which lines the cavities of the proximal portion of the olfactory region and of the cerebral ventricles. DNA synthesis activity has been also found in cells scattered in the telencephalic, diencephalic, mesencephalic and medulla oblongata periventricular grey. This immunoreactivity was not revealable in the cerebellum. Our findings are discussed in the light of bibliographic news.

  11. Bcl-2/caspase 3 mucosal imbalance favors T cell resistance to apoptosis in dogs with inflammatory bowel disease.

    PubMed

    Jergens, A; Young, J; Moore, D; Wang, C; Hostetter, J; Augustine, L; Allenspach, K; Schmitz, S; Mosher, C

    2014-04-15

    Canine idiopathic inflammatory bowel disease (IBD) is believed to result from complex interplay between genetic, microbial, and immunologic factors. Abnormal cell death by apoptosis may result in the persistence of activated intestinal T cells that contribute to mucosal inflammation and clinical severity. To test this hypothesis, we investigated the mucosal expression of pro- and anti-apoptotic proteins in different intestinal compartments and their association with inflammatory indices in dogs with IBD. Apoptosis of lamina propria (LP) T cells in duodenal, ileal, and colonic tissues in control and IBD dogs was analyzed by caspase 3/Bcl-2 immunohistochemistry and TUNEL assays. Densities and distributions of LP caspase 3 and Bcl-2 cells were correlated to histopathologic lesions and the clinical activity index (CIBDAI). Compared to control tissues, IBD dogs had significantly (P<0.01) fewer caspase 3 cells in colonic mucosa. Double immunostaining identified the majority of apoptotic cells as TUNEL(+)/caspase 3(+). Within intestinal mucosa of IBD dogs, there were significantly greater numbers of Bcl-2 cells at the apical and basilar villus in the duodenum as compared to the colon and to the apical and basilar villus in the ileum (P<0.001 for all comparisons). There were significantly greater numbers of Bcl-2 cells at the apical and basilar villus of the duodenum but significantly fewer numbers of Bcl-2 cells at the apical villus of the ileum in IBD dogs compared with controls (P<0.001, P<0.001, and P<0.02, respectively). There was a significant association between the number of Bcl-2 cells in the duodenum of IBD dogs and the CIBDAI (P<0.001 each for mild, moderate and severe clinical IBD). In conclusion, apoptosis of T lymphocytes varies within intestinal compartments of dogs with IBD. Mucosal imbalance of Bcl-2/caspase 3 expression favors T cell resistance to apoptosis which may contribute to T cell accumulation and chronic intestinal inflammation, similar to human

  12. Strophalloside induces apoptosis of SGC-7901 cells through the mitochondrion-dependent caspase-3 pathway.

    PubMed

    Zhang, Xue-Jiao; Mei, Wen-Li; Tan, Guang-Hong; Wang, Cai-Chun; Zhou, Song-Lin; Huang, Feng-Ru; Chen, Bin; Dai, Hao-Fu; Huang, Feng-Ying

    2015-01-01

    Cardenolides with special chemical structures have been considered as effective anti-cancer drugs in clinic trials. Strophalloside is a cardenolide we recently isolated from Antiaris toxicaria obtained from Hainan, China. The aim of this study was to investigate the possible anticancer effects induced by strophalloside and the underlying molecular mechanism. Gastric carcinoma SGC-7901 cells were treated with strophalloside at various concentrations for different times, and resulting cell viability was determined by the MTT assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Apoptosis were measured by Annexin V-FITC/PI and Hoechst staining. The changes of mitochondrial transmembrane potential were examined by a JC-1 kit. The expressions of pro-apoptotic protein cytochrome c, caspase-3 and caspase-9 were detected by western blotting analysis. The results showed that strophalloside was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion in a time- and dose-dependent manner. Mitochondrial membrane potential declined and the concentration of cytochrome c increased in cytoplasm and caspase-3 and caspase-9 were cleaved into activated states, suggesting that cytochrome c was released from the mitochondrion to cytoplasm and finally activated the caspase-dependent apoptosis pathway. Our results indicate that strophalloside is a potential anticancer drug.

  13. Flavonoids of Rosa roxburghii Tratt exhibit radioprotection and anti-apoptosis properties via the Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway.

    PubMed

    Xu, Ping; Cai, Xinhua; Zhang, Wenbo; Li, Yana; Qiu, Peiyong; Lu, Dandan; He, Xiaoyang

    2016-10-01

    The objective of our study was to assess the radioprotective effect of flavonoids extracted from Rosa roxburghii Tratt (FRT) and investigate the role of Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway in radiation-induced apoptosis. Cells and mice were exposed to (60)Co γ-rays at a dose of 6 Gy. The radiation treatment induced significant effects on tissue pathological changes, apoptosis, Ca(2+), ROS, DNA damage, and expression levels of Bcl-2, Caspase-3 (C-Caspase-3), and PARP-1. The results showed that FRT acted as an antioxidant, reduced DNA damage, corrected the pathological changes of the tissue induced by radiation, promoted the formation of spleen nodules, resisted sperm aberration, and protected the thymus. FRT significantly reduced cell apoptosis compared with the irradiation group. The expression of Ca(2+) and C-Caspase-3 was decreased after FRT treatment compared with the radiation-treated group. At the same time, expression of prototype PARP-1 and Bcl-2 increased, leading to a decrease in the percentage of apoptosis cells in FRT treatment groups. We conclude that FRT acts as a radioprotector. Apoptosis signals were activated via the Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway in irradiated cells and FRT inhibited this pathway of apoptosis by down-regulation of C-Caspase-3 and Ca(2+) and up-regulation of prototype PARP-1 and Bcl-2.

  14. Epstein-Barr viral microRNAs target caspase 3.

    PubMed

    Harold, Cecelia; Cox, Diana; Riley, Kasandra J

    2016-01-01

    The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that transforms B cells and causes several malignancies including Burkitt's lymphoma. EBV differentially expresses at least 49 mature microRNAs (miRNAs) during latency in various infected epithelial and B cells. Recent high-throughput studies and functional assays have begun to reveal the function of the EBV miRNAs suggesting roles in latency, cell cycle control, and apoptosis. In particular, the central executioner of apoptosis, Caspase 3 (CASP3), was proposed as a target of select EBV miRNAs. However, whether CASP3 is truly a target of EBV miRNAs, and if so, which specific miRNAs target CASP3 is still under debate. Based on previously published high-throughput biochemical data and a bioinformatic analysis of the entire CASP3 3'-UTR, we identified 12 EBV miRNAs that have one or more seed binding sites in the CASP3 3'-UTR. We individually tested all 12 miRNAs for repression of CASP3 in luciferase reporter assays, and nine showed statistically significant (P < 0.001) repression of a full-length CASP3 reporter. Further, three EBV miRNAs, including BART22, exhibited repression of endogenous CASP3 protein. These data confirm that CASP3 is a direct target of specific EBV BART miRNAs. PMID:27565721

  15. Atorvastatin attenuates cognitive deficits through Akt1/caspase-3 signaling pathway in ischemic stroke.

    PubMed

    Yang, Jie; Pan, Ying; Li, Xuejing; Wang, Xianying

    2015-12-10

    Neuronal damage in the hippocampal formation is more sensitive to ischemic stimulation and easily injured, causing severe learning and memory impairment. Therefore, protection of hippocampal neuronal damage is the main contributor for learning and memory impairment during cerebral ischemia. Atorvastatin has been reported to ameliorate ischemic brain damage after ischemia reperfusion (I/R). However, its molecular mechanism has not been elucidated clearly. In this study, we established four-vessel occlusion model in rats with cerebral ischemia. Here, we demonstrated that atorvastatin significantly improves the behavior of I/R-rat in open field tasks. We also found that atorvastatin significantly shortens the distance and time of loading onto the hidden platform in the positioning navigation process, decreases the latency in the space exploration process when cognitive testing with Morris water maze was performed during ischemic stroke in rats. Furthermore, the survival rate of neurons in the CA1 area of the hippocampus and the phosphorylation of Akt (Ser473) in the neurons are increased, whereas the expression of caspase-3 are inhibited by atorvastatin. However, after an intracerebroventricular injection of LY294002 (an inhibitor of Akt1), the above neuroprotective effects of atorvastatin are attenuated. In summary, our results imply atorvastatin may improve the survival rate of hippocampal neurons and reduce the impairment of learning and memory by downregulating the activation of the caspase-3 via increasing the phosphorylation of Akt1 during ischemia/reperfusion. PMID:26597376

  16. QSAR Analysis for Some 1, 2-Benzisothiazol-3-one Derivatives as Caspase-3 Inhibitors by Stepwise MLR Method

    PubMed Central

    Hajimahdi, Zahra; Safizadeh, Fatemeh; Zarghi, Afshin

    2016-01-01

    Caspase-3 inhibitory activities of some 1, 2-benzisothiazol-3-one derivatives were modeled by quantitative structure–activity relationship (QSAR) using stepwise-multiple linear regression (SW-MLR) method. The built model was robust and predictive with correlation coefficient (R2) of 0.91 and 0.59 for training and test groups, respectively. The quality of the model was evaluated by leave-one out (LOO) cross validation (LOO correlation coefficient, Q2) of 0.80). The results indicate that the descriptors related to the electronegativity, the atomic masses, the atomic van der Waals volumes and R--CX--R Atom-centered fragments play a more significant role in caspase-3 inhibitory activity. PMID:27642314

  17. QSAR Analysis for Some 1, 2-Benzisothiazol-3-one Derivatives as Caspase-3 Inhibitors by Stepwise MLR Method.

    PubMed

    Hajimahdi, Zahra; Safizadeh, Fatemeh; Zarghi, Afshin

    2016-01-01

    Caspase-3 inhibitory activities of some 1, 2-benzisothiazol-3-one derivatives were modeled by quantitative structure-activity relationship (QSAR) using stepwise-multiple linear regression (SW-MLR) method. The built model was robust and predictive with correlation coefficient (R(2)) of 0.91 and 0.59 for training and test groups, respectively. The quality of the model was evaluated by leave-one out (LOO) cross validation (LOO correlation coefficient, Q(2)) of 0.80). The results indicate that the descriptors related to the electronegativity, the atomic masses, the atomic van der Waals volumes and R--CX--R Atom-centered fragments play a more significant role in caspase-3 inhibitory activity. PMID:27642314

  18. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism.

    PubMed

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

  19. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism

    PubMed Central

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

  20. Caspase-3-mediated degradation of condensin Cap-H regulates mitotic cell death.

    PubMed

    Lai, S-K; Wong, C-H; Lee, Y-P; Li, H-Y

    2011-06-01

    Mitotic death is a major form of cell death in cancer cells that have been treated with chemotherapeutic drugs. However, the mechanisms underlying this form of cell death is poorly understood. Here, we report that the loss of chromosome integrity is an important determinant of mitotic death. During prolonged mitotic arrest, caspase-3 is activated and it cleaves Cap-H, a subunit of condensin I. The depletion of Cap-H results in the loss of condensin I complex at the chromosomes, thus affecting the integrity of the chromosomes. Consequently, DNA fragmentation by caspase-activated DNase is facilitated, thus driving the cell towards mitotic death. By expressing a caspase-resistant form of Cap-H, mitotic death is abrogated and the cells are able to reenter interphase after a long mitotic delay. Taken together, we provide new insights into the molecular events that occur during mitotic death.

  1. Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3

    PubMed Central

    Dai, Zhi-Jun; Wang, Xi-Jing; Li, Zong-Fang; Ji, Zong-Zheng; Ren, Hong-Tao; Tang, Wei; Liu, Xiao-Xu; Kang, Hua-Feng; Guan, Hai-Tao; Song, Ling-Qin

    2008-01-01

    AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G1 phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3. PMID:19109865

  2. Nascent histamine induces α-synuclein and caspase-3 on human cells

    SciTech Connect

    Caro-Astorga, Joaquín; Fajardo, Ignacio; Ruiz-Pérez, María Victoria; Sánchez-Jiménez, Francisca; Urdiales, José Luis

    2014-09-05

    Highlights: • Nascent histamine alters cyclin expression pattern. • Nascent histamine increases expression of α-synuclein. • Nascent histamine activates caspase-3. - Abstract: Histamine (Hia) is the most multifunctional biogenic amine. It is synthetized by histidine decarboxylase (HDC) in a reduced set of mammalian cell types. Mast cells and histaminergic neurons store Hia in specialized organelles until the amine is extruded by exocytosis; however, other immune and cancer cells are able to produce but not store Hia. The intracellular effects of Hia are still not well characterized, in spite of its physiopathological relevance. Multiple functional relationships exist among Hia metabolism/signaling elements and those of other biogenic amines, including growth-related polyamines. Previously, we obtained the first insights for an inhibitory effect of newly synthetized Hia on both growth-related polyamine biosynthesis and cell cycle progression of non-fully differentiated mammalian cells. In this work, we describe progress in this line. HEK293 cells were transfected to express active and inactive versions of GFP-human HDC fusion proteins and, after cell sorting by flow cytometry, the relative expression of a large number of proteins associated with cell signaling were measured using an antibody microarray. Experimental results were analyzed in terms of protein–protein and functional interaction networks. Expression of active HDC induced a cell cycle arrest through the alteration of the levels of several proteins such as cyclin D1, cdk6, cdk7 and cyclin A. Regulation of α-synuclein and caspase-3 was also observed. The analyses provide new clues on the molecular mechanisms underlying the regulatory effects of intracellular newly synthetized Hia on cell proliferation/survival, cell trafficking and protein turnover. This information is especially interesting for emergent and orphan immune and neuroinflammatory diseases.

  3. TRAIL, DR5 and caspase 3-dependent apoptosis in vessels of diseased human temporomandibular joint disc. An immunohistochemical study

    PubMed Central

    Loreto, C.; Almeida, L.E.; Migliore, M.R.; Caltabiano, M.; Leonardi, R.

    2010-01-01

    To evaluate the apoptosis involvement in the angiogenesis as a self-limiting process in patients with temporomandibular joint (TMJ) degenerated disc vessels, we assessed, by immunohistochemistry, the detection of TRAIL, its death receptor DR5 and caspase 3. TRAIL, its death receptor DR5 and caspase 3 expression were studied by immunohistochemistry in 15 TMJ discs displaced without reduction and in 4 unaffected discs. These apoptosis molecules were detected in the intima and media layers of newly formed vessels affected discs. In conclusion, vessels apoptosis activation in TMJ disc with ID could be regarded as a self-limiting process that try to leads to vessel regression; in this way an inhibition of angiogenic vessels may prove a key strategy in limiting pathological angiogenesis, by cutting off blood supply to tumors, or by reducing harmful inflammation. PMID:20839416

  4. [Component I from Agkistrodon acutus venom induces apoptosis of K562/A02 cells by promoting caspase 3 expression].

    PubMed

    Zhou, Bing; Zhang, Gen-Bao; Duan, Ting; Zhou, Jue; Wu, Juan

    2012-04-01

    To investigate the effects of component I from Agkistrodon acutus venom (AAVC-I) on the biological features of chronic myeloid leukemia cells, K562/A02 leukemia cells were cultured in the presence of AAVC-I (6.25 - 100 µg/ml) and the proliferation status was assayed by CCK-8 method. Morphological changes were observed by inversed microscope after Giemsa and Hochest 33258 staining, and cell apoptosis was detected by flow cytometry. Caspase 3 activity was tested by using Chromogenic Activity Assay Kit. The results showed that AAVC-I inhibited the growth of K562/A02 cells in time- and concentration-dependant manners, and the IC(50) at 48 h was 30.988 µg/ml. Giemsa and Hochest 33258 staining showed the typical apoptotic features in K562/A02 cells after induction with AAVC-I for 48 h. Flow cytometric analysis revealed that the percentage of the apoptotic cells reached from 0.88 up to 53.66 as the treated concentration was elevated from 0 to 50 µg/ml. Compared with the control group, the expression of caspase 3 in the tested group was enhanced in a dose-dependent manner (P < 0.05). It is concluded that AAVC-I can effectively inhibit the growth and promote apoptosis of K562/A02 cells. Elevated expression of caspase-3 may be attributed to the apoptosis of K562/A02 cells. PMID:22541080

  5. Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.

    PubMed

    Iwasaki, Takahiro; Katayama, Takeshi; Kohama, Kazuhiro; Endo, Yaeta; Sawasaki, Tatsuya

    2013-03-01

    In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

  6. Acteoside Binds to Caspase-3 and Exerts Neuroprotection in the Rotenone Rat Model of Parkinson's Disease

    PubMed Central

    Wang, Ying; He, Xiao; Zhao, Yuwu

    2016-01-01

    Parkinson’s disease (PD) is characterized by the progressive degeneration of the dopaminergic neurons in the substantia nigra (SN) region. Acteoside has displayed multiple biological functions. Its potential role against PD and the underlying signaling mechanisms are largely unknown. Here, we showed that oral administration of acteoside significantly attenuated parkinsonism symptoms in rotenone-induced PD rats. Further, acteoside inhibited rotenone-induced α-synuclein, caspase-3 upregulation and microtubule-associated protein 2 (MAP2) downregulation in PD rats. The molecular docking and molecular dynamics (MD) simulation results indicated that acteoside may directly bind to and inhibit caspase-3. Acteoside formed hydrogen bonds with at least six residues of caspase-3: ThrA177, SerA178, GlyA238, SerB339, ArgB341 and TrpB348. In addition, a pi-pi interaction was formed between acteoside and caspase-3’s HisA237, which might further stabilize the complex. MD simulation results demonstrated that the binding affinity of the caspase-3-acteoside complex was higher than that of caspase-3 and its native ligand inhibitor. Together, we show that acteoside binds to caspase-3 and exerts neuroprotection in the rotenone rat model of PD. PMID:27632381

  7. Acteoside Binds to Caspase-3 and Exerts Neuroprotection in the Rotenone Rat Model of Parkinson's Disease.

    PubMed

    Yuan, Jiawen; Ren, Jinpeng; Wang, Ying; He, Xiao; Zhao, Yuwu

    2016-01-01

    Parkinson's disease (PD) is characterized by the progressive degeneration of the dopaminergic neurons in the substantia nigra (SN) region. Acteoside has displayed multiple biological functions. Its potential role against PD and the underlying signaling mechanisms are largely unknown. Here, we showed that oral administration of acteoside significantly attenuated parkinsonism symptoms in rotenone-induced PD rats. Further, acteoside inhibited rotenone-induced α-synuclein, caspase-3 upregulation and microtubule-associated protein 2 (MAP2) downregulation in PD rats. The molecular docking and molecular dynamics (MD) simulation results indicated that acteoside may directly bind to and inhibit caspase-3. Acteoside formed hydrogen bonds with at least six residues of caspase-3: ThrA177, SerA178, GlyA238, SerB339, ArgB341 and TrpB348. In addition, a pi-pi interaction was formed between acteoside and caspase-3's HisA237, which might further stabilize the complex. MD simulation results demonstrated that the binding affinity of the caspase-3-acteoside complex was higher than that of caspase-3 and its native ligand inhibitor. Together, we show that acteoside binds to caspase-3 and exerts neuroprotection in the rotenone rat model of PD. PMID:27632381

  8. Anti-apoptotic effect of succinyl gelatine in a liver ischaemia-reperfusion injury model (Bcl-2, Bax, Caspase 3)?

    PubMed

    Altunkan, Ali; Aydin, Ozlem; Ozer, Zeliha; Colak, Tahsin; Bilgin, Egemen; Oral, Uğur

    2002-06-01

    Apoptosis of tissues may contribute to ischaemia-reperfusion injury. The aim of the present study was to determine whether administration of a colloid solution would prevent apoptosis after liver ischaemia-reperfusion. New Zealand rabbits, weighing 1.5-2 kg, were randomized to receive either 4% SG (20 ml kg (-1)h(-1) ) by 30 min of intravenous (i.v.) infusion (Group I, n= 7) or equivalent volumes of 0.9% sodium chloride (Group II, n= 6) i.v. before a 45 min interruption of the portal vein blood flow and then 45 min of reperfusion. The animals were killed following the reperfusion period. Their livers were processed for histopathological examination and paraffin sections of these tissues were examined. The expression of Bcl-2, Bax and caspase 3 were analysed by immunohistochemistry. ANOVA and the Wilcoxon W -test were used for statistical analysis, and mean values were expressed +/-sd. Histologically, the foci of ischaemic necrosis were observed in liver specimens of the periportal area in one of the animals in Group I and in two in Group II. Immunhistochemical analysis demonstrated an increase in Bcl-2 protein levels in Group I compared to Group II ( P< 0.05). Bax expression was lower in Group I than in Group II. Immunoreactivity for caspase 3 did not differ significantly between the two groups (47.0 +/- 35.93 in Group I, 32.83 +/- 23.63 in Group II). Our results indicate that gelofusine did not protect the liver tissue against ischaemia-reperfusion-induced apoptosis.

  9. Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

    PubMed

    Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z; Wickrema, Amittha; Yang, Jing; Ji, Peng

    2016-03-01

    Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice.

  10. Sca-1(+) mesenchymal stromal cells inhibit splenic marginal zone B lymphocytes commitment through Caspase-3.

    PubMed

    Chen, Yaozhen; Yang, Jialei; Zhang, Hui-Jie; Fan, Hong; An, Ning; Xin, Jiajia; Li, Na; Xu, Jinmei; Yin, Wen; Wu, Zhongliang; Hu, Xingbin

    2016-05-01

    Mesenchymal stromal cells (MSCs) have been characterized as an important component of hematopoietic niche, which are capable of modulating the immune system through interaction with a wide range of immune cells. Marginal zone B cells, one main type of mature B lymphocytes, play a central role in eliciting antibody response against pathogens. However, how MSCs and its subpopulations regulate marginal zone B cells commitment is unknown yet. In this study, we assessed the contribution of Sca-1(+) MSCs on marginal zone B cells commitment. Our results showed that Sca-1(+) MSCs inhibit the commitment of marginal zone B lymphocytes. The inhibition was exerted through lowered Caspase-3 expression. Furthermore, we found marginal zone B lymphocytes in spleen of Caspase-3 knockout mice decreased and Caspase-3 knockout Sca-1(+) MSCs accounted for the MZB lymphocytes decrease. In conclusion, our investigation provided clues about Sca-1(+) MSCs regulation on the commitment of marginal zone B cells through Caspase-3 gene.

  11. Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge.

    PubMed

    Norden, Diana M; Trojanowski, Paige J; Villanueva, Emmanuel; Navarro, Elisa; Godbout, Jonathan P

    2016-02-01

    Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2-12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2-4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba-1(+)) and astrocytes (GFAP(+)), however, were undetected during this 2-12 h timeframe. Increased Iba-1 immunoreactivity and de-ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba-1 or GFAP immunoreactivity after LPS challenge.

  12. Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling.

    SciTech Connect

    Fang, Bin; Fu, Guoxing; Agniswamy, Johnson; Harrison, Robert W.; Weber, Irene T.

    2009-03-31

    Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9-2.6 {angstrom}. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.

  13. miR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells.

    PubMed

    Xu, Bo; Mao, Zhilei; Ji, Xiaoli; Yao, Mengmeng; Chen, Minjian; Zhang, Xuemei; Hang, Bo; Liu, Yi; Tang, Wei; Tang, Qiusha; Xia, Yankai

    2015-01-01

    Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect. PMID:26263183

  14. miR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells

    PubMed Central

    Xu, Bo; Mao, Zhilei; Ji, Xiaoli; Yao, Mengmeng; Chen, Minjian; Zhang, Xuemei; Hang, Bo; Liu, Yi; Tang, Wei; Tang, Qiusha; Xia, Yankai

    2015-01-01

    Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect. PMID:26263183

  15. miR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells

    NASA Astrophysics Data System (ADS)

    Xu, Bo; Mao, Zhilei; Ji, Xiaoli; Yao, Mengmeng; Chen, Minjian; Zhang, Xuemei; Hang, Bo; Liu, Yi; Tang, Wei; Tang, Qiusha; Xia, Yankai

    2015-08-01

    Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect.

  16. Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

    PubMed Central

    Wu, Yi-Feng; Cao, Ming-Fu; Gao, Yan-Ping; Chen, Fei; Wang, Tao; Zumbika, Edward P.; Qian, Kai-Xian

    2004-01-01

    AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7, -9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event. PMID:15378770

  17. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-05-11

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis.

  18. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis. PMID:27187358

  19. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional “repair and survive, or die” hypothesis. PMID:27187358

  20. Prenatal ethanol exposure alters ethanol-induced Fos immunoreactivity and dopaminergic activity in the mesocorticolimbic pathway of the adolescent brain.

    PubMed

    Fabio, M C; Vivas, L M; Pautassi, R M

    2015-08-20

    Prenatal ethanol exposure (PEE) promotes alcohol intake during adolescence, as shown in clinical and pre-clinical animal models. The mechanisms underlying this effect of prenatal ethanol exposure on postnatal ethanol intake remain, however, mostly unknown. Few studies assessed the effects of moderate doses of prenatal ethanol on spontaneous and ethanol-induced brain activity on adolescence. This study measured, in adolescent (female) Wistar rats prenatally exposed to ethanol (0.0 or 2.0g/kg/day, gestational days 17-20) or non-manipulated (NM group) throughout pregnancy, baseline and ethanol-induced cathecolaminergic activity (i.e., colocalization of c-Fos and tyrosine hydroxylase) in ventral tegmental area (VTA), and baseline and ethanol-induced Fos immunoreactivity (ir) in nucleus accumbens shell and core (AcbSh and AcbC, respectively) and prelimbic (PrL) and infralimbic (IL) prefrontal cortex. The rats were challenged with ethanol (dose: 0.0, 1.25, 2.5 or 3.25g/kg, i.p.) at postnatal day 37. Rats exposed to vehicle prenatally (VE group) exhibited reduced baseline dopaminergic tone in VTA; an effect that was inhibited by prenatal ethanol exposure (PEE group). Dopaminergic activity in VTA after the postnatal ethanol challenge was greater in PEE than in VE or NM animals. Ethanol-induced Fos-ir at AcbSh was found after 1.25g/kg and 2.5g/kg ethanol, in VE and PEE rats, respectively. PEE did not alter ethanol-induced Fos-ir at IL but reduced ethanol-induced Fos-ir at PrL. These results suggest that prenatal ethanol exposure heightens dopaminergic activity in the VTA and alters the response of the mesocorticolimbic pathway to postnatal ethanol exposure. These effects may underlie the enhanced vulnerability to develop alcohol-use disorders of adolescents with a history of in utero ethanol exposure.

  1. Determination of Diamine Oxidase in Lentil Seedlings by Enzymic Activity and Immunoreactivity

    PubMed Central

    Federico, Rodolfo; Angelini, Riccardo; Cesta, Alberinda; Pini, Carlo

    1985-01-01

    A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified 125I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor. PMID:16664402

  2. α6 Integrin Transactivates Insulin-like Growth Factor Receptor-1 (IGF-1R) to Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation*

    PubMed Central

    Basu, Subhasree; Rajakaruna, Suren; De Arcangelis, Adèle; Zhang, Liping; Georges-Labouesse, Elisabeth; Menko, A. Sue

    2014-01-01

    The canonical mitochondrial death pathway was first discovered for its role in signaling apoptosis. It has since been found to have a requisite function in differentiation initiation in many cell types including the lens through low level activation of the caspase-3 protease. The ability of this pathway to function as a molecular switch in lens differentiation depends on the concurrent induction of survival molecules in the Bcl-2 and IAP families, induced downstream of an IGF-1R/NFκB coordinate survival signal, to regulate caspase-3 activity. Here we investigated whether α6 integrin signals upstream to this IGF-1R-mediated survival-linked differentiation signal. Our findings show that IGF-1R is recruited to and activated specifically in α6 integrin receptor signaling complexes in the lens equatorial region, where lens epithelial cells initiate their differentiation program. In studies with both α6 integrin knock-out mice lenses and primary lens cell cultures following α6 integrin siRNA knockdown, we show that IGF-1R activation is dependent on α6 integrin and that this transactivation requires Src kinase activity. In addition, without α6 integrin, activation and expression of NFκB was diminished, and expression of Bcl-2 and IAP family members were down-regulated, resulting in high levels of caspase-3 activation. As a result, a number of hallmarks of lens differentiation failed to be induced; including nuclear translocation of Prox1 in the differentiation initiation zone and apoptosis was promoted. We conclude that α6 integrin is an essential upstream regulator of the IGF-1R survival pathway that regulates the activity level of caspase-3 for it to signal differentiation initiation of lens epithelial cells. PMID:24381169

  3. PCNA immunoreactivity revealing normal proliferative activity in the brain of an adult Elasmobranch, Torpedo marmorata.

    PubMed

    Margotta, Vito

    2007-01-01

    The brain of adult heterothermic vertebrates can be already provided of quiescent cells, scattered ("matrix cells") and/or clustered ("matrix areas"). These typical cells, in some regions located at or near ventricular surfaces and at peri-ependymal layers, in other territories populating their framework, maintain some embryonic properties and are responsible of normal or variously experimentally induced proliferative activities. On these topics there are a great number of reports concerning Teleostean Osteichthyes, Urodele and Anuran Amphibians, Lacertilian Reptiles. At the contrary, only few are the contributions regarding the Petromyzontidae. Involving an immunocytochemical marker, the Proliferating Cell Nuclear Antigen (PCNA), revealing proliferative events, in the last years we have undertaken a reappraisal focused on these encephalic performances in normal adult poikilothermal vertebrates. To provide a valid comparison between our results and the literature data, our choice of the specimens was based on the desire to employ organisms belonging to the same or phylogenetically close species used by previous Authors in similar studies. In our immunocytochemical panorama there is a substantial agreement between our contributions and bibliographic references concerning natural encephalic proliferative phenomena in these vertebrates. At this point of our study, the last missing piece was represented by the Chondrichthyes about which the literature data are lacking. In order to fill this gap, the aim of the present research is to investigate, involving the same PCNA test, whether proliferative events also persist in the brain of adult cartilaginous fishes. The immunostaining images obtained in the Elasmo branch Torpedo marmorata, well-known for the emission of high electrical discharges, exhibit undifferentiated cells in relationship with the ependymal epithelium lining the cavities of all cerebral districts; some other neuroblasts are scattered in the mesencephalic

  4. XIAP immunoreactivity in glial and neuronal cytoplasmic inclusions in multiple system atrophy.

    PubMed

    Kawamoto, Yasuhiro; Ito, Hidefumi; Ihara, Masafumi; Takahashi, Ryosuke

    2014-01-01

    X-linked inhibitor of apoptosis protein (XIAP) selectively binds to caspases-3, -7 and -9, and inhibits the activities of these caspases. To elucidate the role of XIAP in patients with multiple system atrophy (MSA), we performed immunohistochemical studies on XIAP in formalin-fixed, paraffin-embedded sections from 8 normal subjects and 10 patients with MSA. In normal brains, several types of neurons were immunostained for XIAP, and XIAP-immunopositive oligodendrocytes were scattered throughout the cerebral and cerebellar white matter. In the MSA brains, neuronal XIAP immunoreactivity was spared even in the severely-affected lesions, and glial cytoplasmic inclusions (GCIs), neuronal cytoplasmic inclusions (NCIs) and dystrophic neurites were all intensely immunoreactive for XIAP. A semiquantitative analysis of mid-pons sections double-immunostained for XIAP and α-synuclein demonstrated that the average percentages of XIAP-immunopositive GCIs and NCIs in the pontine nucleus were 70.2% and 82.2%, respectively. Our results suggest that a widespread accumulation of XIAP may occur in brains with MSA, and that XIAP may be partially associated with the pathogenesis of MSA. PMID:23993308

  5. Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis-induced salivary gland acinar cell apoptosis.

    PubMed

    Slomiany, B L; Slomiany, A

    2010-06-01

    Recent advances in identifying the salivary constituents capable of influencing the oral mucosal inflammatory responses have brought to focus the importance of a peptide hormone, ghrelin. Here, we report on the involvement of ghrelin in controlling the apoptotic processes induced in sublingual salivary gland acinar cells by the lipopolysaccharide (LPS) of a periodontopathic bacterium, Porphyromonas gingivalis. We show that the countering effect of ghrelin on the LPS-induced acinar cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (iNOS). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, but not the iNOS inhibitor, 1400W. The effect of ghrelin on the LPS-induced changes in cNOS activity, moreover, was reflected in the increased cNOS phosphorylation that was sensitive to PP2 as well as SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by SH-5 and L-NAME. The findings point to the involvement of ghrelin in Src/Akt kinase-mediated cNOS activation and the apoptogenic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

  6. Post-immunization immunohistochemical expression of Caspase 3 and p53 apoptotic markers in experimental hydatidosis.

    PubMed

    El-Aal, Amany Ahmed Abd; El-Gebaly, Naglaa Saad Mahmoud; Al-Antably, Abeer Said; Hassan, Marwa Adel; El-Dardiry, Marwa Ahmed

    2016-01-01

    The aim of this study was to investigate post-immunization apoptotic changes in experimental hydatidosis, using Caspase 3 and p53 immunohistochemical markers. Two groups of rabbits were immunized with a crude antigen (group 1) or a partially purified antigen (group 2) and were compared to an infected non-immunized control group. More effective immune responses were obtained in group 2 than group 1, signified by fewer and smaller cystic lesions and more severe destructive changes. Normal growth of cysts was attained in the control group, with no expression of apoptotic markers. Significantly higher expression of Caspase 3 and p53 were observed in group 1 compared to group 2, as indicated by OD and area percentage, respectively (Group 1 Caspase 3: 0.89±0.21, 93.5%±6.2; Group 1 p53: 0.46±0.18, 53.26%±11.6; Group 2 Caspase 3: 0.52±0.15, 49.23%±11.7; Group 2 p53: 0.19±0.4, 18.17%±7.3). Vaccine-induced immune responses and cellular damage may underlie the expression of apoptotic markers that appeared to result in a degenerative and atrophic course of action upon immunization. The results of the current study emphasize the importance of immunization for the stimulation of protective immune responses and in preventing mechanisms of evasion to ensure normal cell growth. A cost/benefit control program that implements proper vaccine preparations should be further assessed for complete elimination of severe infections in endemic areas. PMID:27683842

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

    PubMed Central

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

    2016-01-01

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

  8. Isatin sulfonamides: potent caspases-3 and -7 inhibitors, and promising PET and SPECT radiotracers for apoptosis imaging.

    PubMed

    Limpachayaporn, Panupun; Schäfers, Michael; Haufe, Günter

    2015-01-01

    Caspases-3 and -7 play an essential role in apoptosis. Isatin sulfonamides have been identified as potent inhibitors of these executing caspases. Besides pharmacological application, these compounds can also serve as recognition units to target caspases using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) when labeled with a positron or a gamma emitter. Fluorinated, alkylated, arylated isatin derivatives, in addition to derivatives modified with heterocycles, have been prepared in order to improve their binding potency, selectivity and metabolic stability. Structural optimization has led to stable, highly active inhibitors, which after labeling have been applied in PET studies in tumor mouse models and for first preclinical and clinical investigations with healthy human volunteers. The results support further development of such radiotracers for clinical apoptosis imaging. PMID:26132525

  9. Enhanced cognitive activity--over and above social or physical activity--is required to protect Alzheimer's mice against cognitive impairment, reduce Abeta deposition, and increase synaptic immunoreactivity.

    PubMed

    Cracchiolo, Jennifer R; Mori, Takashi; Nazian, Stanley J; Tan, Jun; Potter, Huntington; Arendash, Gary W

    2007-10-01

    Although social, physical, and cognitive activities have each been suggested to reduce the risk of Alzheimer's disease (AD), epidemiologic studies cannot determine which activity or combination of activities is most important. To address this question, mutant APP transgenic AD mice were reared long-term in one of four housing conditions (impoverished, social, social+physical, or complete enrichment) from 1(1/2) through 9 months of age. Thus, a stepwise layering of social, physical, and enhanced cognitive activity was created. Behavioral evaluation in a full battery of sensorimotor, anxiety, and cognitive tasks was carried out during the final 5 weeks of housing. Only AD mice raised in complete enrichment (i.e., enhanced cognitive activity) showed: (1) protection against cognitive impairment, (2) decreased brain beta-amyloid deposition, and (3) increased hippocampal synaptic immunoreactivity. The protection provided by enhanced cognitive activity spanned multiple cognitive domains (working memory, reference learning, and recognition/identification). Cognitive and neurohistologic benefits of complete enrichment occurred without any changes in blood cytokine or corticosterone levels, suggesting that enrichment-dependent mechanisms do not involve changes in the inflammatory response or stress levels, respectively. These results indicate that the enhanced cognitive activity of complete enrichment is required for cognitive and neurologic benefit to AD mice-physical and/or social activity are insufficient. Thus, our data suggest that humans who emphasize a high lifelong level of cognitive activity (over and above social and physical activities) will attain the maximal environmental protection against AD.

  10. Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms

    PubMed Central

    Zhang, Juan; Diao, Hua; Li, Guangming; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wenying; Ma, Jia-Li; Yu, Heguo; Wang, Yu-Gang

    2016-01-01

    Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms. PMID:26980707

  11. Gray matter oligodendrocyte progenitors and neurons die caspase-3 mediated deaths subsequent to mild perinatal hypoxic/ischemic insults.

    PubMed

    Rothstein, Raymond P; Levison, Steven W

    2005-01-01

    With significant improvements in neonatal care, fewer infants sustain severe injury as a consequence of hypoxia/ischemia (H/I). However, the majority of experimental studies have inflicted moderate to severe injuries, or they have assessed damage to the caudal forebrain; therefore, to better understand how a mild H/I episode affects the structures and cells of the rostral forebrain, we assessed the relative vulnerabilities of cells in the neocortex, striatum, corpus callosum, choroid plexus and subventricular zone (SVZ). To inflict mild H/I injury, the right common carotid artery was ligated followed by 1 h of hypoxia (8% O(2)) at 37 degrees C. Regional vulnerabilities were assessed using TUNEL, active caspase-3 and hematoxylin and eosin staining at 24 and 48 h of recovery. Scattered columns of cell death were observed in the neocortex with deep-layer neurons more vulnerable than more superficial neurons. The majority of these dying neurons appeared to be dying apoptotic rather than necrotic deaths. In addition, approximately 1/3 of the apoptotic cells in the neocortex were O4+ oligodendrocyte progenitors. We also observed a decrease in NG2 staining within the affected regions of the forebrain. By contrast, active caspase-3+/S-100beta+ astrocytes were not observed. Neurons and O4+ oligodendrocyte progenitors also died apoptotic deaths within the striatum. The lining cells of the choroid plexus also sustained damage. Elevated numbers of apoptotic cells were observed in the most lateral region of the SVZ and some of these dying cells were O4+. The most novel finding of this study, that oligodendrocyte progenitors in the gray matter are damaged and eliminated as a consequence of perinatal H/I, provides new insights into the histopathology and neurological deficits observed in infants who sustain mild H/I brain injuries.

  12. Glutamine supplementation prevents exercise-induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression.

    PubMed

    Lagranha, Claudia J; Hirabara, Sandro M; Curi, Rui; Pithon-Curi, Tania C

    2007-01-01

    We have previously shown that a single session of exercise induces DNA fragmentation, mitochondrial membrane depolarization, increases expression of pro-apoptotic genes (bax and bcl-xS) and decreases expression of anti-apoptotic genes (bcl-xL) in rat neutrophils. Glutamine supplementation had a protective effect in the apoptosis induced by a single session of exercise. The mechanism involved in the effect of single session of exercise to induce apoptosis was investigated by measuring expression of p53 and caspase 3 and phosphorylation of p38 mitogen-activated protein kinases (MAPK) and cJun NH(2)-terminal kinase (JNK) in neutrophils from rats supplemented or not with glutamine. Exercise was carried out on a treadmill for 1 h and the rats were killed by decapitation. Neutrophils were obtained by intraperitoneal (i.p.) lavage with PBS, 4 h after injection of oyster glycogen solution. Glutamine supplementation (1g per Kg b.w.) was given by gavage 1 h before the exercise session. Gene expression and protein phosphorylation were then analyzed by reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. A single session of exercise increased p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression. Glutamine supplementation partially prevented the increase in p38 MAPK and JNK phosphorylation and p53 expression, and fully abolished the increase in caspase 3 expression. Thus, neutrophil apoptosis induced by a single session of exercise is accompanied by increased p53 and caspase 3 expression and p38 MAPK and JNK phosphorylation. Glutamine supplementation prevents these effects of exercise and reduces apoptosis. PMID:17542038

  13. Glutamine supplementation prevents exercise-induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression.

    PubMed

    Lagranha, Claudia J; Hirabara, Sandro M; Curi, Rui; Pithon-Curi, Tania C

    2007-01-01

    We have previously shown that a single session of exercise induces DNA fragmentation, mitochondrial membrane depolarization, increases expression of pro-apoptotic genes (bax and bcl-xS) and decreases expression of anti-apoptotic genes (bcl-xL) in rat neutrophils. Glutamine supplementation had a protective effect in the apoptosis induced by a single session of exercise. The mechanism involved in the effect of single session of exercise to induce apoptosis was investigated by measuring expression of p53 and caspase 3 and phosphorylation of p38 mitogen-activated protein kinases (MAPK) and cJun NH(2)-terminal kinase (JNK) in neutrophils from rats supplemented or not with glutamine. Exercise was carried out on a treadmill for 1 h and the rats were killed by decapitation. Neutrophils were obtained by intraperitoneal (i.p.) lavage with PBS, 4 h after injection of oyster glycogen solution. Glutamine supplementation (1g per Kg b.w.) was given by gavage 1 h before the exercise session. Gene expression and protein phosphorylation were then analyzed by reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. A single session of exercise increased p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression. Glutamine supplementation partially prevented the increase in p38 MAPK and JNK phosphorylation and p53 expression, and fully abolished the increase in caspase 3 expression. Thus, neutrophil apoptosis induced by a single session of exercise is accompanied by increased p53 and caspase 3 expression and p38 MAPK and JNK phosphorylation. Glutamine supplementation prevents these effects of exercise and reduces apoptosis.

  14. Brain caspase-3 and intestinal FABP responses in preterm and term rats submitted to birth asphyxia

    PubMed Central

    Figueira, R.L.; Gonçalves, F.L.; Simões, A.L.; Bernardino, C.A.; Lopes, L.S.; Castro e Silva, O.; Sbragia, L.

    2016-01-01

    Neonatal asphyxia can cause irreversible injury of multiple organs resulting in hypoxic-ischemic encephalopathy and necrotizing enterocolitis (NEC). This injury is dependent on time, severity, and gestational age, once the preterm babies need ventilator support. Our aim was to assess the different brain and intestinal effects of ischemia and reperfusion in neonate rats after birth anoxia and mechanical ventilation. Preterm and term neonates were divided into 8 subgroups (n=12/group): 1) preterm control (PTC), 2) preterm ventilated (PTV), 3) preterm asphyxiated (PTA), 4) preterm asphyxiated and ventilated (PTAV), 5) term control (TC), 6) term ventilated (TV), 7) term asphyxiated (TA), and 8) term asphyxiated and ventilated (TAV). We measured body, brain, and intestine weights and respective ratios [(BW), (BrW), (IW), (BrW/BW) and (IW/BW)]. Histology analysis and damage grading were performed in the brain (cortex/hippocampus) and intestine (jejunum/ileum) tissues, as well as immunohistochemistry analysis for caspase-3 and intestinal fatty acid-binding protein (I-FABP). IW was lower in the TA than in the other terms (P<0.05), and the IW/BW ratio was lower in the TA than in the TAV (P<0.005). PTA, PTAV and TA presented high levels of brain damage. In histological intestinal analysis, PTAV and TAV had higher scores than the other groups. Caspase-3 was higher in PTAV (cortex) and TA (cortex/hippocampus) (P<0.005). I-FABP was higher in PTAV (P<0.005) and TA (ileum) (P<0.05). I-FABP expression was increased in PTAV subgroup (P<0.0001). Brain and intestinal responses in neonatal rats caused by neonatal asphyxia, with or without mechanical ventilation, varied with gestational age, with increased expression of caspase-3 and I-FABP biomarkers. PMID:27356106

  15. Brain caspase-3 and intestinal FABP responses in preterm and term rats submitted to birth asphyxia.

    PubMed

    Figueira, R L; Gonçalves, F L; Simões, A L; Bernardino, C A; Lopes, L S; Castro E Silva, O; Sbragia, L

    2016-06-23

    Neonatal asphyxia can cause irreversible injury of multiple organs resulting in hypoxic-ischemic encephalopathy and necrotizing enterocolitis (NEC). This injury is dependent on time, severity, and gestational age, once the preterm babies need ventilator support. Our aim was to assess the different brain and intestinal effects of ischemia and reperfusion in neonate rats after birth anoxia and mechanical ventilation. Preterm and term neonates were divided into 8 subgroups (n=12/group): 1) preterm control (PTC), 2) preterm ventilated (PTV), 3) preterm asphyxiated (PTA), 4) preterm asphyxiated and ventilated (PTAV), 5) term control (TC), 6) term ventilated (TV), 7) term asphyxiated (TA), and 8) term asphyxiated and ventilated (TAV). We measured body, brain, and intestine weights and respective ratios [(BW), (BrW), (IW), (BrW/BW) and (IW/BW)]. Histology analysis and damage grading were performed in the brain (cortex/hippocampus) and intestine (jejunum/ileum) tissues, as well as immunohistochemistry analysis for caspase-3 and intestinal fatty acid-binding protein (I-FABP). IW was lower in the TA than in the other terms (P<0.05), and the IW/BW ratio was lower in the TA than in the TAV (P<0.005). PTA, PTAV and TA presented high levels of brain damage. In histological intestinal analysis, PTAV and TAV had higher scores than the other groups. Caspase-3 was higher in PTAV (cortex) and TA (cortex/hippocampus) (P<0.005). I-FABP was higher in PTAV (P<0.005) and TA (ileum) (P<0.05). I-FABP expression was increased in PTAV subgroup (P<0.0001). Brain and intestinal responses in neonatal rats caused by neonatal asphyxia, with or without mechanical ventilation, varied with gestational age, with increased expression of caspase-3 and I-FABP biomarkers.

  16. Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer

    PubMed Central

    LEITE, Ana Flávia Schueler de Assumpção; BERNARDO, Vagner Gonçalves; BUEXM, Luisa Aguirre; da FONSECA, Eliene Carvalho; da SILVA, Licínio Esmeraldo; BARROSO, Danielle Resende Camisasca; LOURENÇO, Simone de Queiroz Chaves

    2016-01-01

    ABSTRACT Objective This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. Material and Methods A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. Results From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). Conclusions Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors. PMID:27556207

  17. Proteolytic cleavage of polymeric tau protein by caspase-3: implications for Alzheimer disease.

    PubMed

    Jarero-Basulto, Jose J; Luna-Muñoz, Jose; Mena, Raul; Kristofikova, Zdena; Ripova, Daniela; Perry, George; Binder, Lester I; Garcia-Sierra, Francisco

    2013-12-01

    Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.

  18. Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart

    PubMed Central

    Cardona, Maria; López, Juan Antonio; Serafín, Anna; Rongvaux, Anthony; Inserte, Javier; García-Dorado, David; Flavell, Richard; Llovera, Marta; Cañas, Xavier; Vázquez, Jesús; Sanchis, Daniel

    2015-01-01

    Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart’s cellularity and maturation during development, contributing novel information about caspase biology and heart development. PMID:26121671

  19. Growth inhibitory effect of KYKZL-1 on Hep G{sub 2} cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest

    SciTech Connect

    Cheng, Jing; Du, Yi-Fang; Xiao, Zhi-Yi; Pan, Li-Li; Li, Wei; Huan, Lin; Gong, Zhu-Nan; Wei, Shao-Hua; Huang, Shi-Qian; Xun, Wei; Zhang, Yi; Chang, Lei-Lei; Xie, Meng-Yu; Ao, Gui-Zhen; Cai, Jie; Qiu, Ting; Wu, Hao; Sun, Ting; Xu, Guang-Lin

    2014-01-01

    KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G{sub 2} growth. We found that KYKZL-1 inhibited the growth of Hep G{sub 2} cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE{sub 2} and LTB{sub 4} production in Hep G{sub 2} cells. Accordingly, exogenous addition of PGE{sub 2} or LTB{sub 4} reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S–G{sub 2} checkpoint via the activation of p21{sup CIP1} protein and down-regulation of cyclin A expression. These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest. Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile. - Highlights: • KYKZL-1 is designed to exhibit COX/5-LOX dual inhibition. • KYKZL-1 resulted in apoptosis of Hep G{sub 2} cells. • KYKZL-1 activated caspase-3 through cytochrome c and bcl-2/bax ratio. • KYKZL-1 caused cell cycle arrest via modulation of p21{sup CIP1} and cyclin A level.

  20. Garlic (Allium sativum) Fresh Juice Induces Apoptosis in Human Oral Squamous Cell Carcinoma: The Involvement of Caspase-3, Bax and Bcl-2

    PubMed Central

    Farhadi, Farrokh; Jahanpour, Salar; Hazem, Kameliya; Aghbali, Amirala; Baradran, Behzad; Vahid Pakdel, Seyyed Mahdi

    2015-01-01

    Background and aims. There is no report on the apoptotic impact of Allium sativum L.(Garlic) on the oral squamous cell carcinoma (KB); hence, this study was designed to survey the apoptotic effects of garlic fresh juice (GFJ) on the KB cells. Materials and methods. MTTassay (MicrocultureTetrazolium Assay) was carried out to evaluate the cytotoxicity of GFJ on KB cells. Furthermore, TUNEL(Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)and DNA fragmentation tests were performed to determine if GFJ is able to induce apoptosis in KB cells. Also a standard kit was used to assess caspase-3 activity in KB cells. Also western blotting was employed to evaluate the effect of GFJ on Bax:Bcl-2 ratio. Results. Significant cytotoxic effects were observed for the minimum used concentration (1μg/mL) as calculated to be 77.97±2.3% for 24 h and 818±3.1% for 36h of incubation (P < 0.001). Furthermore, TUNEL and DNA fragmentation tests corroborated the apoptosis inducing activity of GFJ. Consistently, after treating KB cells with GFJ(1μg/mL), caspase-3 activity and Bax:Bcl-2 ratio were raised by 7.3±0.6 and (P <0.001) folds, respectively. Conclusion. The results of this study advanced that GFJ induces apoptosis in the KB cells through increasing caspase-3 activity and Bax:Bcl2 ratio which could be attributed to its organo-sulfurcomponents. PMID:26889365

  1. Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

    PubMed

    Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

    2013-01-01

    Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

  2. Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging.

    PubMed

    Means, John C; Gerdes, Bryan C; Kaja, Simon; Sumien, Nathalie; Payne, Andrew J; Stark, Danny A; Borden, Priscilla K; Price, Jeffrey L; Koulen, Peter

    2016-09-01

    Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain. PMID:27220334

  3. Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

    PubMed Central

    Marissen, Wilfred E.; Lloyd, Richard E.

    1998-01-01

    Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity. PMID:9819442

  4. Differential proteomic analysis of lymphocytes treated with mycophenolic acid reveals caspase 3-induced cleavage of rho GDP dissociation inhibitor 2.

    PubMed

    Heller, Tanja; Asif, Abdul R; Petrova, Darinka Todorova; Doncheva, Yuliana; Wieland, E; Oellerich, Michael; Shipkova, Maria; Armstrong, Victor William

    2009-04-01

    The antiproliferative immunosuppressive drug mycophenolic acid (MPA) is an uncompetitive inhibitor of inosine monophosphate dehydrogenase, a key enzyme in de novo synthesis of purine nucleotides. The latter are not only required for synthesis of DNA and RNA but also are essential for the regulation of numerous cellular signaling pathways modulated by guanine nucleotide binding proteins (G proteins). We undertook an analysis of the influence of MPA on protein expression in a T-lymphoblast cell line (CCRF-CEM), which displays concentration-dependent inhibition of proliferation by MPA to obtain insight into the influence of MPA on the cellular proteome. Cells were stimulated with phorbol myristate acetate/ionomycin and incubated in the presence or absence of MPA. Two-dimensional electrophoresis and densitometric imaging revealed 11 differentially expressed protein spots (P < 0.05) on MPA treatment, 6 with increased and 5 with decreased abundance. After in-gel tryptic digestion, proteins were identified by quadrupole time-of-flight mass spectrometry. Proteins displaying increased abundance after MPA treatment included splicing factor arginine/serine-rich 2, prostaglandin E synthase 3, peptidyl-prolyl cis-trans isomerase A, and deoxyuridine 5'-triphosphate nucleotidohydrolase. Endoplasmin, proliferating cell nuclear antigen, acidic leucine-rich nuclear phosphoprotein 32 family member A, and cofilin 1 showed decreased abundance after MPA treatment. Three separate spots (1 decreased and 2 increased abundance) were identified as Rho guanosine diphosphate dissociation inhibitor 2 (Rho GDI 2) proteins. Western blotting with a monoclonal antibody directed against the Rho GDI 2 site cleaved by caspase 3 demonstrated 1 spot with increased abundance to be the caspase 3-cleaved product of Rho GDI 2 lacking the first 19 amino acids. Rho GDI 2 plays a central regulatory role in the activation of Rho guanosine triphosphatases that function as molecular switches in cell signaling

  5. Distribution of Fos-Like Immunoreactivity, Catecholaminergic and Serotoninergic Neurons Activated by the Laryngeal Chemoreflex in the Medulla Oblongata of Rats.

    PubMed

    Wang, Xiaolu; Guo, Ruichen; Zhao, Wenjing

    2015-01-01

    The laryngeal chemoreflex (LCR) induces apnea, glottis closure, bradycardia and hypertension in young and maturing mammals. We examined the distribution of medullary nuclei that are activated by the LCR and used immunofluorescent detection of Fos protein as a cellular marker for neuronal activation to establish that the medullary catecholaminergic and serotoninergic neurons participate in the modulation of the LCR. The LCR was elicited by the infusion of KCl-HCl solution into the laryngeal lumen of adult rats in the experimental group, whereas the control group received the same surgery but no infusion. In comparison, the number of regions of Fos-like immunoreactivity (FLI) that were activated by the LCR significantly increased in the nucleus of the solitary tract (NTS), the vestibular nuclear complex (VNC), the loose formation of the nucleus ambiguus (AmbL), the rostral ventral respiratory group (RVRG), the ventrolateral reticular complex (VLR), the pre-Bötzinger complex (PrBöt), the Bötzinger complex (Böt), the spinal trigeminal nucleus (SP5), and the raphe obscurus nucleus (ROb) bilaterally from the medulla oblongata. Furthermore, 12.71% of neurons with FLI in the dorsolateral part of the nucleus of the solitary tract (SolDL) showed tyrosine hydroxylase-immunoreactivity (TH-ir, catecholaminergic), and 70.87% of neurons with FLI in the ROb were serotoninergic. Our data demonstrated the distribution of medullary nuclei that were activated by the LCR, and further demonstrated that catecholaminergic neurons of the SolDL and serotoninergic neurons of the ROb were activated by the LCR, indicating the potential central pathway of the LCR. PMID:26087133

  6. Chemotherapy resistance of mouse WAP-SVT/t breast cancer cells is mediated by osteopontin, inhibiting apoptosis downstream of caspase-3.

    PubMed

    Graessmann, M; Berg, B; Fuchs, B; Klein, A; Graessmann, A

    2007-05-01

    Impairment of the complex regulatory network of cell death and survival is frequently the reason for therapy resistance of breast cancer cells and a major cause of tumor progression. We established two independent cell lines from a fast growing mouse breast tumor (WAP-SVT/t transgenic animal). Cells from one line (ME-A cells) are sensitive to apoptotic stimuli such as growth factor depletion or treatment with antitumor agents (e.g. doxorubicin). Cells from the second line (ME-C cells), which carry a missense mutation at the p53 codon 242, are very insensitive to apoptotic stimuli. Co-cultivation experiments revealed that the ME-C cells mediate cell death resistance to the ME-A cells. Microarray and Western blot analysis showed that osteopontin (OPN) is selectively overexpressed by the ME-C cells. This glycoprotein is the most abundant protein secreted by the ME-C cells and we obtained strong indications that OPN is the main antiapoptotic factor. However, the OPN containing ME-C cell medium does not alter the expression level of pro- or antiapoptotic genes or known inhibitors of apoptosis (IAPs). Its signaling involves mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)1/2 as the kinase inhibitor PD98059 restores apoptosis but not the Akt inhibitor. In the ME-A cells, mitochondrial cytochrome c release occurs with and without external apoptotic stimuli. OPN containing ME-C cell medium does not prevent the mitochondrial cytochrome c release and caspase-9 processing. In serum starved ME-A cells, the OPN containing ME-C cell medium prevents caspase-3 activation. However, in doxorubicin-treated cells, although apoptosis is blocked, it does not inhibit caspase-3. This indicates that the ME-A cells distinguish between the initial apoptotic stimuli and that the cells possess a further uncharacterized control element acting downstream from caspase-3. PMID:17160024

  7. Implication of Caspase-3 as a Common Therapeutic Target for Multineurodegenerative Disorders and Its Inhibition Using Nonpeptidyl Natural Compounds

    PubMed Central

    Khan, Saif; Ahmad, Khurshid; Alshammari, Eyad M. A.; Adnan, Mohd; Baig, Mohd Hassan; Lohani, Mohtashim; Haque, Shafiul

    2015-01-01

    Caspase-3 has been identified as a key mediator of neuronal apoptosis. The present study identifies caspase-3 as a common player involved in the regulation of multineurodegenerative disorders, namely, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). The protein interaction network prepared using STRING database provides a strong evidence of caspase-3 interactions with the metabolic cascade of the said multineurodegenerative disorders, thus characterizing it as a potential therapeutic target for multiple neurodegenerative disorders. In silico molecular docking of selected nonpeptidyl natural compounds against caspase-3 exposed potent leads against this common therapeutic target. Rosmarinic acid and curcumin proved to be the most promising ligands (leads) mimicking the inhibitory action of peptidyl inhibitors with the highest Gold fitness scores 57.38 and 53.51, respectively. These results were in close agreement with the fitness score predicted using X-score, a consensus based scoring function to calculate the binding affinity. Nonpeptidyl inhibitors of caspase-3 identified in the present study expeditiously mimic the inhibitory action of the previously identified peptidyl inhibitors. Since, nonpeptidyl inhibitors are preferred drug candidates, hence, discovery of natural compounds as nonpeptidyl inhibitors is a significant transition towards feasible drug development for neurodegenerative disorders. PMID:26064904

  8. Effects of met-enkephalin on the mechanical activity and distribution of met-enkephalin-like immunoreactivity in the cat small intestine.

    PubMed

    Radomirov, R; Venkova, K; Davidoff, M; Pencheva, N

    1990-01-01

    Naloxone-dependent effects of Met-enkephalin (10(-8) M) on the spontaneous and electrically induced mechanical activities were studied in longitudinal and circular preparations isolated from the cat duodenum, jejunum and ileum. Met-Enkephalin changed the spontaneous activity of all preparations tested with the exception of the circular preparations from the ileum. Met-Enkephalin-induced responses of the longitudinal preparations from the ileum were abolished by treatment with tetrodotoxin (10(-7) M), while the responses of both longitudinal and circular preparations from the duodenum and jejunum were only partially depressed, being resistant to tetrodotoxin components. The latter were most pronounced in the duodenum. The neurogenic electrically induced (0.5 msec, 5 Hz, 150 pulses) responses of all the preparations consisted mainly of contractile components which were significantly and naloxone-dependently reduced by Met-enkephalin (10(-8) M). The contractile components of the responses, which were reduced by Met-enkephalin, were entirely abolished by atropine (3 x 10(-6) M). Both Met-enkephalin and atropine inhibitory effects on the neurogenic responses were more pronounced in the ileum. Met-Enkephalin was found in nerve fibers of the myenteric plexus distributed mainly among the circular muscle. Single immunoreactive nerve fibers were observed in the longitudinal muscle layer of the duodenum but not in the jejunum and ileum. The distribution of Met-enkephalin-like immunoreactivity along the small intestine did not show significant differences among the three intestinal regions tested. The results obtained suggest that Met-enkephalin can modulate the mechanical activity of the cat small intestine, inhibiting cholinergic transmission and/or activating smooth muscle opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Bilberry extract (Antho 50) selectively induces redox-sensitive caspase 3-related apoptosis in chronic lymphocytic leukemia cells by targeting the Bcl-2/Bad pathway

    PubMed Central

    Alhosin, Mahmoud; León-González, Antonio J.; Dandache, Israa; Lelay, Agnès; Rashid, Sherzad K.; Kevers, Claire; Pincemail, Joël; Fornecker, Luc-Matthieu; Mauvieux, Laurent; Herbrecht, Raoul; Schini-Kerth, Valérie B.

    2015-01-01

    Defect in apoptosis has been implicated as a major cause of resistance to chemotherapy observed in B cell chronic lymphocytic leukaemia (B CLL). This study evaluated the pro-apoptotic effect of an anthocyanin-rich dietary bilberry extract (Antho 50) on B CLL cells from 30 patients and on peripheral blood mononuclear cells (PBMCs) from healthy subjects, and determined the underlying mechanism. Antho 50 induced concentration- and time-dependent pro-apoptotic effects in B CLL cells but little or no effect in PBMCs. Among the main phenolic compounds of the bilberry extract, delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside induced a pro-apoptotic effect. Antho 50-induced apoptosis is associated with activation of caspase 3, down-regulation of UHRF1, a rapid dephosphorylation of Akt and Bad, and down-regulation of Bcl-2. Antho 50 significantly induced PEG-catalase-sensitive formation of reactive oxygen species in B CLL cells. PEG-catalase prevented the Antho 50-induced induction of apoptosis and related signaling. The present findings indicate that Antho 50 exhibits strong pro-apoptotic activity through redox-sensitive caspase 3 activation-related mechanism in B CLL cells involving dysregulation of the Bad/Bcl-2 pathway. This activity of Antho 50 involves the glucoside and rutinoside derivatives of delphinidin. They further suggest that Antho 50 has chemotherapeutic potential by targeting selectively B CLL cells. PMID:25757575

  10. Morus alba Accumulates Reactive Oxygen Species to Initiate Apoptosis via FOXO-Caspase 3-Dependent Pathway in Neuroblastoma Cells.

    PubMed

    Kwon, Young Hwi; Bishayee, Kausik; Rahman, Ataur; Hong, Jae Seung; Lim, Soon-Sung; Huh, Sung-Oh

    2015-07-01

    Morus alba root extract (MARE) has been used to treat hyperglycaemic conditions in oriental medicine. Here, we studied whether MARE possesses a cytotoxic effect on neuroblastoma. To check the cytotoxicity generated by MARE was whether relatively higher against the cancer cells rather than normal cells, we chose a neuroblastoma cell line (B103) and a normal cell line (Rat-2). A CCK assay revealed that MARE (10 μg/ml) reduced cell viability to approximately 60% compared to an untreated control in B103 cells. But in Rat-2 cells, MARE induced relatively lower cytotoxicity. To investigate the mechanisms underlying the cytotoxic effect of MARE, we used flow cytometry combined with immunoblot analyses. We found that MARE-treatment could accumulate ROS and depolarize mitochondria membrane potential of B103 cells. Further treatment with MARE in B103 cells also could damage DNA and induce apoptosis. An expression study of p-Akt also suggested that there was a reduction in cellular proliferation and transcription along with the process of apoptosis, which was further evidenced by an increase in Bax and cleaved-caspase 3 activity. Together, our findings suggest that MARE produces more cytotoxicity in cancer cells while having a relatively attenuated effect on normal cells. As such, MARE may be a safer option in cancer therapeutics, and it also shows potential for the patients with symptoms of hyperglycemia and cancer.

  11. The Apoptotic Function Analysis of p53, Apaf1, Caspase3 and Caspase7 during the Spermatogenesis of the Chinese Fire-Bellied Newt Cynops orientalis

    PubMed Central

    Wang, Li-Ya; Hu, Yan-Jun; Tan, Fu-Qing; Zhou, Hong; Shao, Jian-Zhong; Yang, Wan-Xi

    2012-01-01

    Background Spontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure. Methodology/Principal Findings TUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h), cold exposure(4°C 12 h), cadmium exposure(Cd 36 h), and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8,-9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h), cold exposure(4°C 12 h), and cadmium exposure(Cd 36 h), while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment. Conclusions/Significance Severe germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of spermatid and sperm

  12. The association between splenocyte apoptosis and alterations of Bax, Bcl-2 and caspase-3 mRNA expression, and oxidative stress induced by dietary nickel chloride in broilers.

    PubMed

    Huang, Jianying; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wu, Bangyuan

    2013-12-01

    Two hundred and forty avian broilers were equally divided into four groups, and raised with a corn-soybean basal diet or the same diet supplemented with 300, 600, 900 mg/kg NiCl2 for 42 days. Numbers or percentages of apoptotic splenocytes by flow cytometry (FCM) and TUNEL were higher (p < 0.05 or p < 0.01) in the 300, 600 and 900 mg/kg groups than those in the control group. Results measured by qRT-PCR and ELISA showed that mRNA expression and contents were significantly higher (p < 0.05 or p < 0.01) in Bax and Caspase-3, and were significantly lower (p < 0.05 or p < 0.01) in Bcl-2 of the 300, 600 and 900 mg/kg groups. Also, the SOD, CAT and GSH-Px activities, and the ability to inhibit hydroxyl radical, and GSH contents were significantly decreased (p < 0.05 or p < 0.01), and MDA contents were increased (p < 0.05 or p < 0.01) in all groups. In conclusion, dietary NiCl2 in excess of 300 mg/kg caused apoptosis, altered Bax, Bcl-2 and Caspase-3 mRNA expression levels and contents, and induced oxidative stress in the spleen. Also, splenocyte apoptosis was closely related to the alternations of Bax, Bcl-2 and Caspase-3 mRNA expression, and oxidative damage. The splenic immunity and blood filtration functions were impaired in broilers. PMID:24351749

  13. Intravenous human umbilical cord blood transplantation for stroke: impact on infarct volume and caspase-3-dependent cell death in spontaneously hypertensive rats.

    PubMed

    Riegelsberger, Ute-Maria; Deten, Alexander; Pösel, Claudia; Zille, Marietta; Kranz, Alexander; Boltze, Johannes; Wagner, Daniel-Christoph

    2011-01-01

    Transplantation of human umbilical cord blood cells (HUCBC) produces reliable behavioral and morphological improvements in animal models of stroke. However, the mechanisms of action still have not been fully elucidated. The aim of the present study is the evaluation of potential neuroprotective effects produced by HUCBC in terms of reduced infarct volume and caspase-3-dependent cell death. Permanent middle cerebral artery occlusion was induced in 90 spontaneously hypertensive rats. The animals were randomly assigned to the control group (n=49) or the verum group (n=41). The cell suspension (8 × 10(6) HUCBC per kilogram bodyweight) or vehicle solution was intravenously administered 24h after stroke onset. Fifty subjects (n=25/25) were sacrificed after 25, 48, 72 and 96h, and brain specimens were removed for immunohistochemistry for MAP2, cleaved caspase-3 (casp3) and GFAP. Another 42 animals (n=26/16) were sacrificed after 0, 6, 24, 36 and 48h and their brains processed for quantitative PCR for casp3 and survivin. The infarct volume remained stable over the entire experimental period. However, cleaved casp3 activity increased significantly in the infarct border zone within the same time frame. Numerous cleaved casp3-positive cells were colocalized with the astrocytic marker GFAP, whereas cleavage of neuronal casp3 was observed rarely. Neither the infarct volume nor casp3 activity was significantly affected by cell transplantation. Delayed systemic transplantation of HUCBC failed to produce neuroprotective effects in a permanent stroke model using premorbid subjects.

  14. Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2 ratio and caspase-3-dependent pathway.

    PubMed

    Abarikwu, Sunny O; Farombi, Ebenezer O

    2015-02-01

    Atrazine (ATZ) is a well known herbicide that is frequently detected in ground and surface water at significant levels. Our objective was to study the toxic effect of ATZ on the human neuroblastoma (SH-SY5Y) cells, and the degree of cytotoxicity and morphological changes were followed during the cell death. Application of cytotoxicity bioassays indicates that ATZ (5-50 µg/mL) decreases cell viability in a dose- and time-dependent manner. The evidence of apoptosis was confirmed by an increase in caspase-3 activity, and cell death was blocked when caspase-3 activity was inhibited. Typical apoptotic phenotype that includes nuclear fragmentation, micro nuclei formation, DNA fragmentation and increase in the expressions apoptosis-associated markers Bax, p53 and p21 and decreased expression of Bcl-2 were observed in treated cells. We also observed dose-dependent increase in reactive oxygen species (ROS) levels in ATZ-treated cells. These results suggest that ATZ-induces apoptosis and ROS levels in SH-SY5Y cells, and could be implicated in human neurodegenerative disorder. PMID:25752436

  15. Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2 ratio and caspase-3-dependent pathway.

    PubMed

    Abarikwu, Sunny O; Farombi, Ebenezer O

    2015-02-01

    Atrazine (ATZ) is a well known herbicide that is frequently detected in ground and surface water at significant levels. Our objective was to study the toxic effect of ATZ on the human neuroblastoma (SH-SY5Y) cells, and the degree of cytotoxicity and morphological changes were followed during the cell death. Application of cytotoxicity bioassays indicates that ATZ (5-50 µg/mL) decreases cell viability in a dose- and time-dependent manner. The evidence of apoptosis was confirmed by an increase in caspase-3 activity, and cell death was blocked when caspase-3 activity was inhibited. Typical apoptotic phenotype that includes nuclear fragmentation, micro nuclei formation, DNA fragmentation and increase in the expressions apoptosis-associated markers Bax, p53 and p21 and decreased expression of Bcl-2 were observed in treated cells. We also observed dose-dependent increase in reactive oxygen species (ROS) levels in ATZ-treated cells. These results suggest that ATZ-induces apoptosis and ROS levels in SH-SY5Y cells, and could be implicated in human neurodegenerative disorder.

  16. Neuronal degeneration and a decrease in laminin-like immunoreactivity is associated with elevated tissue-type plasminogen activator in the rat hippocampus after kainic acid injection.

    PubMed

    Nagai, N; Urano, T; Endo, A; Takahashi, H; Takada, Y; Takada, A

    1999-02-01

    Tissue-type plasminogen activator (tPA) is a serine protease that converts the inactive precursor plasminogen to the active protease plasmin. In the central nervous system, tPA has been suggested to participate in plasticity, memory and the neuronal degeneration caused by excitotoxins, but its precise functions during these processes are still unclear. We show in this report that tPA antigen level and extracellular tPA activity increased in the hippocampus during the early stages of neuronal degeneration in the CA3 region following the injection of kainic acid (KA) into the lateral cerebral ventricles. The increase in tPA antigen level was transient and its peak was at 4 h after the injection. tPA activity was also increased 4 h after the injection, but it remained at a high level for more than 8 h. Histological zymography showed that the increase in tPA activity was mainly localized in the CA3 region. In the same region, the disappearance of interneuronal laminin-like immunoreactivity and atrophic changes in pyramidal neurons were observed 4 h after the injection of KA. These results suggested that such focal and transient increases in tPA synthesis and release, which result in the proteolysis of laminin through plasminogen activation, could be involved in the neuronal degeneration in the CA3 region after the injection of KA.

  17. PLGA-Carbon Nanotube Conjugates for Intercellular Delivery of Caspase-3 into Osteosarcoma Cells

    PubMed Central

    Cheng, Qingsu; Blais, Marc-Olivier; Harris, Greg; Jabbarzadeh, Ehsan

    2013-01-01

    Cancer has arisen to be of the most prominent health care issues across the world in recent years. Doctors have used physiological intervention as well as chemical and radioactive therapeutics to treat cancer thus far. As an alternative to current methods, gene delivery systems with high efficiency, specificity, and safety that can reduce side effects such as necrosis of tissue are under development. Although viral vectors are highly efficient, concerns have arisen from the fact that viral vectors are sourced from lethal diseases. With this in mind, rod shaped nano-materials such as carbon nanotubes (CNTs) have become an attractive option for drug delivery due to the enhanced permeability and retention effect in tumors as well as the ability to penetrate the cell membrane. Here, we successfully engineered poly (lactic-co-glycolic) (PLGA) functionalized CNTs to reduce toxicity concerns, provide attachment sites for pro-apoptotic protein caspase-3 (CP3), and tune the temporal release profile of CP3 within bone cancer cells. Our results showed that CP3 was able to attach to functionalized CNTs, forming CNT-PLGA-CP3 conjugates. We show this conjugate can efficiently transduce cells at dosages as low as 0.05 μg/ml and suppress cell proliferation up to a week with no further treatments. These results are essential to showing the capabilities of PLGA functionalized CNTs as a non-viral vector gene delivery technique to tune cell fate. PMID:24312611

  18. NADPH oxidase 3-associated oxidative stress and caspase 3-dependent apoptosis in the cochleae of D-galactose-induced aged rats

    PubMed Central

    DU, ZHENGDE; LI, SHUO; LIU, LIN; YANG, QIONG; ZHANG, HONGWEI; GAO, CHUNSHENG

    2015-01-01

    Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D-galactose (D-gal), and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D-gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D-gal for 8 weeks. In addition, the levels of 8-hydroxy-2-deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling-positive cells were increased in the cochleae of D-gal-treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3-dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age-associated hearing loss, also termed presbycusis. PMID:26498835

  19. Repeated administration of propofol upregulated the expression of c-Fos and cleaved-caspase-3 proteins in the developing mouse brain

    PubMed Central

    Cui, Yin; Ling-Shan, Gou; Yi, Liu; Xing-Qi, Wang; Xue-Mei, Zhuang; Xiao-Xing, Yin

    2011-01-01

    Objectives and Aim: This study was designed to analyze the relationship between the expression of c-Fos protein and apoptosis in the hippocampus following propofol administration in infant mice. There are reports that certain drugs, including the general anesthetics applied in pediatrics and obstetrics, could block N-methyl-D-aspartate glutamate receptors and activate γ-aminobutyric acid type A receptors. Furthermore, some anesthetics could trigger neuroapoptosis and the expression of c-Fos in the developing rodent brain. Propofol is a general anesthetic increasingly used in pediatrics and obstetrics, and is reported to be able to interact with both γ-aminobutyric acid type A and N-methyl-D-aspartate glutamate receptors. No adequate evaluations have been available as to whether the dosage of propofol to maintain anaesthesia could trigger the expression of c-Fos and apoptosis. Materials and Methods: Intraperitoneal injections of propofol (50, 100 and 150 mg/kg) or vehicle were administered every 90 minutes (4 times) in infant mice (5–7 days old). 30 minutes after the final administration, the protein expressions of c-Fos and cleaved-caspase-3 in the hippocampus were determined by immunohistochemistry and Western blotting. Results: It was demonstrated that the expressions of cleaved-caspase-3 and c-Fos were upregulated in the hippocampal CA3 region in this study. Conclusions: The upregulated c-Fos expression induced by repeated injections of propofol might evoke neuroapoptosis. PMID:22144767

  20. Location of caspase 3-like protease in the development of sieve element and tracheary element of stem in Cucurbita moschata.

    PubMed

    Hao, Xia; Qian, Jie; Xu, Shan; Song, Xin; Zhu, Jian

    2008-12-01

    The casepase is considered to regulate the process of programmed cell death in the development of organisms. In this study, caspase 3-like protease was detected by immunohistochemistry and immunoelectron microscopy during the development of sieve element and tracheary element of stem in Cucurbita moschata Duch. Antibody with brown color (under light microscopy) and gold particles (under transmission electron microscopy) for detecting caspase 3-like protease was mainly displayed in inner phloem, external phloem and xylem in the region close to procambium. From the results it was considered that caspase 3-like protease did exist in vascular elements and played different roles during the development of sieve and tracheary elements, and different types of programmed cell death might be carried out. The caspase 3-like protease mainly participated in making cytoplasmic streaming cease and in degrading P-protein bodies; however, it rarely participated in the function for signal transferring in the developmental sieve element. However, it might induce calcium accumulation for rupturing the tonoplast in the signal of PCD in the developmental tracheary element.

  1. [Changes in the activity of sympathetic-adrenal medullary system and hypothalamic-pituitary-adrenal system in humans exposed to psychogenic stressors and their effects on immunoreactivity].

    PubMed

    Simić, Natasa

    2010-10-01

    This paper gives an account of the functioning of the two systems in different stress induced situations. The activation of the sympathetic-adrenal medullary system is accompanied by the release of catecholamines, while the increased activity of the hypothalamic-pituitary-adrenal system results in the increased release of corticosteroids, especially cortisol. The role of the sympathetic-adrenal medullary system was investigated in immunologic changes induced by laboratory stressors. In the real, as in laboratory conditions, the effects of different stressors on the level of cortisol were studied, as it is the final product of the hypothalamic-pituitary-adrenal system activity. Additional (negative) effects on the functioning of these systems could induce some variables, as an increased consumption of alcohol, smoking, and sleeping disorder. Furthermore, the methodological shortcomings and the selection of subjects in previous studies are discussed. Previous results are also discussed, such as the immunosuppressive effects of cortisol, as well as the mediator and moderator variables in relation to stress and immunoreactivity.

  2. Carnosol increases caspase-3 activation, but delays DNA fragmentation induced by chemotherapeutic drugs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we showed that carnosol from rosemary induced apoptosis in leukemic cells derived from patients with high-risk pre-B acute lymphoblastic leukemia (ALL). In the current study, carnosol was tested for its ability to sensitize leukemia-derived cells to or synergize with conventional chemot...

  3. Preparation and immunoreactivity of high specific activity indium-111-DTPA labeled monoclonal antibody (MoAb) using ultrapure indium-111

    SciTech Connect

    Zoghbi, S.S.; Neumann, R.D.; Gottschalk, A.

    1986-10-01

    The preparation of high-specific activity /sup 111/In-DTPA-MoAb without increasing the number of DTPA molecules per Ab was investigated. Instant thin layer chromatography was used to assay the relationship between labeling efficiencies and specific activities. With ultrapurified /sup 111/In, the specific activity of the radiolabeled MoAb approached the expected theoretic maximum of 100 muCi/microgram. The bioactivity of such high-specific activity preparation showed no degradation as measured by in vitro cell binding assay.

  4. Cyclin D1 and E2F-1 immunoreactivity in bone marrow biopsy specimens of multiple myeloma: relationship to proliferative activity, cytogenetic abnormalities and DNA ploidy.

    PubMed

    Wilson, C S; Butch, A W; Lai, R; Medeiros, L J; Sawyer, J R; Barlogie, B; McCourty, A; Kelly, K; Brynes, R K

    2001-03-01

    Cyclin D1, encoded by the CCND1 gene, is immunohistochemically detectable in up to one-third of cases of multiple myeloma (MM). To examine the mechanism of cyclin D1 overexpression, we compared cyclin D1 immunoreactivity with the results of conventional cytogenetics to determine if the t(11;14)(q13;q32) or other abnormalities of 11q11-14 explained cyclin D1 overexpression. Karyotypic abnormalities were found in 45 out of 67 (67%) MM cases; the t(11;14) was present in seven cases (10%). Additional 11q11-14 abnormalities were not identified. The t(11;14) correlated with cyclin D1 upregulation in low to intermediately proliferative MM, but was not present in highly proliferative tumours (assessed using bromodeoxyuridine labelling index). Cyclin D1 indirectly activates the transcription factor E2F-1. In the bone marrow biopsy specimens of MM cases, E2F-1 was concurrently expressed with cyclin D1 (P = 0.001), indicating that cyclin D1 is functional. However, as neither E2F-1 nor cyclin D1 expression correlated with proliferative activity, the speculation that t(11;14) upregulates the CCND1 gene to induce higher proliferation and possibly more aggressive disease is not supported. We conclude that in low to intermediately proliferative MM cases, cyclin D1 is probably upregulated by t(11;14), but an alternative mechanism is more probable in highly proliferative MM.

  5. Differential regulation of spontaneous and immune complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase-3.

    PubMed

    Ottonello, Luciano; Frumento, Guido; Arduino, Nicoletta; Bertolotto, Maria; Dapino, Patrizia; Mancini, Marina; Dallegri, Franco

    2002-07-01

    Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)-2, IL-6, IL-8, IL-15, GM-CSF, and fMLP on spontaneous and IC-induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM-CSF, IL-6, and IL-15, but only GM-CSF overturned IC-induced apoptosis. No role of oxidants on the modulation of IC-dependent apoptosis was found. Indeed, fMLP or GM-CSF augmented the IC-dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM-CSF-inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18-h aged neutrophils was down-regulated by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a nearly threefold Bax up-regulation, which was completely reversed only by GM-CSF. Accordingly, the spontaneous activity of caspase-3 was inhibited by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM-CSF inhibited the IC-dependent acceleration. Our results show that apoptosis of resting and IC-activated neutrophils is regulated differently, GM-CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant-independent, Bax- and caspase-3-dependent, intracellular pathway regulating neutrophil apoptosis.

  6. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity

    PubMed Central

    Keeratijarut, Angsana; Lohnoo, Tassanee; Rujirawat, Thidarat; Yingyong, Wanta; Kalambaheti, Thareerat; Miller, Shannon; Phuntumart, Vipaporn; Krajaejun, Theerapong

    2015-01-01

    The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called “pythiosis”. Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen. PMID:26263509

  7. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

    PubMed

    Keeratijarut, Angsana; Lohnoo, Tassanee; Rujirawat, Thidarat; Yingyong, Wanta; Kalambaheti, Thareerat; Miller, Shannon; Phuntumart, Vipaporn; Krajaejun, Theerapong

    2015-01-01

    The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen. PMID:26263509

  8. Influence of oxidation on the susceptibility of purified desmin to degradation by μ-calpain, caspase-3 and -6.

    PubMed

    Chen, Qianqian; Huang, Jichao; Huang, Feng; Huang, Ming; Zhou, Guanghong

    2014-05-01

    This study was designed to investigate the effects of desmin oxidation on its degradation by proteolytic enzymes. Desmin was isolated from bovine muscle and exposed to varying oxidative conditions, and then incubated with μ-calpain, caspase-3 or -6, respectively. The extent of protein degradation was subsequently determined using SDS-PAGE and Western-blotting. Furthermore, the oxidative modification of the secondary structure of desmin was measured by circular dichroism (CD). Our results revealed that, compared with the native desmin, degradation of oxidised desmin was enhanced by caspases, but suppressed by μ-calpain. The CD spectra of desmin showed that the content of α-helix decreased from 76.2% to 52% while random coil increased from 8% to 22.4% after oxidation. These findings demonstrated that oxidative modifications of desmin changed their susceptibility to μ-calpain, caspase-3 and -6 as well as their secondary structure.

  9. The effect of resistance exercise on p53, caspase-9, and caspase-3 in trained and untrained men.

    PubMed

    Sharafi, Hossein; Rahimi, Rahman

    2012-04-01

    Apoptosis is a programmed cell death that has been demonstrated in human and animal studies and plays an essential role to remove injured cells after acute strenuous exercise. Protein p53 plays important roles in regulating apoptosis via mitochondrial pathway. Therefore, the aims of this study were to determine the effects of acute resistance exercise (RE) on serum p53, caspase-9, and caspase-3, markers of apoptosis, and whether resistance training status influences the magnitude of the RE-induced apoptosis. Nine resistance-trained (RT) (age, 22.37 ± 1.99 years; height, 174 ± 5.04 cm; body weight, 71.32 ± 5.57 kg; and body mass index [BMI] 23.58 ± 2.05 kg·m(-2)) and 9 untrained (UT) college-age men (age, 22.25 ± 2.13 years; height, 171 ± 3.4 cm; body weight, 68.45 ± 3.23 kg; and BMI, 23.41 ± 1.08 kg·m(-2)) volunteered to participate in this study. Resistance-trained and UT men completed an RE bout consisting of 4 sets of 6 exercise at 80% of 1 repetition maximum until failure. Serum levels of p53, caspase-9, and caspase-3 were examined at preexercise (pre), immediately post (IP), 3 hours post (3 hours post), and 24 hours post RE (24 hours post). In UT, serum levels of p53, caspase-9, and caspase-3 were significantly increased at IP compared with RT. However, plasma insulin-like growth factor 1 level was higher for RT compared with UT at IP. Collectively, our data suggest the role of p53 in regulating apoptosis through mitochondrial pathway as measured by caspase-9 and caspase-3 after acute RE in UT. Our results also revealed that regular RT alters apoptosis biomarkers, especially the intrinsic pathway of apoptosis. PMID:22446679

  10. Human caspase-3 inhibition by Z-tLeu-Asp-H: tLeu(P{sub 2}) counterbalances Asp(P{sub 4}) and Glu(P{sub 3}) specific inhibitor truncation

    SciTech Connect

    Colantonio, Patrizia; Leboffe, Loris; Bolli, Alessandro; Marino, Maria; Ascenzi, Paolo; Luisi, Grazia

    2008-12-19

    Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H are reported. Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with K{sub i}{sup 0} = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 and 25 deg. C). Moreover, Z-tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z-tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P{sub 2} position.

  11. Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry.

    PubMed

    Yang, Jing-Feng; Gao, Rong-Chun; Wu, Hai-Tao; Li, Peng-Fei; Hu, Xian-Shu; Zhou, Da-Yong; Zhu, Bei-Wei; Su, Yi-Cheng

    2015-11-01

    The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.

  12. Inhibitory effects of hyperoside on lung cancer by inducing apoptosis and suppressing inflammatory response via caspase-3 and NF-κB signaling pathway.

    PubMed

    Lü, Ping

    2016-08-01

    Lung cancer is one of the most common malignancies in the world and the most threatening cancer to human health. Effective therapies based on non-cytotoxic induction in cell inflammation- and apoptosis-responsive pathways are thought to represent a novel advance in treating lung cancer. However, many studies are still required for effective pharmaceutical to induce cancer cell death. Hyperoside (Hyp) is the chief component of some Chinese herbs with anticancer effect. Here, we investigated the role of hyperoside on the lung cancer cell migration, invasion, inflammation and apoptosis in A549 cells in vitro and xenografts of nude mice in vivo. A549 cells were injected in nude mice for establishing tumors. Our results showed that hyperoside suppressed the proliferation, migration and invasion. Additionally, apoptosis was induced by hyperoside via Bcl-2/Bax-regulated Caspase3 activation, suggesting that hyperoside might inhibit lung cancer progression through apoptotic induction. And also, hyperoside could prevent progression and development of lung cancer through inactivating NF-κB signaling pathway. Subsequently, inflammatory cytokines, including TNF-α, IL-6, IL-1β and IL-18, were down-regulated significantly. And animal experiments also illustrated that the tumor volume and weight were reduced after hyperoside administration, which was also through apoptosis induction and prevention of inflammation response by Caspase3 activation and NF-κB inactivation. To our knowledge, it was the first time to evaluate the effects of hyperoside on preventing progression and development of lung cancer in vivo and in vitro to assess the possible therapies of hyperoside as a future approach for preventing lung cancer progression and development. PMID:27470358

  13. Curcumin reverses cis-platin resistance and promotes human lung adenocarcinoma A549/DDP cell apoptosis through HIF-1α and caspase-3 mechanisms.

    PubMed

    Ye, Ming-Xiang; Zhao, Yi-Lin; Li, Yan; Miao, Qing; Li, Zhi-Kui; Ren, Xin-Ling; Song, Li-Qiang; Yin, Hong; Zhang, Jian

    2012-06-15

    Curcumin, a yellow pigment derived from Curcuma longa Linn, has been favored by the Eastern as dietary ingredients for centuries. During the past decade, extensive investigations have revealed curcumin sensitized various chemotherapeutic agents in human breast, colon, pancreas, gastric, liver, brain and hematological malignant disorders in vivo and in vitro. Several pathways and specific targets including NF-κB, STAT3, COX-2, Akt and multidrug resistant protein have been identified to facilitate curcumin as a chemosensitizer. Recent studies suggest HIF-1α participated in the development of drug resistance in cancer cells and targeting HIF-1α either by RNAi or siRNA successfully overcame chemotherapeutic resistance. To investigate the mechanism basis of curcumin as a chemosensitizer in lung cancer, we examined curcumin's effects on HIF-1α in cis-platin (DDP) sensitive A549 and resistant A549/DDP cell lines by RT-PCR and Western blot. HIF-1α in A549/DDP cells was found to be overexpressed at both mRNA and protein levels together with a poor response to DDP. Results from transient transfection and flow cytometry showed the HIF-1α abnormality contributed to DDP resistance in A549/DDP lung cancer cells. Combined curcumin and DDP treatment markedly inhibited A549/DDP cells proliferation, reversed DDP resistance and triggered apoptotic death by promoting HIF-1α degradation and activating caspase-3, respectively. Expression of HIF-1α-dependent P-gp also seemed to decrease as response to curcumin in a dose-dependent manner. Our findings shed light on drug resistant reversing effect of curcumin in lung cancer cells by inhibiting HIF-1α expression and activating caspase-3. PMID:22483553

  14. Quercetin ameliorates ischemia/reperfusion-induced cognitive deficits by inhibiting ASK1/JNK3/caspase-3 by enhancing the Akt signaling pathway.

    PubMed

    Pei, Bing; Yang, Miaomiao; Qi, Xiaoyan; Shen, Xin; Chen, Xing; Zhang, Fayong

    2016-09-01

    Cerebral ischemia/reperfusion (I/R) is a major cause of severe disability and death all worldwide. However, therapeutic options to minimize the detrimental effects of cerebral I/R injury are limited. Recent research has demonstrated that quercetin mediates neuroprotective effects associated with the activation of the Akt signaling pathway in the cerebral I/R brain. Therefore, the aim of this study was to further investigate the mechanisms of cognitive deficits induced by cerebral I/R injury and the effects of quercetin on these mechanisms. First, we assessed anxiety-like behavioral and cognitive impairment using the open field test and the Morris water maze test, respectively. Next, we examined the severity of apoptosis by staining hippocampal neurons by the Cresyl violet method. Third, we used western blot analysis to investigate the expression of total and phosphorylated Akt, ASK1, JNK3, c-Jun and caspase-3 after I/R injury. Our results revealed that mice subjected to bilateral common carotid occlusion exhibited severe anxiety-like behavior, learning and memory impairment, cell damage and apoptosis. These severe effects were attenuated by administration of quercetin. Further, western blot analysis revealed that quercetin increased p-Akt expression and decreased p-ASK1, p-JNK3 and cleaved caspase-3 expression after cerebral I/R injury and led to inhibition of neuronal apoptosis. Conversely, treatment with LY294002 (a selective inhibitor of Akt1) reversed the effects of quercetin. In conclusion, these findings highlight the important role of quercetin in protecting against cognitive deficits and inhibiting neuronal apoptosis via the Akt signaling pathway. We believe that quercetin might prove to be a useful therapeutic component in treating cerebral I/R diseases in the near future. PMID:27450812

  15. Meta-analysis of the relationship between single nucleotide polymorphism rs72689236 of caspase-3 and Kawasaki disease.

    PubMed

    Xing, Yanlin; Wang, Hong; Liu, Xiaomei; Yu, Xianyi; Chen, Rui; Wang, Ce; Yu, Xuexin; Sun, Le

    2014-10-01

    Kawasaki disease is a pediatric systemic vasculitis of unknown etiology, for which a genetic influence is suspected. But whether single nucleotide polymorphism (SNP) of caspase-3 rs72689236 is associated with Kawasaki disease is controversial. The aim of our study is to assess the association between the SNP of caspase-3 and risk for Kawasaki disease. We searched PubMed, MEDLINE, EMBASE, Springer, Elsevier Science Direct, Cochrane Library Google scholar, CNKI (China National Knowledge Infrastructure, in Chinese) and Wanfang database (in Chinese) to identify studies investigating the association between rs72689236 polymorphism and Kawasaki disease occurrence. There were five eligible studies, which included 4,241 (case group 1,560; control group 2,681) participants in this meta-analysis. Pooled odds ratios (ORs) and 95 % confidence intervals (95 % CIs) were calculated in a fixed-effects model (the Mantel-Haenszel method) or a random-effects model (the DerSimonian and Laird method) when appropriate. Significant associations were found under the overall ORs for A-allele comparison (A vs. G, pooled OR 1.33, 95 % CI 1.21-1.46), AA versus GG comparison (pooled OR 1.64, 95 % CI 1.35-2.00), GA versus GG comparison (pooled OR 1.42, 95 % CI 1.24-1.63), recessive model (AA vs. GG + GA, pooled OR 1.37, 95 % CI 1.15-1.64) and dominant model (AA + GA vs. GG, pooled OR 1.47, 95 % CI 1.29-1.67). This meta-analysis suggested that SNP rs72689236 of caspase-3 might be associated with susceptibility of Kawasaki disease and the allele A might increase the risk of Kawasaki disease in Asian samples such as Japanese and Chinese. In addition, individual studies with large sample size are needed to further evaluate the associations in various ethnic populations.

  16. Mesenchymal stem cells from rat bone marrow down regulate Caspase-3 mediated apoptotic pathway after spinal cord injury in rats

    PubMed Central

    Dasari, Venkata Ramesh; Spomar, Daniel G.; Cady, Craig; Gujrati, Meena; Rao, Jasti S.; Dinh, Dzung H.

    2007-01-01

    Mesenchymal stem cells have been intensively studied for their potential use in reparative strategies for neurodegenerative diseases and traumatic injuries. We used mesenchymal stem cells (rMSC) from rat bone marrow to evaluate the therapeutic potential after spinal cord injury (SCI). Immunohistochemistry confirmed a large number of apoptotic neurons and oligodendrocytes in caudal segments 2mm away from the lesion site. Expression of caspase-3 on both neurons and oligodendrocytes after SCI was significantly downregulated by rMSC. Caspase-3 downregulation by rMSC involves increased expression of FLIP and XIAP in the cytosol and inhibition of PARP cleavage in the nucleus. Animals treated with rMSC had higher BBB scores and better recovery of hind limb sensitivity. Treatment with rMSC had a positive effect on behavioral outcome and histopathological assessment after SCI. The ability of rMSC to incorporate into the spinal cord, differentiate and to improve locomotor recovery hold promise for a potential cure after SCI. PMID:17564836

  17. Dietary flavonoid fisetin targets caspase-3-deficient human breast cancer MCF-7 cells by induction of caspase-7-associated apoptosis and inhibition of autophagy.

    PubMed

    Yang, Pei-Ming; Tseng, Ho-Hsing; Peng, Chih-Wen; Chen, Wen-Shu; Chiu, Shu-Jun

    2012-02-01

    The outcome of producing apoptotic defects in cancer cells is the primary obstacle that limits the therapeutic efficacy of anticancer agents, and hence the development of novel agents targeting novel non-canonical cell death pathways has become an imperative mission for clinical research. Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid commonly found in fruits and vegetables. In this study, we investigated the potential anticancer effects of fisetin on breast cancer cells. The result showed fisetin induced higher cytotoxicity in human breast cancer MCF-7 than in MDA-MB-231 cells otherwise it did not exert any detectable cytotoxicity in non-tumorigenic MCF-10A cells. We found fisetin can trigger a novel form of atypical apoptosis in caspase-3-deficient MCF-7 cells, which was characterized by several apoptotic features, including plasma membrane rupture, mitochondrial depolarization, activation of caspase-7, -8 and -9, and PARP cleavage; however, neither DNA fragmentation and phosphotidylserine (PS) externalization was observed. Although p53 was also activated by fisetin, the fisetin-induced apoptosis was not rescued by the p53 inhibitor pifithrin-α. In contrast, the fisetin-induced apoptosis was abrogated by pan-caspase inhibitor z-VAD-fmk. Furthermore, inhibition of autophagy by fisetin was shown as additional route to prompt anticancer activity in MCF-7 cells. These data allow us to propose that fisetin appears as a new potential anticancer agent which can be applied to develop a clinical protocol of human breast cancers.

  18. Effective Targeting Survivin, Caspase-3 and MicroRNA-16-1 Expression by Methyl-3-pentyl-6-methoxyprodigiosene Triggers Apoptosis in Colorectal Cancer Stem-Like Cells.

    PubMed

    Sam, Sohrab; Sam, Mohammad Reza; Esmaeillou, Mohammad; Safaralizadeh, Reza

    2016-10-01

    Over-expression of the proto-oncogene survivin in colorectal cancer stem cells (CCSCs) is thought to be one the primary causes for therapy failure. It has also been reported that tumor suppressor miR-16-1 is down-regulated in colorectal cancer (CRC) cells. Therefore, the search for new anti-proliferative agents which target survivin or miR-16-1 in CCSCs is warranted. Several studies have shown that prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in different kinds of cancer cells. Here, we investigated the effects of prodigiosin on HCT-116 cells that serve as a model for CRC initiating cells with stem-like cells properties. HCT-116 cells were treated with 100, 200 and 400 nM prodigiosin after which cell number, viability, growth-rate, survivin and miRNA-16-1 expression, caspase-3 activation and apoptotic rate were evaluated. Prodigiosin decreased significantly growth-rate in a dose-and time-dependent manner. After a 48 h treatment with 100, 200 and 400 nM prodigiosin, growth-rates were measured to be 84.4 ± 9.2 %, 58 ± 6.5 % and 46.3 ± 5.2 %, respectively, compared to untreated cells. We also found that treatment for 48 h with indicated concentrations of prodigiosin resulted in 41 %, 54.5 % and 63 % decrease in survivin mRNA levels and induced 32 %, 48 % and 61 % decrease in survivin protein levels as well as resulted in 128.3 ± 10 %, 178.7 ± 6.1 % and 205 ± 7.6 % increase in caspase-3 activation respectively compared to untreated cells. Prodigiosin caused a significant increase in miRNA-16-1 expression at a concentration of 100 nM and treatment with different concentrations of prodigiosin resulted in 2.2- to 3-fold increase in miRNA-16-1/survivin ratios compared to untreated cells. An increase in number of apoptotic cells ranging from 28.2 % to 86.8 % was also observed with increasing prodigiosin concentrations. Our results provide the first evidence that survivin and miRNA-16-1 as potential

  19. Effects of ischemic preconditioning on myocardium Caspase-3, SOCS-1, SOCS-3, TNF-α and IL-6 mRNA expression levels in myocardium IR rats.

    PubMed

    Ma, Jiangwei; Qiao, Zengyong; Xu, Biao

    2013-10-01

    The aim of this study was to characterise the effects of ischemic preconditioning (IP) on heart function parameters (ΔST and ΔT), activities of serum creatine kinase (CK), lactate dehydrogenase (LDH), and levels of serum nitric oxide (NO), malondialdehyde (MDA), and myocardium Caspase-3 mRNA, SOCS-1, SOCS-3, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression levels and Apoptosis index in myocardium IR rats. Results showed that ΔST and ΔST values in IP group were markedly lower than those in IR group. Compared with IR group, IP significantly (p < 0.01) decreased serum CK (0.83 ± 0.09 vs 1.36 ± 0.15), LDH (5613 ± 462 vs 7106 ± 492) activities and MDA (11.32 ± 1.05 vs 15.49 ± 1.26) level, increased the serum NO (86.39 ± 7.03 vs 53.77 ± 4.27) level in IR group. The IP induced a significant decreased in myocardium Caspase-3 mRNA (0.303 ± 0.021 vs 0.515 ± 0.022) gene expression (p < 0.01) compared to IR model group. The IP induced a significant decreased in myocardium SOCS-1 (0.241 ± 0.031 vs 0.596 ± 0.036), SOCS-3 (0.258 ± 0.031 vs 0.713 ± 0.057), TNF-α (0.137 ± 0.011 vs 0.427 ± 0.035) and IL-6 (0.314 ± 0.021 vs 0.719 ± 0.064) mRNA gene expression (p < 0.01) compared to IR model group. We conclude that IP is effective in the therapy of heart disease. These findings may have implications for the clinical development of preconditioning-based therapies for ischemic heart disease.

  20. TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

    PubMed Central

    Espín, Raquel; Roca, Francisco J.; Candel, Sergio; Sepulcre, María P.; González-Rosa, Juan M.; Alcaraz-Pérez, Francisca; Meseguer, José; Cayuela, María L.; Mercader, Nadia; Mulero, Victoriano

    2013-01-01

    SUMMARY Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis. PMID:22956347

  1. Mesenchymal stromal cells protect against caspase 3-mediated apoptosis of CD19(+) peripheral B cells through contact-dependent upregulation of VEGF.

    PubMed

    Healy, Marc E; Bergin, Ronan; Mahon, Bernard P; English, Karen

    2015-10-15

    The immune suppressive and anti-inflammatory capabilities of bone marrow-derived mesenchymal stromal cells (MSCs) represent an innovative new tool in regenerative medicine and immune regulation. The potent immune suppressive ability of MSC over T cells, dendritic cells, and natural killer cells has been extensively characterized, however, the effect of MSC on B cell function has not yet been clarified. In this study, the direct effect of MSC on peripheral blood B cell function is defined and the mechanism utilized by MSC in enhancing B cell survival in vitro identified. Human MSC supported the activation, proliferation, and survival of purified CD19(+) B cells through a cell contact-dependent mechanism. These effects were not mediated through B cell activating factor or notch signaling. However, cell contact between MSC and B cells resulted in increased production of vascular endothelial growth factor (VEGF) by MSC facilitating AKT phosphorylation within the B cell and inhibiting caspase 3-mediated apoptosis. Blocking studies demonstrated that this cell contact-dependent effect was not dependent on signaling through CXCR4-CXCL12 or through the epidermal growth factor receptor (EGFR). These results suggest that direct cell contact between MSC and B cells supports B cell viability and function, suggesting that MSC may not represent a suitable therapy for B cell-mediated disease. PMID:26076727

  2. Copper induced apoptosis in Caco-2 and Hep-G2 cells: Expression of caspases 3, 8 and 9, AIF and p53.

    PubMed

    Santos, Stefanie; Silva, Amélia M; Matos, Manuela; Monteiro, Sandra M; Álvaro, Ana R

    2016-01-01

    Copper (Cu) is an essential trace metal needed to ensure cell function. However, when present at high concentrations it becomes toxic to organisms. Cell death, induced by toxic levels of copper, was previously observed in in vitro studies. However, there is no consensus about the cell death pathway induced by Cu and it is still not known whether this occurs as a result of the direct action of the metal or by indirect effects. In the present work, we intend to identify the influence of different Cu concentrations in the induction of apoptosis and to explore the potential signaling pathways, using two different in vitro cell culture models (Caco-2 and Hep-G2). Cells were exposed, during 6, 12, 24 and 48h, to Cu concentrations corresponding to IC50 and 1/8 of IC50, according to the viability assays. Then, considering the different apoptosis pathways, the expression of caspases 3, 8 and 9, apoptosis inducing factor (AIF) and p53 genes was analyzed by quantitative real time PCR. The results suggested that different Cu concentrations could trigger different apoptotic pathways, at different times of exposure. In both cell lines, apoptosis seems to be initiated by caspase independent pathway and intrinsic pathway, followed by extrinsic pathway. In conclusion, this study demonstrates that Cu induces the activation of apoptosis through caspase dependent and independent pathways, also suggesting that apoptosis activation mechanism is dependent on the concentration, time of exposure to Cu and cell type.

  3. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells

    PubMed Central

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  4. miR-30e controls DNA damage-induced stress responses by modulating expression of the CDK inhibitor p21WAF1/CIP1 and caspase-3

    PubMed Central

    Sohn, Dennis; Peters, Dominik; Piekorz, Roland P.; Budach, Wilfried; Jänicke, Reiner U.

    2016-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3′-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value. PMID:26895377

  5. miR-30e controls DNA damage-induced stress responses by modulating expression of the CDK inhibitor p21WAF1/CIP1 and caspase-3.

    PubMed

    Sohn, Dennis; Peters, Dominik; Piekorz, Roland P; Budach, Wilfried; Jänicke, Reiner U

    2016-03-29

    MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3'-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value.

  6. Subacute Zinc Administration and L-NAME Caused an Increase of NO, Zinc, Lipoperoxidation, and Caspase-3 during a Cerebral Hypoxia-Ischemia Process in the Rat

    PubMed Central

    Blanco-Alvarez, Victor Manuel; Lopez-Moreno, Patricia; Soto-Rodriguez, Guadalupe; Martinez-Fong, Daniel; Rubio, Hector; Gonzalez-Barrios, Juan Antonio; Piña-Leyva, Celia; Torres-Soto, Maricela; Gomez-Villalobos, María de Jesus; Hernandez-Baltazar, Daniel; Eguibar, José Ramon; Ugarte, Araceli; Cebada, Jorge

    2013-01-01

    Zinc or L-NAME administration has been shown to be protector agents, decreasing oxidative stress and cell death. However, the treatment with zinc and L-NAME by intraperitoneal injection has not been studied. The aim of our work was to study the effect of zinc and L-NAME administration on nitrosative stress and cell death. Male Wistar rats were treated with ZnCl2 (2.5 mg/kg each 24 h, for 4 days) and N-ω-nitro-L-arginine-methyl ester (L-NAME, 10 mg/kg) on the day 5 (1 hour before a common carotid-artery occlusion (CCAO)). The temporoparietal cortex and hippocampus were dissected, and zinc, nitrites, and lipoperoxidation were assayed at different times. Cell death was assayed by histopathology using hematoxylin-eosin staining and caspase-3 active by immunostaining. The subacute administration of zinc before CCAO decreases the levels of zinc, nitrites, lipoperoxidation, and cell death in the late phase of the ischemia. L-NAME administration in the rats treated with zinc showed an increase of zinc levels in the early phase and increase of zinc, nitrites, and lipoperoxidation levels, cell death by necrosis, and the apoptosis in the late phase. These results suggest that the use of these two therapeutic strategies increased the injury caused by the CCAO, unlike the alone administration of zinc. PMID:23997853

  7. Dual vulnerability of TDP-43 to calpain and caspase-3 proteolysis after neurotoxic conditions and traumatic brain injury

    PubMed Central

    Yang, Zhihui; Lin, Fan; Robertson, Claudia S; Wang, Kevin K W

    2014-01-01

    Transactivation response DNA-binding protein 43 (TDP-43) proteinopathy has recently been reported in chronic traumatic encephalopathy, a neurodegenerative condition linked to prior history of traumatic brain injury (TBI). While TDP-43 appears to be vulnerable to proteolytic modifications under neurodegenerative conditions, the mechanism underlying the contribution of TDP-43 to the pathogenesis of TBI remains unknown. In this study, we first mapped out the calpain or caspase-3 TDP-43 fragmentation patterns by in vitro protease digestion. Concurrently, in cultured cerebrocortical neurons subjected to cell death challenges, we identified distinct TDP-43 breakdown products (BDPs) of 35, 33, and 12 kDa that were indicative of dual calpain/caspase attack. Cerebrocortical culture incubated with calpain and caspase-fragmented TDP-43 resulted in neuronal injury. Furthermore, increased TDP-43 BDPs as well as redistributed TDP-43 from the nucleus to the cytoplasm were observed in the mouse cortex in two TBI models: controlled cortical impact injury and overpressure blast-wave-induced brain injury. Finally, TDP-43 and its 35 kDa fragment levels were also elevated in the cerebrospinal fluid (CSF) of severe TBI patients. This is the first evidence that TDP-43 might be involved in acute neuroinjury and TBI pathology, and that TDP-43 and its fragments may have biomarker utilities in TBI patients. PMID:24917042

  8. Osteocyte expression of caspase-3, COX-2, IL-6 and sclerostin are spatially and temporally associated following stress fracture initiation.

    PubMed

    Wu, Andy C; Kidd, Lisa J; Cowling, Nicholas R; Kelly, Wendy L; Forwood, Mark R

    2014-01-01

    Stress fractures (SFxs) are debilitating injuries and exact mechanisms that initiate their repair incompletely understood. We hypothesised that osteocyte apoptosis and expression of cytokines and proteins such as sclerostin, VEGF, TGF-β, COX-2 and IL-6 were early signalling events to facilitate the formation of periosteal woven bone and recruitment of osteoclast precursors to the site of remodelling. A SFx was created in the right ulna of mature female wistar rats using cyclic end loading. Rats were killed 1, 4 and 7 days after loading (n=5 per group). Standard histological staining was used to examine SFx morphology and immunohistochemistry to detect the localisation of these proteins and in situ hybridisation to detect mRNA along the SFx line or gene expression to quantify the target genes. Unloaded ulnae served as controls. The labelling index of caspase-3, COX-2 and IL-6 was significantly elevated in the region of SFxs at all time points compared with controls (P<0.001). In addition, the labelling index of sclerostin protein was significantly reduced in osteocytes adjacent to the SFx region when compared with controls at all three time points (P<0.001). Both VEGF and TGF-β expressions were only localised in the woven bone. These data reinforce the involvement of osteocyte apoptosis in the healing of fatigue damage in bone, and demonstrate that local regulation of sclerostin, COX-2 and IL-6 are important signalling events associated with new bone formation and SFx remodelling. PMID:25228984

  9. Huangzhi Oral Liquid Prevents Arrhythmias by Upregulating Caspase-3 and Apoptosis Network Proteins in Myocardial Ischemia-Reperfusion Injury in Rats

    PubMed Central

    Ran, Xu; Sun, Xue Gang; Wang, Ming; An, Hui; Huang, Guo Qiang; Zhao, Xiao Shan; Zhou, Feng Hua; Yang, Yun Gao; Miao, Can Ming

    2015-01-01

    To study the effect of Huangzhi oral liquid (HZOL) on I/R after 2 h and 4 h and determine its regulatory function on caspase-3 and protein networks. 70 SD male rats were randomly divided into seven groups and established myocardial I/R injury model by ligating the left anterior descending coronary artery. Myocardial infarction model was defined by TTC staining and color of the heart. The levels of CK-MB, CTnI, C-RPL, SOD, and MDA were tested at 2 h and 4 h after reperfusion. HE staining and ultramicrostructural were used to observe the pathological changes. The apoptotic index (AI) of cardiomyocyte was marked by TUNEL. The expression levels of caspase-3, p53, fas, Bcl-2, and Bax were tested by immunohistochemistry and western blot. HZOL corrected arrhythmia, improved the pathologic abnormalities, decreased CK-MB, CTnI, C-RPL, MDA, AI, caspase-3, p53, fas, and Bax, and increased SOD ans Bcl-2 with different times of myocardial reperfusion; this result was similar to the ISMOC (P > 0.05). HZOL could inhibit arrhythmia at 2 and 4 h after I/R and ameliorate cardiac function, which was more significant at 4 h after reperfusion. This result may be related to decreased expression of caspase-3, p53, and fas and increased Bcl-2/Bax ratio. PMID:26074995

  10. Caffeine decreases phospho-Chk1 (Ser317) and increases mitotic cells with cyclin B1 and caspase 3 in tumors from UVB-treated mice.

    PubMed

    Lu, Yao-Ping; Lou, You-Rong; Peng, Qing-Yun; Nghiem, Paul; Conney, Allan H

    2011-07-01

    Oral administration of caffeine to mice inhibits UVB-induced carcinogenesis, and these results are paralleled by epidemiology studies indicating that caffeinated coffee and tea intake (but not decaffeinated beverage intake) is associated with decreased incidence of nonmelanoma skin cancer. Topical applications of caffeine to the skin of SKH-1 mice that had previously been treated with UVB inhibited subsequent skin tumor development and stimulated apoptosis in tumors but not in nontumor areas of the epidermis. This study sought to determine the basis of these differential effects on tumor versus nontumor sites that can be induced by caffeine, long after all UVB treatment has ceased. The activation status of the ATR/Chk1 pathway in UVB-induced tumors and uninvolved skin was determined by quantitating phospho-Chk1 (Ser317) and induction of lethal mitosis in vivo in the presence and absence of topical caffeine treatment. In the absence of caffeine, we found that UVB-induced tumors often had islands of phospho-Chk1 (Ser317) staining cells that were not present in nontumor areas of the epidermis. Treatment of mice with topical caffeine significantly diminished phospho-Chk1 (Ser317) staining and increased the number of mitotic cells that expressed cyclin B1 and caspase 3 in tumors, consistent with caffeine-induced lethal mitosis selectively in tumors. We hypothesize that compared with adjacent uninvolved skin, UVB-induced skin tumors have elevated activation of, and dependence on, the ATR/Chk1 pathway long after UVB exposure has ceased and that caffeine can induce apoptosis selectively in tumors by inhibiting this pathway and promoting lethal mitosis. PMID:21505179

  11. Acceleration of pro-caspase-3 maturation and cell migration inhibition in human breast cancer cells by phytoconstituents of Rheum emodi rhizome extracts

    PubMed Central

    Naveen Kumar, D.R.; George, V. Cijo; Suresh, P.K.; Kumar, R. Ashok

    2013-01-01

    The aggressive nature of estrogen receptor (ER)-negative breast cancer subtype obligates for innovative targeted therapies. The present study aimed to investigate the phytoconstituents and specific anticancer activities of Rheum emodi rhizome, a known food source used locally to treat various ailments. Petroleum ether extracts (hot [PHR] and cold [PCR]) of R. emodi, exhibited significant free radical scavenging potentials through DPPH and reducing power assays, rendering them as good sources of antioxidants. The extracts, PHR and PCR had shown significant (P < 0.05) cancer-cell-specific cytotoxicity in the assayed cells (MDA-MB-231 [breast carcinoma] and WRL-68 [non-tumoral]) at 100 μg/ml, and 50 and 100 μg/ml concentrations respectively. Extracts also induced fervent apoptosis in ER-negative cells (MDA-MB-231) compared to ER-positive subtype (MCF-7), and found to involve CPP32/caspase-3 in its apoptosis induction mechanism. Moreover, extracts had an inevitable potential to inhibit the migration of metastatic breast cancer cells (MDA-MB-231) in vitro. Further, the active principles of extracts were identified through HPLC and GC-MS analysis to reveal major polyphenolics, 4,7-Dimethyl-(octahydro)indolo[4,3-fg]quinolin-10-one, 5-Oxo-isolongifolene, Valencene-2, and other quinone, quinoline and anthraquinone derivatives. The extracts are thus good candidates to target malignant ER-negative breast cancer, and the identified phytoconstituents could be eluted in further exploratory studies for use in dietary-based anti-breast cancer therapies. PMID:26417238

  12. Basal plasma immunoreactive calcitonin in postmenopausal osteoporosis.

    PubMed

    Chesnut, C H; Baylink, D J; Sisom, K; Nelp, W B; Roos, B A

    1980-06-01

    Calcitonin (CT) deficiency has been suggested as an etiologic factor in postmenopausal osteoporosis (PM-OP). Basal immunoreactive calcitonin (iCT) was measured with a sensitive radioimmunoassay (RIA) in 62 PM-OP women with compression fractures (CF) and in 28 normal age-matched women. Mean iCT values in the two groups were not significantly different (43.5 and 45.1 pg/ml, p greater than 0.10). In the 62 PM-OP females, no significant correlation was noted between basal plasma iCT levels and (1) age; (2) severity of disease as assessed by number of CF; (3) serum calcium, phosphorus, alkaline phosphatase, and immunoreactive parathyroid hormone; and (4) total bone mass as assessed by neutron activation analysis determinations of total body calcium (TBC). In 20 PM-OP patients treated for 24 mo with 100 Medical Research Council (MRC) units daily of synthetic salmon CT, no correlation was observed between basal plasma iCT and response of bone mass (TBC) to therapy. These data suggest that basal CT is not decreased in women with PM-OP, and that the level of circulating CT does not influence therapeutic changes in bone mass during CT therapy. CT is probably not a major etiologic or pathogenetic factor in PM-OP.

  13. The effect of concentration and duration of normobaric oxygen in reducing caspase-3 and -9 expression in a rat-model of focal cerebral ischaemia.

    PubMed

    Chen, Suyan; Peng, Huizhen; Rowat, Anne; Gao, Feng; Zhang, Zhenxiang; Wang, Peng; Zhang, Weihong; Wang, Xianyuan; Qu, Lixia

    2015-08-27

    The aim of this study was to determine the effect of different concentrations of normobaric oxygen (NBO) on neurological function and the expression of caspase-3 and -9 in a rat model of acute cerebral ischaemia. Sprague-Dawley rats (n=120) were randomly divided into four groups (n=30 per group), including 3 groups given NBO at concentrations of 33%, 45% or 61% and one control group given air (21% oxygen). After 2h of ischaemic occlusion, each group was further subdivided into six subgroups (n=5) during reperfusion according to the duration (3, 6, 12, 24, 48 or 72h) and concentration of NBO (33%, 45% or 61%) or air treatment. The Fluorescence Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to detect caspase-3 and -9 mRNA and protein relative expression respectively. The Neurologic Impairment Score (NIS) was significantly lower in rats given 61% NBO ≥3h after reperfusion when compared to the control group (P<0.05, Mann-Whitney U). NBO significantly reduced caspase-3 and -9 mRNA and protein expression when compared to the control group at all NBO concentrations and time points (P<0.05, ANOVA). The expression of caspase-3 and -9 was lower in the group given 61% NBO compared any other group, and this difference was statistically significant when compared to the group given 33% NBO for ≥48h and the control group (both P<0.05, ANOVA). These findings indicate that NBO may inhibit the apoptotic pathway by reducing caspase-3 and -9 expression, thereby promoting neurological functional recovery after stroke.

  14. Conserved miR-26b enhances ovarian granulosa cell apoptosis through HAS2-HA-CD44-Caspase-3 pathway by targeting HAS2

    PubMed Central

    Liu, Jiying; Tu, Fei; Yao, Wang; Li, Xinyu; Xie, Zhuang; Liu, Honglin; Li, Qifa; Pan, Zengxiang

    2016-01-01

    The hyaluronan synthase 2 (HAS2)-hyaluronic acid (HA)-CD44-Caspase-3 pathway is involved in ovarian granulosa cell (GC) functions in mammals. HAS2 is a key enzyme required for HA synthesis and is the key factor in this pathway. However, the regulation of HAS2 and the HAS2-mediated pathway by microRNAs in GCs is poorly understood. Here, we report that miR-26b regulates porcine GC (pGC) apoptosis through the HAS2-HA-CD44-Caspase-3 pathway by binding directly to the 3′- untranslated region of HAS2 mRNA. Knockdown of miR-26b reduced pGC apoptosis. Luciferase reporter assays demonstrated that HAS2 is a direct target of miR-26b in pGCs. Knockdown and overexpression of miR-26b increased and decreased, respectively, HA content, and HAS2 and CD44 expression in pGCs. At the same time, inhibition and overexpression of miR-26b decreased and increased the expression of Caspase-3, a downstream factor in the HAS2-HA-CD44 pathway. Moreover, knockdown of HAS2 enhanced pGC apoptosis, reduced the inhibitory effects of a miR-26b inhibitor on pGC apoptosis, repressed HA content and CD44 expression, and promoted Caspase-3 expression. In addition, overexpression of HAS2 has a opposite effect. Collectively, miR-26b positively regulates pGC apoptosis via a novel HAS2-HA-CD44-Caspase-3 pathway by targeting the HAS2 gene. PMID:26887530

  15. Induction of apoptosis and growth arrest in human breast carcinoma cells by a snake (Walterinnesia aegyptia) venom combined with silica nanoparticles: crosstalk between Bcl2 and caspase 3.

    PubMed

    Al-Sadoon, Mohamed K; Abdel-Maksoud, Mostafa A; Rabah, Danny M; Badr, Gamal

    2012-01-01

    We recently demonstrated that the snake venom extracted from Walterinnesia aegyptia (WEV) either alone or combined with silica nanoparticles (WEV+NP) enhanced the proliferation of mice immune cells and simultaneously decreased the proliferation of human breast carcinoma cell line (MDA-MB-231). However, the molecular mechanism of how this venom induced growth arrest of breast cancer cells has not been studied. In this context, we extended our study to evaluate the anti-tumor potential of WEV and WEV+NP on the human breast carcinoma cell lines MDA-MB-231 and MCF-7, as well as their effects on non-tumorigenic normal breast epithelial cells (MCF-10). The IC(50 )values of WEV alone and WEV+NP in these cell lines were determined to be 50 ng/ml and 20 ng/ml, respectively. Interestingly, at these concentrations, the venom did not affect the viability of normal MCF-10 cells and treatment of all these cell lines with NP alone did not affect their viability. Using annexin-V binding assay followed by flow cytometry analysis, we found that combination of WEV with NP strongly induced apoptosis in MDA-MB-231 and MCF-7 cancer cells without significant effect on normal MCF-10 cells. Furthermore, we found that WEV+NP decreased the expression of Bcl2 and enhanced the activation of caspase 3 in MDA-MB-231 and MCF-7 cells. Most importantly, WEV+NP-treated breast cancer cells, but not normal MCF-10 cells, exhibited a significant (P<0.05) reduction in actin polymerization and cytoskeletal rearrangement in response to CXCL12. Our data reveal biological effects of WEV or WEV+NP and the underlying mechanisms to fight breast cancer cells. PMID:22854437

  16. PANDER-induced cell-death genetic networks in islets reveal central role for caspase-3 and cyclin-dependent kinase inhibitor 1A (p21).

    PubMed

    Burkhardt, Brant R; Greene, Scott R; White, Peter; Wong, Ryan K; Brestelli, John E; Yang, Jichun; Robert, Claudia E; Brusko, Todd M; Wasserfall, Clive H; Wu, Jianmei; Atkinson, Mark A; Gao, Zhiyong; Kaestner, Klaus H; Wolf, Bryan A

    2006-03-15

    PANcreatic DERived factor is an islet-specific cytokine that promotes apoptosis in primary islets and islet cell lines. To elucidate the genetic mechanisms of PANDER-induced cell death we performed expression profiling using the mouse PancChip version 5.0 in conjunction with Ingenuity Pathway Analysis. Murine islets were treated with PANDER and differentially expressed genes were identified at 48 and 72 h post-treatment. 64 genes were differentially expressed in response to PANDER treatment. 22 genes are associated with cell death. In addition, the genes with the highest fold change were linked with cell death or apoptosis. The most significantly affected gene at 48 h was the downregulated cyclin-dependent kinase inhibitor 1A (CDKN1A or p21). Approximately half of the genes impacted at 72 h were linked to cell death. Cell death differentially expressed genes were confirmed by quantitative RT-PCR. Further analysis identified cell death genetic networks at both time points with 21 of the 22 cell death genes related in various biological pathways. Caspase-3 (CASP3) was biologically linked to CDKN1A in several genetic networks and these two genes were further examined. Elevated cleaved CASP3 levels in PANDER-treated beta-TC3 insulinoma cells were found to abrogate CDKN1A expression. Levels of CDKN1A were not affected in the absence of cleaved CASP3. PANDER-induced downregulation of CDKN1A expression coupled with induced CASP3-activation may serve a central role in islet cell death and offers further insight into the mechanisms of cytokine-induced beta-cell apoptosis.

  17. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

    PubMed

    Zhao, Yuwan; Jing, Zuolei; Li, Yan; Mao, Weifeng

    2016-07-01

    Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50% inhibitory concentration (IC50) value of 52.178±1.593 µM and the IC50 value of cisplatin was 49.541±1.618 µM, while in combination with 26 µM BBR, the IC50 value of cisplatin was 5.759±0.76 µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis. PMID:27177238

  18. Apoptotic and anti-adhesion effect of ajoene, a garlic derived compound, on the murine melanoma B16F10 cells: possible role of caspase-3 and the alpha(4)beta(1) integrin.

    PubMed

    Ledezma, Eliades; Apitz-Castro, Rafael; Cardier, José

    2004-03-31

    In this study we evaluated the hypothesis that the antitumor activity of ajoene could be associated with its apoptosis-inducing effect, and with its ability to block the expression of the alpha(4)beta(1) integrin, in the murine melanoma B16F10 cells. Ajoene induced a significant reduction in B16F10 viability (IC(50)=62 microM), in a dose-dependent manner. Flow cytometric analysis showed that the cytotoxic effect of this compound was associated with caspase-3 activation. Ajoene at 25 microM altered the alpha(4)beta(1) integrin expression on B16F10, and induced a significant reduction in the adhesion of these cells to an endothelial cell monolayer.

  19. Combined effect of 17β-estradiol and resveratrol against apoptosis induced by interleukin-1β in rat nucleus pulposus cells via PI3K/Akt/caspase-3 pathway

    PubMed Central

    2016-01-01

    Background: In previous studies, both 17β-estradiol (E2) and resveratrol (RES) were reported to protect intervertebral disc cells against aberrant apoptosis. Given that E2 has a better anti-apoptotic effect with more cancer risk and RES has an anti-apoptotic effect with less cancer risk, the combined use of E2 with RES is promising in developing clinical therapies to treat apoptosis-related diseases such as intervertebral disc degeneration in the future. Objective: The purpose of this study was to explore the combined effect of E2 with RES on rat nucleus pulposus cells and the underlying mechanisms. Methods: TUNEL assay and FACS analysis were used to determine apoptotic incidence of nucleus pulposus cells. MTS assay was used to determine cell viability, and cellular binding assay was used to determine cell-ECM (extracellular matrix) ability. Real-time quantitative RT-PCR was to determine mRNA level of target genes. And Western blot was used to determine the protein level. Results: Both E2 and RES decreased apoptotic incidence when used singly; interestingly, they decreased apoptosis more efficiently when used combinedly. Meanwhile, E2 and RES combined together against the decrease of cell viability and binding ability resulting from IL-1β cytotoxicity. As well, activated caspase-3 was suppressed by the combined effect. Furthermore, IL-1β downregulated expression level of type II collagen and aggrecan (standing for anabolism), while upregulated MMP-3 and MMP-13 (standing for catabolism). However, the combined use of E2 with RES effectively abolished the above negative effects caused by IL-1β, better than either single use. Finally, it turned out to be that E2 and RES combined together against apoptosis via the activation of PI3K/Akt/caspase-3 pathway. Conclusion: This study presented that IL-1β induced aberrant apoptosis, which was efficiently resisted by the combined use of E2 with RES via PI3K/Akt/caspase-3 pathway. PMID:26824000

  20. Angiotensin II-Induced Apoptosis of Human Umbilical Vein Endothelial Cells was Inhibited by Blueberry Anthocyanin Through Bax- and Caspase 3-Dependent Pathways.

    PubMed

    Du, Jian; Leng, Jiyan; Zhang, Li; Bai, Guangxin; Yang, Di; Lin, Huan; Qin, Junjie

    2016-01-01

    BACKGROUND This study aimed to investigate the inhibitory effect of blueberry anthocyanin (BBA) on Angiotensin II (Ang II)-induced apoptosis of human umbilical vein endothelial cells (HUVECs), and its regulation mechanisms involving Bax and Caspase 3. MATERIAL AND METHODS HUVECs were first treated by different concentrations of Ang II (10-9, 10-8, 10-7, 10-6, 10-5, and 10-4 mol/L) and BBA (80, 40, 20, 10, 5, and 2.5 μg/ml). After 24 h and 48 h of treatment, MTT was performed to detect the viability of HUVECs. Then, HUVECs were randomly divided into the Ang II group (10-6 mol/L Ang II) and Ang II + BBA group (10-6 mol/L Ang II and 20 μg/ml BBA), and the apoptosis rate was detected by flow cytometry. Western blot analysis was performed to detect the expression of Bax and Caspase 3 in these 2 groups. During the whole process, HUVECs without any treatments served as the control group. RESULTS The cell viability of HUVECs was significantly reduced by Ang II in a time- and concentration-dependent manner (P<0.05), while BBA significantly elevated the cell viability of HUVECs until a peak of 20.0 μg/ml. The apoptosis rate of HUVECs was significantly increased by Ang II (P<0.01) and reduced by the BBA intervention (P<0.05). Ang II significantly elevated the expression of Bax and Caspase 3 in HUVECs, but their expression was significantly inhibited by BBA. CONCLUSIONS BBA increased cell viability and reduced apoptosis rate of HUVECs induced by Ang II through Bax- and Caspase 3-dependent pathways. PMID:27616275

  1. Angiotensin II-Induced Apoptosis of Human Umbilical Vein Endothelial Cells was Inhibited by Blueberry Anthocyanin Through Bax- and Caspase 3-Dependent Pathways

    PubMed Central

    Du, Jian; Leng, Jiyan; Zhang, Li; Bai, Guangxin; Yang, Di; Lin, Huan; Qin, Junjie

    2016-01-01

    Background This study aimed to investigate the inhibitory effect of blueberry anthocyanin (BBA) on Angiotensin II (Ang II)-induced apoptosis of human umbilical vein endothelial cells (HUVECs), and its regulation mechanisms involving Bax and Caspase 3. Material/Methods HUVECs were first treated by different concentrations of Ang II (10−9, 10−8, 10−7, 10−6, 10−5, and 10−4 mol/L) and BBA (80, 40, 20, 10, 5, and 2.5 μg/ml). After 24 h and 48 h of treatment, MTT was performed to detect the viability of HUVECs. Then, HUVECs were randomly divided into the Ang II group (10−6 mol/L Ang II) and Ang II + BBA group (10−6 mol/L Ang II and 20 μg/ml BBA), and the apoptosis rate was detected by flow cytometry. Western blot analysis was performed to detect the expression of Bax and Caspase 3 in these 2 groups. During the whole process, HUVECs without any treatments served as the control group. Results The cell viability of HUVECs was significantly reduced by Ang II in a time- and concentration-dependent manner (P<0.05), while BBA significantly elevated the cell viability of HUVECs until a peak of 20.0 μg/ml. The apoptosis rate of HUVECs was significantly increased by Ang II (P<0.01) and reduced by the BBA intervention (P<0.05). Ang II significantly elevated the expression of Bax and Caspase 3 in HUVECs, but their expression was significantly inhibited by BBA. Conclusions BBA increased cell viability and reduced apoptosis rate of HUVECs induced by Ang II through Bax- and Caspase 3-dependent pathways. PMID:27616275

  2. Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity.

    PubMed

    Ichijo, Hiroyuki; Hamada, Michito; Takahashi, Satoru; Kobayashi, Makoto; Nagai, Takeharu; Toyama, Tomoko; Kawaguchi, Masahumi

    2015-06-01

    We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response. PMID:25637311

  3. Oxymatrine protects against sepsis-induced myocardial injury via inhibition of the TNF-α/p38-MAPK/caspase-3 signaling pathway.

    PubMed

    Zhang, Minghao; Wang, Xiuyu; Bai, Bin; Zhang, Rui; Li, Yunhong; Wang, Yin

    2016-07-01

    tissue. The present study concluded that OMT may offer substantial therapeutic potential for the treatment of septic shock‑induced myocardial injury by inhibiting the TNF-α/p38-MAPK/caspase-3 signaling pathway. PMID:27177246

  4. Dietary vitamin A supplementation improved reproductive performance by regulating ovarian expression of hormone receptors, caspase-3 and Fas in broiler breeders.

    PubMed

    Chen, Fang; Jiang, Zongyong; Jiang, Shouqun; Li, Long; Lin, Xiajing; Gou, Zongyong; Fan, Qiuli

    2016-01-01

    The effects of dietary vitamin A (VA) supplementation on reproductive performance, VA deposition, and potential mechanisms of action were studied in Chinese yellow-feathered broiler breeders. A total of 528 yellow-feathered broiler breeders that were 46 wk old were fed a corn-soybean meal basal diet supplemented with 0; 5,400; 10,800; or 21,600 IU/kg VA for 9 wk. Each dietary treatment had 6 replicates with 22 birds per replicate. After 7 wk of treatment, 60 settable eggs per replicate were collected for hatching. The results showed that dietary VA improved the laying rate, egg-to-feed ratio, and hatch weight of offspring (P < 0.05). Hepatic retinyl palmitate in broiler breeders and hatchlings (within 12 h) increased with increasing VA (P < 0.05). VA supplementation increased insulin-like growth factor 1 (IGF-I) receptor transcripts in the ovarian stroma and the walls of yellow follicles, follicle stimulating hormone (FSH) receptor expression in the walls of white and yellow follicles, and luteinizing hormone (LH) receptor and growth hormone (GH) receptor transcripts in the walls of yellow follicles (P < 0.05). Caspase-3 and Fas mRNA levels in the ovarian stroma and the walls of white and yellow follicles decreased with VA supplementation (P < 0.05). The relative expression of retinol dehydrogenase 10 (RDH10) transcripts in the walls of white follicles increased with 5,400 IU/kg VA supplementation (P < 0.05). Supplemental 21,600 IU/kg VA increased cytochrome P450 26A1 (CYP26A1) transcripts in the ovarian stroma and the walls of white follicles (P < 0.05). Dietary VA elevated retinoic acid receptor α (RARα) expression in the ovarian stroma and the walls of yellow follicles and retinoid X receptor α (RXRα) expression in the walls of yellow follicles. It was concluded that VA supplementation improved reproductive performance and hepatic storage of VA, and this was associated with the regulation of ovarian hormone receptor expression and suppression of apoptosis

  5. Fos and serotonin immunoreactivity in the raphe nuclei of the cat during carbachol-induced active sleep: a double-labeling study.

    PubMed

    Yamuy, J; Sampogna, S; López-Rodríguez, F; Luppi, P H; Morales, F R; Chase, M H

    1995-07-01

    The microinjection of carbachol into the nucleus pontis oralis produces a state which is polygraphically and behaviorally similar to active sleep (rapid eye movement sleep). In the present study, using double-labeling techniques for serotonin and the protein product of c-fos (Fos), we sought to examine whether immunocytochemically identified serotonergic neurons of the raphe nuclei of the cat were activated, as indicated by their expression of c-fos, during this pharmacologically-induced behavioral state (active sleep-carbachol). Compared with control cats, which were injected with saline, active sleep-carbachol cats exhibited a significantly greater number of c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus. Whereas most of the c-fos-expressing neurons in the raphe dorsalis were small, those in the raphe magnus were medium-sized and in the raphe pallidus they were small and medium-sized. The mean number of serotonergic neurons that expressed c-fos (i.e. double-labeled cells) was similar in control and active sleep-carbachol cats. These data indicate that there is an increased number of non-serotonergic, c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus during the carbachol-induced state.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7477901

  6. FMRFamide-like immunoreactivity and arylamidase activity in turbellarians and nemerteans--evidence for a novel neurovascular coordinating system in nemerteans.

    PubMed

    Jennings, J B; Davenport, T R; Varndell, I M

    1987-01-01

    The tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) has been immunolocalized in the nervous systems of seven species of Turbellaria and four species of Nemertea. The 11 species represent all the major turbellarian and nemertean taxa, and illustrate most of the various life styles found in these animals. The FMRFamide-like reactivity coincides with histochemically demonstrable arylamidase activity in the nervous systems. It is suggested that the FMRFamide-like reactivity demonstrates the presence in these lower invertebrates of one or more biologically active peptides, analogous to those of higher invertebrates and chordates and acting as putative neurotransmitters and coordinators of growth, maturation and muscular activities. The arylamidases occurring with the peptides are probably an integral part of these peptide-mediated control systems. The nemertean vascular system is especially rich in arylamidases and is believed to be concerned primarily with peptidergic control of bodily functions, rather than with transport of metabolites.

  7. Curcumin improves the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cells via the NF-κB-p53-caspase-3 pathway

    PubMed Central

    DANG, YU-PING; YUAN, XIAO-YING; TIAN, RONG; LI, DONG-GUANG; LIU, WEI

    2015-01-01

    Paclitaxel, isolated from Taxus brevifolia, is considered to be an efficacious agent against a wide spectrum of human cancers, including human cervical cancer. However, dose-limiting toxicity and high cost limit its clinical application. Curcumin, a nontoxic food additive, has been reported to improve paclitaxel chemotherapy in mouse models of cervical cancer. However, the underlying mechanisms remain unclear. In this study, two human cervical cancer cell lines, CaSki [human papilloma virus (HPV)16-positive] and HeLa (HPV18-positive), were selected in which to investigate the effect of curcumin on the anticancer action of paclitaxel and further clarify the mechanisms. Flow cytometry and MTT analysis demonstrated that curcumin significantly promoted paclitaxel-induced apoptosis and cytotoxicity in the two cervical cell lines compared with that observed with paclitaxel alone (P<0.05). Reverse transcription-polymerase chain reaction indicated that the decline of HPV E6 and E7 gene expression induced by paclitaxel was also assisted by curcumin. The expression levels of p53 protein and cleaved caspase-3 were increased significantly in the curcumin plus paclitaxel-treated HeLa and CaSki cells compared with those in the cells treated with paclitaxel alone (P<0.01). Significant reductions in the levels of phosphorylation of IκBα and the p65-NF-κB subunit in CaSki cells treated with curcumin and paclitaxel were observed compared with those in cells treated with paclitaxel alone (P<0.05). This suggests that the combined effect of curcumin and paclitaxel was associated with the NF-κB-p53-caspase-3 pathway. In conclusion, curcumin has the ability to improve the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cell lines via the NF-κB-p53-caspase-3 pathway. Curcumin in combination with paclitaxel may provide a superior therapeutic effect on human cervical cancer. PMID:25780454

  8. The effect of aloe emodin-encapsulated nanoliposome-mediated r-caspase-3 gene transfection and photodynamic therapy on human gastric cancer cells.

    PubMed

    Li, Kai-Ting; Duan, Qin-Qin; Chen, Qing; He, Juan-Wen; Tian, Si; Lin, Hai-Dan; Gao, Qing; Bai, Ding-Qun

    2016-02-01

    Gastric carcinoma (GC) has high incidence and mortality rates in China. Surgery and chemotherapy are the main treatments. Photodynamic therapy (PDT) has become a new treatment modality, appearing in recent experimental studies and clinical trials in various tumors. This study explores the combined effect of gene transfection with PDT on GC cells using aloe emodin (AE)-encapsulated nanoliposomes, which acted as gene carrier as well as one photosensitizer (PS). AE-encapsulated nanoliposomes (nano-AE) were prepared by reverse evaporation method. Electron microscopy and nano-ZS90 analyzer were used to detect its morphology, size, and wavelength. Western blot was used to detect the expression of the caspase-3 after transfection. MTT assay and flow cytometry were employed to determine the cytotoxic and apoptotic rates, respectively. Hoechst 33342 staining was adopted to detect the morphological changes in death gastric cancer cells. Cellular reactive oxygen species (ROS) contents were measured by DCFH-DA staining. Outcomes demonstrated that the nano-AE has good properties as gene delivery carriers as well as a PS. The group in which the recombinant plasmid of r-caspase-3 was transfected had higher protein expression of the caspase-3 than controls, meanwhile the proliferation rates of the transfected cells were inhibited by the nano-AE-mediated PDT in an energy-dependent manner. In addition, in the transfected cells, the death rate increased to 77.3% as assessed 12 h after PDT (6.4 J/cm(2) ). Hochest 33342 staining also revealed that the death rate increased significantly in the transfected group compared with other groups. Compared to control groups, the production of ROS in nano-AE PDT group had quadrupled in SGC-7901 cells as early as 1 h after PDT, while it is similar to the group of nano-AE transfection and PDT. Nano-AE-mediated r-caspase-3 gene transfection coupled with PDT could inhibit the proliferation rate and increase the apoptotic rate remarkably in human

  9. Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom.

    PubMed

    Gutiérrez, José María; Sanz, Libia; Escolano, José; Fernández, Julián; Lomonte, Bruno; Angulo, Yamileth; Rucavado, Alexandra; Warrell, David A; Calvete, Juan J

    2008-10-01

    The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase

  10. Beacon immunoreactivity in the rat hypothalamus.

    PubMed

    Ng, Y K; Brailoiu, G C; Dun, S L; Ling, E A; Yang, J; Chang, J K; Dun, N J

    2006-05-01

    Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.

  11. Post-ischaemic mild hypothermia inhibits apoptosis in the penumbral region by reducing neuronal nitric oxide synthase activity and thereby preventing endothelin-1-induced hydroxyl radical formation.

    PubMed

    Van Hemelrijck, An; Hachimi-Idrissi, Said; Sarre, Sophie; Ebinger, Guy; Michotte, Yvette

    2005-09-01

    Previously, we showed that treatment with resuscitative, post-ischaemic mild hypothermia (34 degrees C for 2 h) reduced apoptosis in the penumbra (cortex), but not in the core (striatum) of an endothelin-1 (Et-1)-induced focal cerebral infarct in the anaesthetized rat. Therefore, the purpose of this study was to investigate by which pathways resuscitative mild hypothermia exerts its neuroprotective effect in this model. The amino acids glutamate, serine, glutamine, alanine, taurine, arginine and the NO-related compound citrulline were sampled from the striatum and cortex of the ischaemic hemisphere using in vivo microdialysis. The in vivo salicylate trapping method was applied for monitoring hydroxyl radical formation via 2,3 dihydroxybenzoic acid (2,3 DHBA) detection. Caspase-3, neuronal nitric oxide synthase (nNOS) immunoreactivity and the volume of ischaemic damage were determined 24 h after the insult. In both the striatum and the cortex, Et-1-induced increases in glutamate, taurine and alanine were refractory to mild hypothermia. However, mild hypothermia significantly attenuated the ischaemia-induced 2,3 DHBA levels and the nNOS immunoreactivity in the cortex, but not in the striatum. These observations were associated with a decreased caspase-3 immunoreactivity. These results suggest that mild hypothermia exerts its neuroprotective effect in the penumbra partially by reducing nNOS activity and thereby preventing oxidative stress. Furthermore, we confirm our previous findings that the neuroprotective effect of resuscitative hypothermia is not mediated by changes in ischaemia-induced amino acid release as they could not be associated with the ischaemia-induced damage in the Et-1 rat model.

  12. Catestatin-like immunoreactivity in the rat eye.

    PubMed

    Gramlich, Oliver W; Lorenz, Katrin; Grus, Franz H; Kriechbaum, Maren; Ehrlich, Daniela; Humpel, Christian; Fischer-Colbrie, Reiner; Bechrakis, Nikolaos E; Troger, Josef

    2014-02-01

    The aim of the study was to investigate the presence and distribution of the chromogranin A-derived peptide catestatin in the rat eye and trigeminal ganglion by immunofluorescence using an antibody which recognizes not only free catestatin but also larger fragments containing the sequence of catestatin. Western blots were performed in an attempt to characterize the immunoreactivities detected by the catestatin antiserum. Sparse immunoreactive nerve fibers were visualized in the corneal stroma, in the chamber angle, in the sphincter muscle but also in association with the dilator muscle, in the stroma of the ciliary body and processes, but dense in the irideal stroma, around blood vessels at the limbus and in the choroid and in cells of the innermost retina representing amacrine cells as identified by colocalization with substance P. Furthermore, catestatin-immunoreactivity was detected in the trigeminal ganglion in small to medium-sized cells and there were abundant catestatin-positive nerve fibers stained throughout the stroma of the ganglion. Double immunofluorescence of catestatin with substance P revealed colocalization both in cells of the trigeminal ganglion as well as in nerve fibers in the choroid. The immunoreactivities are present obviously as free catestatin and/or small-sized catestatin-containing fragments in the retina and ocular nerves but as large processed fragments as well, weak in the retina and more prominent in remaining ocular tissues, possibly in endothelial cells. This indicates that this peptide is a constituent of sensory neurons innervating the rat eye and the presence in amacrine cells in the retina is typical for neuropeptides. Catestatin is biologically highly active and might be of significance in the pathophysiology of the eye.

  13. New avenue in the treatment of temporal lobe epilepsy by classical anti-epileptics: A hypothetical establishment of executioner Caspase 3 inactivation by molecular modeling

    PubMed Central

    Aanandhi, M. Vijey; Bhattacherjee, Debojit; Ray, Anirban; George, P. Samuel Gideon

    2015-01-01

    Patients with temporal lobe epilepsy (TLE) are prescribed first-line antiepileptic drugs and surgery to the management of this disorder. Unfortunately, the surgical treatment has been shown to be beneficial for the selected patients but fails to provide a seizure-free outcome in 20–30% of TLE patients. In our present study, we investigate the possibilities of marketed antiepileptic drugs in a different manner to improve the present situation in TLE. Molecular docking simulation study and various open source computational tools were used to perform the study. AutoDock 4.2 MGL tools, Pymol visualize tools, Patch dock server, and Swarm Dock servers (protein-protein docking) were used to perform the molecular modeling. FTsite and computed atlas of surface topography of protein open source server were used to understand the pocket and ligand binding information respectively. Toxtree application was used to determine the toxicity profile of the drug by Cramers rule. The obtained molecular docking models (Caspase 3, Procaspase 8, and Fas-associated death domain [FADD]) with selected compounds (Clonazepam, Clobazepam, and Retigabine) showed promising trio blocking event of FADD, Caspase 3, and Procaspase 8 (−6.66 kcal, −8.1 kcal, 6.46 kcal) by Clonazepam respectively. Protein-protein interaction study (Swarm Dock, Patch Dock server) indicated promising results that helped to establish our hypothesis. Toxtree showed a quantitative structure toxicity relationship report that helps to clarify the toxicity of the selected compounds. Clonazepam showed a trio inhibition property that may lead to develop a new era of the new generation benzodiazepine prototype drugs in the future. Filtered compounds will further process for higher in vitro, in vivo models for better understanding of the mechanism. PMID:25878976

  14. Targeted suppression of μ-calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle.

    PubMed

    Yang, You Bing; Pandurangan, Muthuraman; Hwang, InHo

    2012-09-01

    The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ-calpain and caspase 9, using RNAi (RNA interference)-mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ-calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)-siRNA2 and CAPN1-siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1-siRNA1 and CAPN1-siRNA4 transfected cells respectively. CAPN1-siRNA4 and CAPN1-siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)-siRNA1 and CARD9-siRNA2-treated cells showed reduction of 40 and 49% respectively. CARD9-siRNA1 and CARD9-siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9-siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross-talk between μ-calpain and the caspase enzyme systems. Suppression of target genes, such as μ-calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy. PMID:22657938

  15. Abeta[25-35] peptide and iron promote apoptosis in lymphocytes by an oxidative stress mechanism: involvement of H2O2, caspase-3, NF-kappaB, p53 and c-Jun.

    PubMed

    Velez-Pardo, Carlos; Ospina, Gloria Garcia; Jimenez del Rio, Marlene

    2002-09-01

    The Abeta deposition in the neuritic plaques is one of the major neuropathological hallmarks of the Alzheimer disease (AD). Studies in vitro have demonstrated that the Abeta[25-35] fragment, which contains the cytotoxic functional sequence of the amyloid peptide, induces neurotoxicity and cell death by apoptosis. Despite intense investigations, a complete picture of the precise molecular cascade leading to cell death in a single cellular model is still lacking. In this study, we provide evidence that Abeta[25-35] induce apoptosis either alone or in presence of iron in peripheral blood lymphocytes cells (PBL) in a concentration-dependent fashion by an oxidative stress mechanism involving: (1) the production of hydrogen peroxide (H2O2), reflected by rhodamine-positive fluorescent cells, (2) activation and/or translocation of NF-kappaB, p53 and c-Jun transcription factors showed by immunocytochemical diaminobenzidine positive nuclei, (3) activation of NF-kappaB complex by electrophoretic mobility shift assay/immuno-blotting/and ammonium pyrrolidinedithiocarbamate (PDTC) inhibition, (4) caspase-3 activation, reflected by caspase Ac-DEVD-cho inhibition, (5) mRNA synthesis de novo according to actinomycin D cell death inhibition. These results are consistent with the notion that the Abeta[25-35]/H2O2 generation precede the apoptotic process and that once H2O2 is generated, it is able to trigger a specific cell death signalisation. Thus, taken together these results, we present a well-ordered cascade of the major molecular events leading PBL to apoptosis. These results may contribute to explain the importance of Abeta alone or in the presence of redox-available iron in association with Abeta plaques (and neurofibrillary tangles) in AD brains and the significant role played by H2O2 as a second messenger of death signal in some degenerative diseases linked to oxidative stress stimuli.

  16. Aloe-Emodin Protects RIN-5F (Pancreatic β-cell) Cell from Glucotoxicity via Regulation of Pro-Inflammatory Cytokine and Downregulation of Bax and Caspase 3

    PubMed Central

    Alshatwi, Ali A; Subash-Babu, P.

    2016-01-01

    To determine the protective effect of aloe-emodin (AE) from high glucose induced toxicity in RIN-5F (pancreatic β-cell) cell and restoration of its function was analyzed. RIN-5F cells have been cultured in high glucose (25 mM glucose) condition, with and without AE treatment. RIN-5F cells cultured in high glucose decreased cell viability and increased ROS levels after 48 hr compared with standard medium (5.5 mM glucose). Glucotoxicity was confirmed by significantly increased ROS production, increased pro-inflammatory (IFN-γ, IL-1β,) & decreased anti-inflammatory (IL-6&IL-10) cytokine levels, increased DNA fragmentation. In addition, we found increased Bax, caspase 3, Fadd, and Fas and significantly reduced Bcl-2 expression after 48 hr. RIN-5F treated with both high glucose and AE (20 μM) decreased ROS generation and prevent RIN-5F cell from glucotoxicity. In addition, AE treated cells cultured in high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8hr and normal basal insulin release within 24 hr was achieved when compared to high glucose. PMID:26759701

  17. MicroRNA-421 regulated by HIF-1α promotes metastasis, inhibits apoptosis, and induces cisplatin resistance by targeting E-cadherin and caspase-3 in gastric cancer

    PubMed Central

    Li, Peng; Liu, Kaiyi; Geng, Ruixuan; Dai, Congqi; Lin, Ying; Tang, Wenbo; Wu, Zheng; Chang, Jinjia; Lu, Jianwei; Li, Jin

    2016-01-01

    Hypoxia and dysregulation of microRNAs (miRNAs) have been identified as crucial factors in carcinogenesis. However, the potential mechanisms of HIF-1α and miR-421 in gastric cancer have not been well elucidated. In this study, we found that miR-421 was up-regulated by HIF-1α. Overexpression of miR-421 promoted metastasis, inhibited apoptosis, and induced cisplatin resistance in gastric cancer in vivo and in vitro. E-cadherin and caspase-3 were identified as targets of miR-421. Besides, relative mRNA expression of miR-421 was significantly increased in gastric cancer tumor tissues compared with non-tumor tissues in a cohort of gastric cancer specimens (n=107). The expression of miR-421 was higher in advanced gastric cancers compared with localized ones. Moreover, Kaplan–Meier analysis illustrated that those patients with low levels of miR-421 had a significant longer overall survival (p = 0.006) and time to relapse (p = 0.007). Therefore, miR-421 could serve as an important prognostic marker and a potential molecular target for therapy in gastric cancer. PMID:27016414

  18. Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein-ligand interactions based on a study on caspase 3 inhibitors.

    PubMed

    Erdas, Ozlem; Andac, Cenk A; Gurkan-Alp, A Selen; Alpaslan, Ferda Nur; Buyukbingol, Erdem

    2015-01-01

    The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein-ligand complexes. CIFAP-2 method is established based on a protein-ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein-ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy ınference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein-ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3-ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC(50)) of the CASP3 inhibitors. PMID:25578823

  19. The effect of adenosine A1 receptor agonist and antagonist on p53 and caspase 3, 8, and 9 expression and apoptosis rate in MCF-7 breast cancer cell line

    PubMed Central

    Dastjerdi, Mehdi Nikbakht; Rarani, Mohammad Zamani; Valiani, Ali; Mahmoudieh, Mohsen

    2016-01-01

    Adenosine receptor family especially A1 type is expressed in breast cancer cells in which P53 and caspase genes are wild-type. The aim of this study was to investigate the correlation between A1 receptor and either cell apoptosis or proliferation and also to recognize the relationship between this receptor and P53 and the expression of caspases 3, 8 and 9 in MCF-7 cell line. MCF-7 cells were treated intermittently with A1 receptor agonist N6-Cyclopentyladenosine (CPA) and A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in different times to measure the expression of p53, caspase 3, 8 and 9 besides apoptosis and survival rate. Our findings indicated that DPCPX significantly induced apoptosis in MCF-7 cells while the cell viability was reduced specially 72 h after the treatment and the expression of p53 gene and caspase expressions was dramatically up-regulated. On the other hand, CPA increased the cell viability and reduced apoptosis in MCF-7 cells. Our results indicated a significant down-regulation in the MCF-7 mRNA expression of p53 and caspases 3, 8 and 9. Furthermore, DPCPX induced p53 and caspase 3, 8 and 9 expressions that consequently promotes the cell apoptosis in MCF-7 cells. Therefore, DPCPX can be considered as an anti-cancer drug. PMID:27651810

  20. The effect of adenosine A1 receptor agonist and antagonist on p53 and caspase 3, 8, and 9 expression and apoptosis rate in MCF-7 breast cancer cell line.

    PubMed

    Dastjerdi, Mehdi Nikbakht; Rarani, Mohammad Zamani; Valiani, Ali; Mahmoudieh, Mohsen

    2016-07-01

    Adenosine receptor family especially A1 type is expressed in breast cancer cells in which P53 and caspase genes are wild-type. The aim of this study was to investigate the correlation between A1 receptor and either cell apoptosis or proliferation and also to recognize the relationship between this receptor and P53 and the expression of caspases 3, 8 and 9 in MCF-7 cell line. MCF-7 cells were treated intermittently with A1 receptor agonist N6-Cyclopentyladenosine (CPA) and A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in different times to measure the expression of p53, caspase 3, 8 and 9 besides apoptosis and survival rate. Our findings indicated that DPCPX significantly induced apoptosis in MCF-7 cells while the cell viability was reduced specially 72 h after the treatment and the expression of p53 gene and caspase expressions was dramatically up-regulated. On the other hand, CPA increased the cell viability and reduced apoptosis in MCF-7 cells. Our results indicated a significant down-regulation in the MCF-7 mRNA expression of p53 and caspases 3, 8 and 9. Furthermore, DPCPX induced p53 and caspase 3, 8 and 9 expressions that consequently promotes the cell apoptosis in MCF-7 cells. Therefore, DPCPX can be considered as an anti-cancer drug. PMID:27651810

  1. Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

    PubMed

    Dos Santos, Maísa Pavani; Batistela, Emanuele; Pereira, Mayara Peron; Paula-Gomes, Silvia; Zanon, Neusa Maria; Kettelhut, Isis do Carmo; Karatzaferi, Christina; Andrade, Claudia Marlise Balbinotti; de França, Suélem Aparecida; Baviera, Amanda Martins; Kawashita, Nair Honda

    2016-08-01

    Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis.

  2. Monoamine neurotoxins-induced apoptosis in lymphocytes by a common oxidative stress mechanism: involvement of hydrogen peroxide (H(2)O(2)), caspase-3, and nuclear factor kappa-B (NF-kappaB), p53, c-Jun transcription factors.

    PubMed

    Del Rio, Marlene Jimenez; Velez-Pardo, Carlos

    2002-02-15

    The destruction of dopaminergic and serotonergic nerve cells by selective 6-hydroxydopamine (6-OHDA), 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT), respectively, is a commonly used tool to investigate the mapping of neuronal pathways, elucidation of function and to mimic human neurodegenerative disease such as Parkinson's and Alzheimer's diseases. Despite intense investigations, a complete picture of the precise molecular cascade leading to cell death in a single cellular model is still lacking. In this study, we provide evidence that 6-OHDA, 5,6- and 5,7-DHT toxins-induced apoptosis in peripheral blood lymphocytes cells in a concentration-dependent fashion by a common oxidative mechanism involving: (1) the oxidation of toxins into quinones and production of the by-product hydrogen peroxide, reflected by desipramine-a monoamine uptake blocker-and antioxidants inhibition, (2) activation and/or translocation of nuclear factor-kappaB, p53 and c-Jun transcription factors, showed by immunocytochemical diaminobenzidine-positive stained nuclei, (3) caspase-3 activation, reflected by caspase Ac-DEVD-CHO inhibition, (4) mRNA and protein synthesis de novo according to cycloheximide and actinomycin D cell death inhibition. These results are consistent with the notion that uptake and intracellular autoxidation of those toxins precede the apoptotic process and that once H(2)O(2) is generated, it is able to trigger a specific cell death signalisation. Thus, taken together these results, we present an ordered cascade of the major molecular events leading peripheral blood lymphocytes to apoptosis. These results may contribute to explain the importance of H(2)O(2) as a second messenger of death signal in some degenerative diseases linked to oxidative stress stimuli.

  3. Effect of Chitosan Properties on Immunoreactivity

    PubMed Central

    Ravindranathan, Sruthi; Koppolu, Bhanu prasanth; Smith, Sean G.; Zaharoff, David A.

    2016-01-01

    Chitosan is a widely investigated biopolymer in drug and gene delivery, tissue engineering and vaccine development. However, the immune response to chitosan is not clearly understood due to contradicting results in literature regarding its immunoreactivity. Thus, in this study, we analyzed effects of various biochemical properties, namely degree of deacetylation (DDA), viscosity/polymer length and endotoxin levels, on immune responses by antigen presenting cells (APCs). Chitosan solutions from various sources were treated with mouse and human APCs (macrophages and/or dendritic cells) and the amount of tumor necrosis factor-α (TNF-α) released by the cells was used as an indicator of immunoreactivity. Our results indicate that only endotoxin content and not DDA or viscosity influenced chitosan-induced immune responses. Our data also indicate that low endotoxin chitosan (<0.01 EU/mg) ranging from 20 to 600 cP and 80% to 97% DDA is essentially inert. This study emphasizes the need for more complete characterization and purification of chitosan in preclinical studies in order for this valuable biomaterial to achieve widespread clinical application. PMID:27187416

  4. Resistance exercise decreases beta-endorphin immunoreactivity.

    PubMed

    Pierce, E F; Eastman, N W; McGowan, R W; Tripathi, H; Dewey, W L; Olson, K G

    1994-09-01

    Previous research investigating the response of plasma beta-endorphins (beta-EP) to resistance exercise has resulted in equivocal findings. To examine further the effects of resistance exercise on beta-EP immunoreactivity, 10 male and 10 female college-age students participated in a series of controlled isotonic resistance exercises. The session consisted of three sets of eight repetitions at 80% of one repetition maximum (1-RM) for each of the following exercises: (1) bench press; (2) lateral pull-downs; (3) seated arm curls; and (4) military press. Blood plasma was sampled both before and after the lifting routine and beta-endorphin levels were determined by radioimmunoassay. A Students t test for paired samples indicated that mean(s.e.) plasma beta-endorphin levels after exercise (10.5(1.3) pg beta-EP ml-1) were significantly decreased as compared with pre-exercise (control) levels (16.5(1.2), P < 0.05). While the mechanism(s) contributing to the decrease in immunoreactivity is unclear, it may be the result of the synergistic effect of beta-EP clearance during rest intervals and changes in psychological states between sampling.

  5. Changes of ribosomal protein S3 immunoreactivity and its new expression in microglia in the mice hippocampus after lipopolysaccharide treatment.

    PubMed

    Lee, Hui Young; Park, Joon Ha; Lee, Choong Hyun; Yan, Bingchun; Ahn, Ji Hyeon; Lee, Young Joo; Park, Chan Woo; Cho, Jun Hwi; Choi, Soo Young; Won, Moo-Ho

    2012-05-01

    Lipopolysaccharide (LPS) has been used as a reagent for a model of systemic inflammatory response. Ribosomal protein S3 (rpS3) is a multi-functional protein that is involved in transcription, metastasis, DNA repair, and apoptosis. In the present study, we examined the changes of rpS3 immunoreactivity in the mouse hippocampus after systemic administration of 1 mg/kg of LPS. From 6 h after LPS treatment, rpS3 immunoreactivity was decreased in pyramidale cells of the hippocampus proper (CA1-CA3 regions) and in granule cells of the dentate gyrus. At this point in time, rpS3 immunoreactivity began to increase in non-pyramidal cells and non-granule cells. From 1 day after LPS treatment, rpS3 immunoreactivity in pyramidal and granule cells was hardly detected; however, strong rpS3 immunoreactivity was shown in non-pyramidal and non-granule cells. Based on double immunofluorescence staining for rpS3/ionized calcium-binding adapter 1 (Iba-1, a marker for microglia) and glial fibrillary acidic protein (GFAP, a marker for astrocytes), strong rpS3 immunoreactivity was expressed in Iba-1-immunoreactive microglia, not in GFAP-immunoreactive astrocytes, at 1 and 2 days after LPS treatment. These results indicate that rpS3 immunoreactivity changes only in pyramidal and granule cells, and rpS3 is expressed only in activated microglia after LPS treatment: this may be associated with the neuroinflammatory responses in the brain.

  6. Estrogen receptor immunoreactivity in prepubertal and adult male Syrian hamsters.

    PubMed

    Romeo, R D; Diedrich, S L; Sisk, C L

    1999-04-23

    Estrogen and estrogen receptors (ER) are involved in the expression of steroid-dependent male sexual behavior and negative feedback regulation of the hypothalamic-pituitary-gonadal axis. The purpose of the present experiment was to determine whether there are pubertal changes in ER expression in brain that are correlated with pubertal changes in responsiveness to steroid negative feedback and behavioral activation. We found equivalent numbers of ER-immunoreactive (ER-ir) cells in castrated prepubertal and adult male hamsters in nuclei that comprise the neural circuit that mediate male sexual behavior. Therefore, increases in the number of cells in these nuclei that express ER are not correlated with the increased behavioral responsiveness to steroid hormone shown by hamsters after puberty. The number of ER-ir cells in the ventral medial hypothalamus was less in adults compared with juveniles. This pubertal decrease in ER expression is correlated with the decreased responsiveness to steroid negative feedback in the adult.

  7. Immunoreactive pattern of Staphylococcus epidermidis biofilm against human whole saliva.

    PubMed

    Carvalhais, Virginia; Amado, Francisco; Cerveira, Frederico; Ferreira, Rita; Vilanova, Manuel; Cerca, Nuno; Vitorino, Rui

    2015-05-01

    Saliva is essential to interact with microorganisms in the oral cavity. Therefore, the interest in saliva antimicrobial properties is on the rise. Here, we used an immunoproteomic approach, based on protein separation of Staphylococcus epidermidis biofilms by 2DE, followed by Western-blotting, to compare human serum and saliva reactivity profile. A total of 17 proteins were identified by MALDI-TOF/TOF. Serum and saliva presented a distinct pattern of immunoreactive proteins. Our results suggest that saliva seems to have higher propensity to react against S. epidermidis proteins with oxidoreductase activity and proteins involved with L-serine metabolic processes. We show that saliva was a powerful tool for the identification of potential S. epidermidis biofilms proteins. PMID:25782040

  8. Immunoreactive pattern of Staphylococcus epidermidis biofilm against human whole saliva.

    PubMed

    Carvalhais, Virginia; Amado, Francisco; Cerveira, Frederico; Ferreira, Rita; Vilanova, Manuel; Cerca, Nuno; Vitorino, Rui

    2015-05-01

    Saliva is essential to interact with microorganisms in the oral cavity. Therefore, the interest in saliva antimicrobial properties is on the rise. Here, we used an immunoproteomic approach, based on protein separation of Staphylococcus epidermidis biofilms by 2DE, followed by Western-blotting, to compare human serum and saliva reactivity profile. A total of 17 proteins were identified by MALDI-TOF/TOF. Serum and saliva presented a distinct pattern of immunoreactive proteins. Our results suggest that saliva seems to have higher propensity to react against S. epidermidis proteins with oxidoreductase activity and proteins involved with L-serine metabolic processes. We show that saliva was a powerful tool for the identification of potential S. epidermidis biofilms proteins.

  9. Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN HELMUT; VILA, ALEJANDRO; BRECHA, NICHOLAS C.

    2009-01-01

    Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in

  10. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.

    PubMed

    Mantena, Sudheer K; Sharma, Som D; Katiyar, Santosh K

    2006-10-01

    Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P < 0.05-0.001) and induced cell death (3-51%, P < 0.01-0.001) in a dose (5-75 microM)- and time (12-72 h)-dependent manner, which was associated with an increase in G(1) arrest. G(0)/G(1) phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin kinase inhibitors (Cdki) and cyclins. Our western blot analysis showed that berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P < 0.05-0.001) than non-berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

  11. Somatostatin-like immunoreactivity in the retina.

    PubMed Central

    Yamada, T; Marshak, D; Basinger, S; Walsh, J; Morley, J; Stell, W

    1980-01-01

    A substance with somatostatin-like immunoreactivity (SLI) was found in extracts of goldfish, frog, and cow retina. Dilutions of retinal SLI parallel the standard curve for radioimmunoassay obtained with synthetic somatostatin. Chromatography of goldfish retinal extract on Sephadex G-50 revealed two peaks of SLI, one that coeluted with synthetic somatostatin and one that eluted as a larger molecule. Incubation in 8 M urea did not alter the chromatographic pattern of the extract. SLI was present in extracts of frog optic nerve and tectum in concentrations higher than those found in the retina. In goldfish retina, SLI was localized by immunofluorescence to four types of processes in the inner plexiform layer; their origins could be traced to three classes of SLI-containing cell bodies in the proximal row of the inner nuclear layer and one class in the ganglion cell layer. Localization of SLI to cells of the retina and characterizations of the molecular forms of retinal SLI suggest that the retina is a promising model system for studies on the potential neurotransmitter function of somatostatin. Images PMID:6103539

  12. Serum immunoreactive prolyl hydroxylase in inflammatory rheumatic diseases.

    PubMed Central

    Kuutti-Savolainen, E R; Kivirikko, K I; Laitinen, O

    1980-01-01

    Serum immunoreactive prolyl hydroxylase protein (S-IRPH) was measured in 56 patients with inflammatory rheumatic diseases, and the values were compared with those in 32 control subjects. S-IRPH was above the 95% confidence limit of the controls in about 70% of the patients with active systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Reiter's syndrome, Sjögren's syndrome, polyarteritis nodosa, or polymyositis. Raised values were observed in about half of the patients with an erythrocyte sedimentation rate (ESR) of 21-50 and in about 90% of those with ESR of over 50, whereas only about 10% of the patients with an inactive disease had an S-IRPH concentration exceeding this limit. Only 1 out of 8 patients with active ankylosing spondylitis had a raised S-IRPH value. The results support previous data indicating that significant changes in collagen metabolism occur in active connective tissue diseases. Assays of S-IRPH might be of some value in assessing the activity of these diseases and in monitoring the treatment provided. PMID:6251755

  13. Rewarding brain stimulation induces only sparse Fos-like immunoreactivity in dopaminergic neurons.

    PubMed

    Hunt, G E; McGregor, I S

    1998-03-01

    In this study, c-fos immunohistochemistry was used to identify the brain regions activated by rewarding brain stimulation in rats. Rats had monopolar electrodes implanted in the medial forebrain bundle and were allocated to either a self-stimulation (n = 4), yoked stimulation (n = 4) or no stimulation (n = 6) group. In a single 1 h test session, each rat in the self-stimulation group made 1000 nose poke responses with each response followed by a 0.5 s train of brain stimulation. Rats in the yoked-stimulation group were paired with a partner in the self-stimulation group and received brain stimulation whenever their partner did. However, their nose poke responses did not trigger stimulation. This yoked procedure was thus used to identify any Fos-like immunoreactivity due to operant responding. Rats in the no stimulation group were placed in the same apparatus as the other rats but received no brain stimulation and were thus used to assess baseline Fos-like immunoreactivity. Results showed that stimulation increased Fos-like immunoreactivity in many areas of the brain in both the self-stimulation and yoked groups. The areas with the highest Fos-like immunoreactivity were ipsilateral to the electrode site and included the medial prefrontal cortex, lateral septum, nucleus accumbens (shell), the medial and lateral preoptic areas, bed nucleus of the stria terminalis, central amygdala, lateral habenula, dorsomedial hypothalamus, lateral hypothalamus and the anterior ventral tegmental area. Bilateral Fos-like immunoreactivity was evident in the nucleus accumbens core, paraventricular nucleus of the hypothalamus, the retrorubral fields and the locus coeruleus. A double-labelling procedure identifying both Fos and tyrosine hydroxylase was used to show that very few (< 5%) of the A10 dopamine cell bodies in the ventral tegmental area expressed Fos following brain stimulation. In contrast, most of the noradrenergic neurons of the locus coeruleus (A6), rubrospinal tract (A5

  14. Direct reticular projections of trigeminal sensory fibers immunoreactive to CGRP: potential monosynaptic somatoautonomic projections

    PubMed Central

    Panneton, W. Michael; Gan, Qi

    2014-01-01

    Few trigeminal sensory fibers project centrally beyond the trigeminal sensory complex, with only projections of fibers carried in its sensory anterior ethmoidal (AEN) and intraoral nerves described. Fibers of the AEN project into the brainstem reticular formation where immunoreactivity against substance P and CGRP are found. We investigated whether the source of these peptides could be from trigeminal ganglion neurons by performing unilateral rhizotomies of the trigeminal root and looking for absence of label. After an 8–14 days survival, substance P immunoreactivity in the trigeminal sensory complex was diminished, but we could not conclude that the sole source of this peptide in the lateral parabrachial area and lateral reticular formation arises from primary afferent fibers. Immunoreactivity to CGRP after rhizotomy however was greatly diminished in the trigeminal sensory complex, confirming the observations of others. Moreover, CGRP immunoreactivity was nearly eliminated in fibers in the lateral parabrachial area, the caudal ventrolateral medulla, both the peri-ambiguus and ventral parts of the rostral ventrolateral medulla, in the external formation of the nucleus ambiguus, and diminished in the caudal pressor area. The nearly complete elimination of CGRP in the lateral reticular formation after rhizotomy suggests this peptide is carried in primary afferent fibers. Moreover, the arborization of CGRP immunoreactive fibers in these areas mimics that of direct projections from the AEN. Since electrical stimulation of the AEN induces cardiorespiratory adjustments including an apnea, peripheral vasoconstriction, and bradycardia similar to those seen in the mammalian diving response, we suggest these perturbations of autonomic behavior are enhanced by direct somatic primary afferent projections to these reticular neurons. We believe this to be first description of potential direct somatoautonomic projections to brainstem neurons regulating autonomic activity. PMID

  15. Development of calretinin immunoreactivity in the mouse inner ear.

    PubMed

    Dechesne, C J; Rabejac, D; Desmadryl, G

    1994-08-22

    Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and a small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretinin were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs. PMID:7983242

  16. Kir7.1 immunoreactivity in canine choroid plexus tumors.

    PubMed

    Choi, Eun Ju; Sloma, Erica A; Miller, Andrew D

    2016-07-01

    Choroid plexus neoplasms are uncommon brain tumors in dogs. Choroid plexus carcinomas often spread diffusely throughout the ventricular system and subarachnoid space and, in aggressive forms, can mimic histologic patterns of other carcinomas, including being embedded in a desmoplastic reaction. Although choroid plexus tumors (CPTs) heterogeneously express pan-cytokeratin, little is known about other markers to identify choroid plexus and their associated tumors. Kir7.1, an inward-rectifier potassium channel, is reported to have high diagnostic utility in human neuropathology to distinguish CPTs from other primary brain tumors and cerebral metastases. To determine Kir7.1 expression in the dog brain, we analyzed the immunoreactivity of Kir7.1 in normal brain, gliomas, ependymomas, CPTs, meningiomas, and carcinomas. In normal brain tissue, the immunostaining was restricted to the choroid plexus where there was robust membrane immunoreactivity along the apical border of the cells with less intense cytoplasmic staining. Similar strong immunoreactivity was detected in 12 of 12 CPTs, whereas 5 of 5 gliomas, 4 of 5 ependymomas, 5 of 5 meningiomas, and 5 of 6 carcinomas had no immunoreactivity. One ependymoma and 1 nasal carcinoma with squamous metaplasia were up to 75% immunopositive, with moderate cytoplasmic and membranous immunoreactivity, but lacking the robust apical immunoreactivity pattern. Analysis for immunoreactivity in a tissue microarray failed to yield any other locations in which immunoreactivity was detected. These results, including the distinctive pattern of immunostaining in CPTs, suggest that Kir7.1 is an excellent marker for CPTs in the dog. PMID:27216721

  17. Selenium reduces the proapoptotic signaling associated to NF-kappaB pathway and stimulates glutathione peroxidase activity during excitotoxic damage produced by quinolinate in rat corpus striatum.

    PubMed

    Santamaría, Abel; Vázquez-Román, Beatriz; La Cruz, Verónica Pérez-De; González-Cortés, Carolina; Trejo-Solís, Ma Cristina; Galván-Arzate, Sonia; Jara-Prado, Aurelio; Guevara-Fonseca, Jorge; Ali, Syed F

    2005-12-15

    Quinolinate (QUIN) neurotoxicity has been attributed to degenerative events in nerve tissue produced by sustained activation of N-methyl-D-aspartate receptor (NMDAr) and oxidative stress. We have recently described the protective effects that selenium (Se), an antioxidant, produces on different markers of QUIN-induced neurotoxicity (Santamaría et al., 2003, J Neurochem 86:479-488.). However, the mechanisms by which Se exerts its protective actions remain unclear. Since some of these events are thought to be related with inhibition of deadly molecular cascades through the activation of antioxidant selenoproteins, in this study we investigated the effects of Se on QUIN-induced cell damage elicited by the nuclear factor kappaB (NF-kappaB) pathway, as well as the time-course response of striatal glutathione peroxidase (GPx) activity. Se (sodium selenite, 0.625 mg/kg/day, i.p.) was administered to rats for 5 days, and 120 min after the last administration, animals received a single striatal injection of QUIN (240 nmol/mul). Twenty-four hours later, their striata were tested for the expression of IkappaB-alpha (the NF-kappaB cytosolic binding protein), the immunohistochemical expression of NF-kappaB (evidenced as nuclear expression of P65), caspase-3-like activation, and DNA fragmentation. Additional groups were killed at 2, 6, and 24 h for measurement of GPx activity. Se reduced the QUIN-induced decrease in IkappaB-alpha expression, evidencing a reduction in its cytosolic degradation. Se also prevented the QUIN-induced increase in P65-immunoreactive cells, suggesting a reduction of NF-kappaB nuclear translocation. Caspase-3-like activation and DNA fragmentation produced by QUIN were also inhibited by Se. Striatal GPx activity was stimulated by Se at 2 and 6 h, but not at 24 h postlesion. Altogether, these data suggest that the protective effects exerted by Se on QUIN-induced neurotoxicity are partially mediated by the inhibition of proapoptotic events underlying Ikappa

  18. MicroRNA-148b Acts as a Tumor Suppressor in Cervical Cancer by Inducing G1/S-Phase Cell Cycle Arrest and Apoptosis in a Caspase-3-Dependent Manner

    PubMed Central

    Mou, Zongmei; Xu, Xiangting; Dong, Mei; Xu, Jin

    2016-01-01

    Background The purpose of our study was to investigate the role of microRNA (miR)-148b in cervical cancer. Material/Methods The expression of miR-148b was determined in HPV-16-immortalized cervical epithelial cell line CRL-2614 cells and in cervical cancer cell line HeLa cells. The miR-148b mimics or scrambled RNA were then transfected into Hela cells. Forty-eight hours after transfection, the mRNA expression of miR-148b and DNA methyltransferase 1 (DNMT1) were confirmed. Cell proliferation ability (cell viability and colony formation ability), invasion ability, and apoptosis were assessed after transfection with miR-148b mimics or scrambled RNA, as well as the protein expression of cyclin D1 and caspase-3. Results The expression of miR-148b was significantly downregulated in HeLa cells compared with CRL2614 cells (P<0.05), but was statistically upregulated by transfection with miR-148b mimics compared with the cells transfected with scrambled RNA (P<0.05). Also, we found that the expression of DNMT1 was significantly decreased by transfection with miR-148b mimics (P<0.05). Additionally, miR-148b mimics significantly decreased the cell proliferation ability and invasion ability, and statistically induced apoptosis. Furthermore, the expression of cyclin D1 protein was significantly decreased and the expression of caspase-3 protein was significantly increased by miR-148b mimics compared with that in the cells transfected with scrambled RNA (P<0.05). Conclusions Our results suggest that overexpression of miR-148b protects against cervical cancer by inducing G1/S-phase cell cycle arrest and apoptosis through caspase-3-dependent manner, and overexpression of miR-148b might develop a therapeutic intervention for cervical cancer. PMID:27505047

  19. Properties of immunoreactive glucagon fractions of canine stomach and pancreas.

    PubMed

    Srikant, C B; McCorkle, K; Unger, R H

    1977-03-25

    The present study was designed to identify the physicochemical, immunologic, and biologic properties of the immunoreactive glucagon (IRG) moieties of canine gastric fundus and to compare them with those of the canine pancreas. Acid-alcohol extracts of the gastric fundus and pancreas of dogs were subjected to Bio-Gel P-10 chromatography, The elution profiles of extracts of both organs revealed IRG peaks in the Mr = 2,000 3,500, and 9,000 zones; in the gastric extracts, a void volume peak was also present. On the basis of Sephadex G-150 rechromatography and sucrose density gradient ultracentrifugation the latter IRG was estimated to have a Mr = 65,000. Incubation of fundic IRG65,000 in 8 M urea failed to alter its elution position. Its pI was 6.4, while fundic IRG3,500 had a pI of 6.15 and pancreatic glucagon 6.25. Fundic IRG9,000 had a pI of 4.5 and pancreatic IRG9,000 4.65. Dilution curves of these three fundic and two pancreatic IRGs were parallel to crystalline beef-pork glucagon. The glycogenolytic activity of fundic IRG3,500 and IRG65,000, measured in the isolated rat liver system, was not different from that of immunoequivalent amounts of dog pancreatic glucagon or crystalline beef-pork glucagon. Both fundic and pancreatic IRG9,000 were devoid of glycogenolytic activity and lacker adenylate cyclase stimulating activity and 125I-glucagon displacing activity when tested on partially purified rat liver membranes. Fundic IRG65,000, however, stimulated adenylate cyclase and displaced 125I-glucagon to the same degree as immunoequivalent amounts of pancreatic glucagon. Fundic IRG3,500 was more active than pancreatic glucagon in stimulating adenylate cyclase activity. This was not clearly attributable to differences in binding to liver cell membranes.

  20. Localization of tachykinin receptors and Fos-like immunoreactivity induced by substance P in guinea-pig brain.

    PubMed

    Yip, J; Chahl, L A

    2000-11-01

    1. In the present study, a comparison was made between the distribution of tachykinin NK1 and NK3 receptor immunoreactivity and the distribution of Fos-like immunoreactivity induced by the tachykinin agonist substance P (SP) in the guinea-pig brain. 2. In agreement with results from previous studies in rat brain, NK1 receptor-immunoreactive neurons were found to be widely distributed throughout the brain in the striatum and in diencephalic and mesencephalic structures, while NK3 receptor-immunoreactive neurons were mainly in telencephalic structures. Considerable overlap was observed between NK1 and NK3 receptor distributions. 3. Substance P induced Fos-like immunoreactivity (Fos-LI) in extensive areas of the guinea-pig brain. The induction of Fos-LI was markedly inhibited in many areas by pretreatment with the NK1 receptor antagonist SR 140333. The NK3 receptor antagonist SR 142801 reduced Fos-LI staining in fewer areas, although a reduction was observed in the cortex, striatum and hypothalamus. 4. In general, tachykinin receptors were located at sites corresponding to areas of functional activation by SP, as shown by Fos-LI. These results extend previous studies by adding a functional dimension to tachykinin receptor localization studies.

  1. Distribution of beacon immunoreactivity in the rat brain.

    PubMed

    Wang, Fei; Tian, De-Run; Tian, Nan; Chen, Hui; Shi, Yu-Shun; Chang, Jaw-Kang; Yang, Jun; Yuan, Lan; Han, Ji-Sheng

    2006-01-01

    Beacon is a novel peptide isolated from the hypothalamus of Israeli sand rat. In the present study, we determined the distribution of beacon in the rat brain using immunohistochemical approach with a polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73). The hypothalamus represented the major site of beacon-immunoreactive (IR) cell bodies that were concentrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Additional immunostained cells were found in the septum, bed nucleus of the stria terminalis, subfornical organ and subcommissural organ. Beacon-IR fibers were seen with high density in the internal layer of the median eminence and low to moderate density in the external layer. Significant beacon-IR fibers were also seen in the nucleus of the solitary tract and lateral reticular formation. The beacon neurons found in the PVN were further characterized by double label immunohistochemistry. Several beacon-IR neurons that resided in the medial PVN were shown to coexpress corticotrophin-releasing hormone (CRH) and most labeled beacon fibers in the external layer of median eminence coexist with CRH. The topographical distribution of beacon-IR in the brain suggests multiple biological activities for beacon in addition to its proposed roles in modulating feeding behaviors and pituitary hormone release.

  2. Effects of Intraduodenal Administration of HCl and Glucose on Circulating Immunoreactive Secretin and Insulin Concentrations

    PubMed Central

    Boden, Guenther; Essa, Noorjehan; Owen, Oliver E.; Reichle, Frederick A.; Saraga, Walter

    1974-01-01

    A new radioimmunoassay for secretin was used to investigate (a) serum secretin responses to intraduodenally infused HCl and glucose, (b) the metabolic half-life and the volume of distribution of exogenous secretin and (c) the effect of endogenously released secretin on insulin secretion in 25 anesthetized dogs. Portal and femoral venous blood samples were taken simultaneously before, during, and after intraduodenal infusion of HCl (21 meq/30 min) and glucose (131 ml/30 min). Control experiments were performed with intraduodenal infusion of saline. Mean portal venous immunoreactive secretin concentration of six dogs rose from 313 μU/ml before to 1,060 μU/ml 10 min after initiation of the intestinal acidification (P < 0.005). Femoral venous immunoreactive secretin concentration rose from 220 μU/ml before to 567 μU/ml 15 min after intestinal acidification (P < 0.01). Secretin concentrations remained elevated during the remainder of the infusion. In the same six dogs mean portal venous immunoreactive insulin concentration rose from 38 μU/ml before to 62 μU/ml at the end of the infusion (P < 0.05). Peripheral immunoreactive insulin, glucose, and free fatty acid concentrations, however, did not change significantly. Pancreatic exocrine function was studied in four dogs. The rise in secretin concentration was followed promptly by a highly significant increase in exocrine pancreatic flow rate and bicarbonate secretion, indicating biological activity of the circulating immunoreactive secretin. The effect of intraduodenal infusion of glucose on immunoreactive secretin concentration was studied in 12 dogs. Glucose in concentrations ranging from 2.5% to 10% had no detectable influence on portal or peripheral secretin concentration. Infusion of 50% glucose caused a slight decline in secretin concentration. The metabolic clearance rate, half-life of disappearance, and volume of distribution of exogenous secretin was studied in three dogs by the constant infusion technic

  3. Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.

    PubMed Central

    Rothenberg, S P; Iqbal, M P

    1982-01-01

    A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of [3H]methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme. Images PMID:6952216

  4. Protective Effects of Carvedilol and Vitamin C against Azithromycin-Induced Cardiotoxicity in Rats via Decreasing ROS, IL1-β, and TNF-α Production and Inhibiting NF-κB and Caspase-3 Expression

    PubMed Central

    El-Shitany, Nagla A.; El-Desoky, Karema

    2016-01-01

    The Food and Drug Administration recently warned of the fatal cardiovascular risks of azithromycin in humans. In addition, a recently published study documented azithromycin-induced cardiotoxicity in rats. This study aimed to justify the exact cardiovascular events accompanying azithromycin administration in rats, focusing on electrocardiographic, biochemical, and histopathological changes. In addition, the underlying mechanisms were studied regarding reactive oxygen species production, cytokine release, and apoptotic cell-death. Finally, the supposed protective effects of both carvedilol and vitamin C were assessed. Four groups of rats were used: (1) control, (2) azithromycin, (3) azithromycin + carvedilol, and (4) azithromycin + vitamin C. Azithromycin resulted in marked atrophy of cardiac muscle fibers and electrocardiographic segment alteration. It increased the heart rate, lactate dehydrogenase, creatine phosphokinase, malondialdehyde, nitric oxide, interleukin-1 beta (IL1-β), tumor necrosis factor alpha (TNF-α), nuclear factor kappa beta (NF-κB), and caspase-3. It decreased reduced glutathione, glutathione peroxidase, and superoxide dismutase. Carvedilol and vitamin C prevented most of the azithromycin-induced electrocardiographic and histopathological changes. Carvedilol and vitamin C decreased lactate dehydrogenase, malondialdehyde, IL1-β, TNF-α, NF-κB, and caspase-3. Both agents increased glutathione peroxidase. This study shows that both carvedilol and vitamin C protect against azithromycin-induced cardiotoxicity through antioxidant, immunomodulatory, and antiapoptotic mechanisms. PMID:27274777

  5. Protein profiles and immunoreactivities of Acanthamoeba morphological groups and genotypes.

    PubMed

    Pumidonming, Wilawan; Koehsler, Martina; Leitsch, David; Walochnik, Julia

    2014-11-01

    Acanthamoeba is a free-living protozoan found in a wide variety of habitats. A classification of Acanthamoeba into currently eighteen genotypes (T1-T18) has been established, however, data on differences between genotypes on the protein level are scarce. The aim of this study was to compare protein and immunoreactivity profiles of Acanthamoeba genotypes. Thirteen strains, both clinical and non-clinical, from genotypes T4, T5, T6, T7, T9, T11 and T12, representing three morphological groups, were investigated for their protein profiles and IgG, IgM and IgA immunoreactivities. It was shown that protein and immunoreactivity profiles of Acanthamoeba genotypes T4, T5, T6, T7, T9, T11 and T12 are clearly distinct from each other, but the banding patterns correlate to the morphological groups. Normal human sera revealed anti-Acanthamoeba antibodies against isolates of all investigated genotypes, interestingly, however only very weak IgM and virtually no IgA immunoreactivity with T7 and T9, both representing morphological group I. The strongest IgG, IgM and IgA immunoreactivities were observed for genotypes T4, T5 and T6. Differences of both, protein and immunological patterns, between cytopathic and non-cytopathic strains, particularly within genotype T4, were not at the level of banding patterns, but rather in expression levels.

  6. Effect of pulsed light on structure and immunoreactivity of gluten.

    PubMed

    Panozzo, Agnese; Manzocco, Lara; Lippe, Giovanna; Nicoli, Maria Cristina

    2016-03-01

    The effect of pulsed light (from 1.75 to 26.25Jcm(-2)) on selected properties of wheat gluten powder and aqueous suspension (absorbance, particle size and microstructure, free sulfhydryl content, protein fractions, protein electrophoretic mobility and immunoreactivity) was investigated. Gluten photoreactivity was strongly affected by hydration. While minor photo-induced structure modifications were observed in gluten powder, pulsed light induced the development of browning and promoted partial depolymerisation of hydrated gluten proteins by disulphide exchange. These changes were associated with a significant decrease in immunoreactivity, suggesting that pulsed light could be exploited to efficiently modify structure and thus functionality of gluten.

  7. Determination of immunoreactive fraction of radiolabeled monoclonal antibodies: what is an appropriate method?

    PubMed

    Konishi, Shota; Hamacher, Klaus; Vallabhajosula, Shankar; Kothari, Paresh; Bastidas, Diago; Bander, Neil; Goldsmith, Stanley

    2004-12-01

    Determination of the immunoreactive fraction (IF) of radiolabeled monoclonal antibodies (MAb) is essential to the understanding of the effects of radiolabeling and subsequent target-specific tumor localization. There has been generally no accepted method of determining the IF of MAbs. The conventional method is based on a radioimmunoassay technique in which the fraction of radiolabeled MAb bound to antigen under conditions of "antigen excess" is determined. Lindmo et al. introduced a modified method in which the IF is determined by extrapolation to conditions representing "infinite antigen excess." Although the Lindmo method, in principle, is insensitive to experimental parameters, it does not always provide a reliable estimate of IF. We, therefore, evaluated an alternate method in which percent cell bound fraction is measured under conditions of fixed antigen concentration and various dilutions of radiolabeled MAb. We developed a mathematical equation to estimate immunoreactivity. J591 MAb specific for prostate-specific membrane antigen was radiolabeled with (111)In, (90)Y and (177)Lu to specific activities of 1-20 mCi/mg. We compared the effect of several experimental conditions on the determination of IF using all three different methods. The Lindmo method requires careful optimization of experimental conditions for each radiolabeled MAb. The alternate method, based on a fixed antigen concentration, appears to be practical and may provide a more reliable measure of immunoreactivity.

  8. Orexin (hypocretin)-like immunoreactivity in the cat hypothalamus: a light and electron microscopic study.

    PubMed

    Zhang, J H; Sampogna, S; Morales, F R; Chase, M H

    2001-02-01

    Orexin-A-like immunoreactive (OrA-ir) neurons and terminals in the cat hypothalamus were examined using immunohistochemical techniques. OrA-ir neurons were found principally in the lateral hypothalamic area (LHA) at the level of the tuberal cinereum and in the dorsal and posterior hypothalamic areas. In the LHA the majority of the neurons were located dorsal and lateral to the fornix; a small number of OrA-ir neurons were also present in other regions of the hypothalamus. OrA-ir fibers with varicose terminals were detected in almost all hypothalamic regions. The high density of fibers was located in the suprachiasmatic nucleus, the infundibular nucleus (INF), the tuberomamillary nucleus (TM) and the supra- and pre-mamillary nuclei. Ultrastructural analysis revealed that OrA-ir neurons in the LHA receive abundant input from non-immunoreactive terminals. These terminals, which contained many small, clear, round vesicles with a few large, dense core vesicles, made asymmetrical synaptic contacts with OrA-ir dendrites, indicating that the activity of orexin neurons is under excitatory control. On the other hand, the terminals of OrA-ir neurons also made asymmetrical synaptic contact with dendrites in the LHA, the INF and the TM. The dendrites in the LHA were both non-immunoreactive and OrA-ir; conversely, the dendrites in the INF and the TM were non-immunoreactive. In these regions, OrA-ir terminals contained many small, clear, round vesicles with few large, dense core vesicles, suggesting that orexinergic neurons also provide excitatory input to other neurons in these regions. PMID:11204055

  9. 21 CFR 862.1405 - Immunoreactive insulin test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunoreactive insulin test system. 862.1405 Section 862.1405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... diabetes mellitus, and hypoglycemia. (b) Classification. Class I (general controls). The device is...

  10. 21 CFR 862.1405 - Immunoreactive insulin test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunoreactive insulin test system. 862.1405 Section 862.1405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... diabetes mellitus, and hypoglycemia. (b) Classification. Class I (general controls). The device is...

  11. 21 CFR 862.1405 - Immunoreactive insulin test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunoreactive insulin test system. 862.1405 Section 862.1405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... diabetes mellitus, and hypoglycemia. (b) Classification. Class I (general controls). The device is...

  12. 21 CFR 862.1405 - Immunoreactive insulin test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunoreactive insulin test system. 862.1405 Section 862.1405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  13. Endogenous morphine-like compound immunoreactivity increases in parkinsonism.

    PubMed

    Charron, Giselle; Doudnikoff, Evelyne; Laux, Alexis; Berthet, Amandine; Porras, Gregory; Canron, Marie-Hélène; Barroso-Chinea, Pedro; Li, Qin; Qin, Chuan; Nosten-Bertrand, Marika; Giros, Bruno; Delalande, François; Van Dorsselaer, Alain; Vital, Anne; Goumon, Yannick; Bezard, Erwan

    2011-08-01

    Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for endogenous morphine formation. As Parkinson's disease results from the loss of dopamine and is associated with central pain, we considered how endogenous morphine is regulated in the untreated and l-DOPA-treated parkinsonian brain. However, as the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinson's disease-like and l-DOPA-treated Parkinson's disease-like conditions in experimental (rat and monkey) and human Parkinson's disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunoreactivity and levels in experimental and human Parkinson's disease, only partially normalized by l-DOPA treatment. Our data suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain experiences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinson's disease.

  14. Assessment of expressions of Bcl-XL, b-FGF, Bmp-2, Caspase-3, PDGFR-α, Smad1 and TGF-β1 genes in a rat model of lung ischemia/reperfusion

    PubMed Central

    Şimşek, Hasan; Demiryürek, Şeniz; Demir, Tuncer; Atabay, Hüsne Didem; Çeribasi, Ali Osman; Bayraktar, Recep; Kaplan, Davut Sinan; Öztuzcu, Serdar; Cengiz, Beyhan

    2016-01-01

    Objective(s): Ischemia is described as organs and tissues are destitute of oxygen due to decreased arterial or venous blood flow. Many mechanisms play role in cell death happened as a consequence of a new blood flow is needed for both cell regeneration and to clean toxic metabolites during ischemia and later. Lung damage induced by ischemia/reperfusion (I/R) is a frequent problem in lung transplantation. Apoptosis (programmed cell death) is known as cell suicide, and plays a key role in embryonic developmental and in maintain adult tissue’s life. Materials and Methods: It is investigated expressions of Smad1, Bmp-2, Bcl-XL, b-FGF, Caspase-3, TGF-β1, PDGFR-α genes for molecular changes in lung tissues, after I/R is formed, in this study. For this, we included 40 Wistar albino rats to this study and divided 4 groups (n=10). The Groups were determined as Control (C), Group 1= 1 hr ischemia (I), Group 2= 1 hr ischemia+2 hr reperfusion (I+2R), Group 3= 1 hr ischemia+4 hr reperfusion (I+4R). Besides, molecular analysis and histopathologic examinations of tissues were performed, and the results were evaluated by normalization and statistics analysis. Results: We have found a significant increase in expression of Bcl-XL (P=0.046) and Caspase-3 (P=0.026) genes of group 1, and it was not monitored any significant difference in Group 2 and Group 3. In all groups, the changes in b-FGF (P=0.087), Bmp-2 (P=0.457), TGF-β1 (P=0.201) and PDGFR-α (P=0.116) were not significant compared to control group. We did not see any mRNA expression of Smad1 gene in all groups include control. Conclusion: These findings suggest that I/R injury may trigger apoptotic mechanism in lung. PMID:27081467

  15. Localization of Neuropeptide Y1 Receptor Immunoreactivity in the Rat Retina and the Synaptic Connectivity of Y1 Immunoreactive Cells

    PubMed Central

    D'Angelo, Iona; Oh, Su-Ja; Chun, Myung-Hoon; Brecha, Nicholas C.

    2010-01-01

    Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1–y6) G-protein–coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells. PMID:12455004

  16. Streptozotocin induced activation of oxidative stress responsive splenic cell signaling pathways: Protective role of arjunolic acid

    SciTech Connect

    Manna, Prasenjit; Ghosh, Jyotirmoy; Das, Joydeep

    2010-04-15

    Present study investigates the beneficial role of arjunolic acid (AA) against the alteration in the cytokine levels and simultaneous activation of oxidative stress responsive signaling pathways in spleen under hyperglycemic condition. Diabetes was induced by injection of streptozotocin (STZ) (at a dose of 70 mg/kg body weight, injected in the tail vain). STZ administration elevated the levels of IL-2 as well as IFN-gamma and attenuated the level of TNF-alpha in the sera of diabetic animals. In addition, hyperglycemia is also associated with the increased production of intracellular reactive intermediates resulting with the elevation in lipid peroxidation, protein carbonylation and reduction in intracellular antioxidant defense. Investigating the oxidative stress responsive cell signaling pathways, increased expressions (immunoreactive concentrations) of phosphorylated p65 as well as its inhibitor protein phospho IkappaBalpha and phosphorylated mitogen activated protein kinases (MAPKs) have been observed in diabetic spleen tissue. Studies on isolated splenocytes revealed that hyperglycemia caused disruption of mitochondrial membrane potential, elevation in the concentration of cytosolic cytochrome c as well as activation of caspase 3 leading to apoptotic cell death. Histological examination revealed that diabetic induction depleted the white pulp scoring which is in agreement with the reduced immunological response. Treatment with AA prevented the hyperglycemia and its associated pathogenesis in spleen tissue. Results suggest that AA might act as an anti-diabetic and immunomodulatory agent against hyperglycemia.

  17. Localization of cholecystokinin-like immunoreactivity in isolated nerve terminals.

    PubMed Central

    Pinget, M; Straus, E; Yalow, R S

    1978-01-01

    Subcellular fractionation of the rat cerebral cortex demonstrated the presence of immunoreactive cholecystokinin in the pellet identified by electron microscopy as containing a high proportion of synaptic vesicles. The recovery in this pellet of 40% of the total immunoreactivity in the initial cortical extract is quite comparable to the recovery of other peptides such as vasoactive intestinal polypeptide and somatostatin, which are also located in synaptosomes and for which roles as neuroregulators or transmitters have been suggested. The evidence of concentration of cholecystokinin-like peptides in the synaptosomal pellet is consistent with our earlier demonstration by immunohistochemical techniques of cholecystokinin's presence in rabbit cerebral cortical neurons. These observations and the evidence for diminished concentration of cholecystokinin-like peptides in the brains of hyperphagic mice are consistent with cholecystolinin's suggested role as a neuroregulator for appetite. Images PMID:282649

  18. Growth-hormone-releasing factor immunoreactivity in human endocrine tumors.

    PubMed Central

    Bostwick, D. G.; Quan, R.; Hoffman, A. R.; Webber, R. J.; Chang, J. K.; Bensch, K. G.

    1984-01-01

    Seventy-three human tumors and adjacent nonneoplastic tissues were analyzed immunohistochemically for the presence of growth-hormone-releasing factor (GRF). Four of 9 pancreatic endocrine tumors, 2 of 3 appendiceal carcinoids, and 1 of 5 cecal carcinoids were immunoreactive for GRF. One of the GRF-containing pancreatic tumors was associated with acromegaly. Histologically, the growth patterns of these tumors were variable, and the distribution of immunoreactive cells was patchy and irregular. There were no normal cells that contained GRF. These results indicate that GRF production by human tumors is more common than previously thought, although clinical acromegaly may not be apparent in patients who harbor such neoplasms. Images Figure 1 PMID:6093542

  19. gamma. sub 2 -MSH immunoreactivity in the human heart

    SciTech Connect

    Ekman, R.; Bjartell, A.; Lisander, J.; Edvinsson, L. )

    1989-01-01

    In patients undergoing aorto-coronary by-pass surgery, we found a 26% arterial-venous difference of immunoreactive {gamma}{sub 2}-melanocytostimulating hormone (MSH), a proopiomelanocortin (POMC) derived peptide known to possess profound hemodynamic effects. These results prompted an investigation of the presence of {gamma}{sub 2}-MSH in the human heart. Using a two-step extraction procedure, regions of human hearts were examined by sensitive and specific radioimmunoassays to determine their {gamma}{sub 2}-MSH content. Mean ({plus minus} SEM) concentrations of 0.14 {plus minus} 0.023 pmol/g and 0.12 {plus minus} 0.017 were found in right atrium and right ventricle, respectively. High performance liquid chromatography indicated that 80-90 % of the total immunoreactivity eluted in a single sharp peak in a position identical to that of synthetic {gamma}{sub 2}-MSH.

  20. Tail pinch induces fos immunoreactivity within several regions of the male rat brain: effects of age.

    PubMed

    Smith, W J; Stewart, J; Pfaus, J G

    1997-05-01

    Brief, intermittent stressors, such as low-level foot shock or tail pinch, induce a general excitement and autonomic arousal in rats that increases their sensitivity to external incentives. Such stimulation can facilitate a variety of behaviors, including feeding, aggression, sexual activity, parental behavior, and drug taking if the appropriate stimuli exist in the environment. However, the ability of tail pinch to induce general arousal and incentive motivation appears to diminish with age. Here we report on the ability of tail pinch to induce Fos immunoreactivity within several brain regions as a function of age. Young (2-3 months) and middle-aged (12-13 months) male rats were administered either five tail pinches (one every 2 min), one tail pinch, or zero (sham) tail pinches (n = 4 per stimulation condition). Rats were sacrificed 75 min following the onset of stimulation, and their brains were prepared for immunocytochemical detection of Fos protein. Fos immunoreactivity was induced by one and five tail pinches in several brain regions, including the anterior medial preoptic area (mPOA), paraventricular nucleus of the hypothalamus (PVN), paraventricular nucleus of the thalamus (PV-Thal), medial amygdala (MEA), basolateral amygdala (BLA), lateral habenula (LHab), and ventral tegmental area (VTA), of young rats compared with those that received zero tail pinches. In contrast to young rats, middle-aged rats had significantly less Fos induced by one and five tail pinches in the mPOA, PVN, MEA, BLA, and VTA, but an equivalent amount induced in the LHab. Fos immunoreactivity was not found within the medial prefrontal cortex, nucleus accumbens, striatum, lateral septum, or locus coeruleus in either young or old rats. Tail pinch appears to activate regions of the brain known to be involved in behavioral responses to both incentive cues and stressors. The lower level of cellular reactivity to tail pinch in middle-aged rats suggests a diminished neural responsiveness to

  1. Immunoreactivity for sex steroid hormone receptors in pulmonary hamartomas.

    PubMed

    Pelosi, Giuseppe; Rosai, Juan; Viale, Giuseppe

    2006-07-01

    Sex steroid hormone [ie, estrogen (ER), progesterone (PgR), and androgen (AR)] receptors have been identified previously in normal salivary glands and, more variably, in salivary gland and salivary gland-type tumors. No data are available, however, on their expression in pulmonary hamartoma, a benign biphasic tumor consisting of reactive epithelial cells and neoplastic fibromyxoid stroma, cartilage and fat, which shares some morphologic, immunophenotypic, and genotypic features to pleomorphic adenoma of major salivary glands. Thirty pulmonary hamartomas (15 in male patients and 15 in age-matched female patients), were evaluated for ER, PgR, and AR immunoreactivity, and also for mesenchymal, epithelial, and myoepithelial markers, in the fibromyxoid, epithelial, and chondroid components. ER immunoreactivity was encountered in 90% of hamartomas, PgRs in 90%, and ARs in 53% (P<0.001), but not in normal lung tissues. ARs were confined to males (P<0.001), with a marginal prevalence in the fibromyxoid component (P=0.067). PgRs and ERs were instead present in both sex, with the former being restricted to the fibromyxoid stromal component (P<0.001) and the latter preferentially located in epithelial cells (P=0.107). In most cases, fibromyxoid stroma and spindle cells surrounding the chondroid foci displayed simultaneous immunoreactivity for ERs, PgRs, and ARs, along with immunoreactivity for vimentin, S-100 protein, glial fibrillary acid protein, smooth muscle actin, and calponin but lack of staining for cytokeratins. This profile is consistent with an incomplete myoepithelial differentiation of the receptor-expressing mesenchymal cells. In conclusion, sex steroid hormone receptor expression is a nonrandom event in pulmonary hamartoma, and may be related to the development and growth of this tumor.

  2. Substance P immunoreactivity increases following human traumatic brain injury.

    PubMed

    Zacest, Andrew C; Vink, Robert; Manavis, Jim; Sarvestani, Ghafar T; Blumbergs, Peter C

    2010-01-01

    Recent experimental evidence suggests that neuropeptides, and in particular substance P (SP), are released following traumatic brain injury (TBI) and may play a significant role in the aetiology of cerebral edema and increased intracranial pressure. Whether SP may play a similar role in clinical TBI remains unknown and was investigated in the current study. Archival post-mortem material was selected from patients who had sustained TBI, had died and had undergone post-mortem and detailed neuropathological examination (n = 13). A second cohort of patients who had died, but who showed no neuropathological abnormality (n = 10), served as case controls. Changes in SP immunoreactivity were examined in the cerebral cortex directly beneath the subdural haematoma in 7 TBI cases and in proximity to contusions in the other 6 cases. Increased SP perivascular immunoreactivity was observed after TBI in 10/13 cases, cortical neurones in 12/13 and astrocytes in 10/13 cases. Perivascular axonal injury was observed by amyloid precursor protein (APP) immunoreactivity in 6/13 TBI cases. Co-localization of SP and APP in a small subset of perivascular fibres suggests perivascular axonal injury could be a mechanism of release of this neuropeptide. The abundance of SP fibres around the human cerebral microvasculature, particularly post capillary venules, together with the changes observed following TBI in perivascular axons, cortical neurones and astrocytes suggest a potentially important role for substance P in neurogenic inflammation following human TBI. PMID:19812951

  3. Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy.

    PubMed

    Serrano, J; Uttenthal, L O; Martínez, A; Fernández, A P; Martínez de Velasco, J; Alonso, D; Bentura, M L; Santacana, M; Gallardo, J R; Martínez-Murillo, R; Cuttitta, F; Rodrigo, J

    2000-01-24

    Adrenomedullin is a peptide of marked vasodilator activity first isolated from human pheochromocytoma and subsequently demonstrated in other mammalian tissues. Using a polyclonal antiserum against human adrenomedullin-(22-52) amide and the avidin-biotin peroxidase complex technique, we have demonstrated by light and electron microscopy that adrenomedullin-like immunoreactivity is widely distributed in the rat central nervous system. Western blotting of extracts of different brain regions demonstrated the fully processed peptide as the major form in the cerebellum, whereas a 14-kDa molecular species and a small amount of the 18-kDa propeptide were present in other brain regions. Immunoreactive neurons and processes were found in multipolar neurons and pyramidal cells of layers IV-VI of the cerebral cortex and their apical processes, as well as in a large number of telencephalic, diencephalic, mesencephalic, pontine and medullary nuclei. Cerebellar Purkinje cells and mossy terminal nerve fibers as well as neurons of the cerebellar nuclei were immunostained, as were neurons in area 9 of the anterior horn of the spinal cord. Immunoreactivity was also found in some vascular endothelial cells and surrounding processes that probably originated from perivascular glial cells. Electron microscopy confirmed the light microscopy findings and showed the reaction product in relation to neurofilaments and the external membrane of small mitochondria. Immunoreactive terminal boutons were occasionally seen. The distribution of adrenomedullin-like immunoreactivity in the central nervous system suggests that it has a significant role in neuronal function as well as in the regulation of regional blood flow.

  4. Cytoplasmic p53 and Activated Bax Regulate p53-dependent, Transcription-independent Neural Precursor Cell Apoptosis

    PubMed Central

    Geng, Ying; Walls, K.C.; Ghosh, Arindam P.; Akhtar, Rizwan S.; Klocke, Barbara J.; Roth, Kevin A.

    2010-01-01

    The prodeath effects of p53 are typically mediated via its transcriptional upregulation of proapoptotic Bcl-2 family members, including PUMA, Noxa, and/or Bax. We previously reported that staurosporine (STS), a broad-spectrum kinase inhibitor and prototypical apoptosis-inducing agent, produced p53-dependent, Bax-dependent, neural precursor cell (NPC) apoptosis, but that this effect occurred independently of new gene transcription and PUMA expression. To further characterize the mechanism by which p53 regulates NPC death, we used primary cerebellar NPCs derived from wild-type, p53-deficient, and Bax-deficient neonatal mice and the mouse cerebellar neural stem cell line, C17.2. We found that STS rapidly increased p53 cytoplasmic immunoreactivity in neuritic-like processes in C17.2 cells, which preceded Bax activation and caspase-3 cleavage. Confocal microscopy analysis of STS-treated cells revealed partial colocalization of p53 with the mitochondrial marker pyruvate dehydrogenase as well as with conformationally altered “activated” Bax, suggesting an interaction between these proapoptotic molecules in triggering apoptotic death. Nucleophosmin (NPM), a CRM1-dependent nuclear chaperone, also exhibited partial colocalization with both activated Bax and p53 following STS treatment. These observations suggest that cytoplasmic p53 can trigger transcription-independent NPC apoptosis through its potential interaction with NPM and activated Bax. (J Histochem Cytochem 58:265–275, 2010) PMID:19901272

  5. Relation of pontine choline acetyltransferase immunoreactive neurons with cells which increase discharge during REM sleep.

    PubMed

    Shiromani, P J; Armstrong, D M; Bruce, G; Hersh, L B; Groves, P M; Gillin, J C

    1987-03-01

    The purpose of this study was to determine whether neurons in the medial pontine reticular formation with high discharge rates during REM sleep could be localized in regions of the brainstem having neurons displaying choline acetyltransferase immunoreactivity. Six cats were implanted with sleep recording electrodes and microwires to record extracellular potentials of neurons in the pontine reticular formation. Single-units with a S:N ratio greater than 2:1 were recorded for at least two REM sleep cycles. A total of 49 units was recorded from the pontine reticular formation at medial-lateral planes ranging from 0.8 to 3.7 mm. The greatest proportion of the units (28.6%) showed highest discharge during active waking and phasic REM sleep compared to quiet waking, non-REM sleep, transition into REM sleep or quiet REM sleep periods. A percentage (20.4%) of the cells had high discharge associated with phasic REM sleep periods while 8.2% of the cells showed a progressive increase in discharge from waking to REM sleep. Subsequent examination of the distribution of choline acetyltransferase immunoreactive cells in the PRF revealed that cells showing high discharge during REM sleep were not localized near presumed cholinergic neurons. Indeed, we did not find any ChAT immunoreactive somata in the medial PRF, an area which has traditionally been implicated in the generation of REM sleep. These results suggest that while increased discharge of PRF cells may be instrumental to REM sleep generation, these cells are not cholinergic.

  6. Immunoreactive oestrogens and progesterone in amniotic fluid in twin pregnancies.

    PubMed

    Norman, R J; Joubert, S M

    1985-03-01

    Amniotic fluid concentrations of immunoreactive oestrogens and progesterone were measured at the time of caesarean section in 32 twin pregnancies; 25 women had an elective section and seven were in labour at the time of operation. No significant differences between concentrations in the amniotic fluid of the first and second twin were found in respect of conjugated and unconjugated oestrone, oestradiol, oestriol, oestetrol and unconjugated progesterone either before or during labour. It is unlikely that changes in oestrogens or progesterone in the amniotic fluid are responsible for the selective changes seen in prostaglandins and fetal adrenal steroid during labour in the first twin. PMID:3978052

  7. Calretinin immunoreactivity in normal and carbon tetrachloride-induced nephrotoxic rats.

    PubMed

    Kang, Ki Young; Kim, Jin Nam; Chang, In Youb; Park, Sung Ho; Yoon, Sang Pil

    2011-11-01

    Carbon tetrachloride (CCl(4)) is a potent hepatotoxic and nephrotoxic chemical. Little, however, is known about the association of CCl(4)-induced nephrotoxicity and calretinin. We hypothesized that calretinin might be localized in the proximal tubule cells and play a role against CCl(4)-induced nephrotoxicity, since the target of CCl(4) is the brush border-bearing tubule cells. CCl(4) (1 ml/kg) was administrated by oral gavage to 8-week old male Sprague-Dawley rats once a week for 4 weeks. A significant increase in serum blood urea nitrogen and creatinine was confirmed by serum analysis. Calretinin immunolocalization was compared with the calbindin D-28k immunoreactivity in normal and CCl(4)-treated kidneys. Calretinin was clearly immunolocalized in the apical surface of proximal convoluted tubule in the deeper cortex of normal kidney and blurred after CCl(4) administration, with only minor changes of calbindin D-28k immunoreactivity in the distal convoluted tubules and collecting ducts, irrelevant to the CCl(4) treatment. These findings might have significance since decreased immunolocalization of calretinin with CCl(4)-induced nephrotoxicity may contribute to the toxicity-related decrease in calcium transport or calcium buffering activity in the kidney. PMID:20947139

  8. Seasonal changes in immunoreactivity of activin signaling component proteins in wild ground squirrel testes.

    PubMed

    Sheng, Xia; Zhang, Haolin; Zhang, Mengyuan; Zhang, Wei; Hu, Xiao; Song, Moshi; Zhou, Jiao; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2012-01-01

    The seasonal spermatogenesis and localization of inhibin/activin subunits (alpha, betaA, betaB) in the testes of wild ground squirrel has been previously described; however, the expression pattern of activin receptors and cytoplasmic signaling SMADs has not been detected in any seasonal breeders. The objective of this study was to investigate the abundance and cellular localization of activin signaling components in testes of the wild ground squirrel during the breeding and nonbreeding seasons. The immunolocalizations of ActRIIB (activin type II receptor B) and activin-related SMADs (phospho-SMAD2/3, SMAD4 and SMAD7) were observed by immunohistochemistry. Total proteins were extracted from testicular tissues in the breeding and nonbreeding seasons and were used for Western blotting analysis for ActRIIB and SMADs. Immunoreactivities of activin signaling components were greater in the testes of the breeding season, and then decreased to a relatively low level in the nonbreeding season. ActRIIB and related SMADs were widely spread in the active testes, while spermatogonia were the predominant cellular sites of activin signal transduction during arrested spermatogenesis. The dynamic regulation of activin type II receptor and SMADs indicated that the activin signal pathway played an important paracrine role in seasonal spermatogenesis of the wild ground squirrel. Furthermore, the distinct localizations and immunoreactivity of ActRIIB and SMADs might suggest different functions of activin in seasonal spermatogenesis.

  9. Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

    PubMed

    Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

    1998-01-01

    hippocampal formation. Only calretinin and somatostatin showed an appreciable degree of co-localization with m2 (20% and 15%, respectively). Using retrograde tracing, some of the m2-positive cells in stratum oriens were shown to project to the medial septum, accouting for 38% of all projection neurons. The present results demonstrate that there is a differential distribution of m2 receptor immunoreactivity on the axonal vs the somadendritic membranes of distinct interneuron types and suggest that acetylcholine via m2 receptors may reduce GABA release presynaptically from the terminals of perisomatic inhibitory cells, while it may act to increase the activity of another class of interneuron, which innervates the dendritic region of pyramidal cells.

  10. The Potential Role of MT and Vimentin Immunoreactivity in the Remodeling of the Microenvironment of Parotid Adenocarcinoma.

    PubMed

    Dutsch-Wicherek, Magdalena; Lazar, Agata; Tomaszewska, Romana

    2010-12-15

    A tumor stimulates the remodeling of its microenvironment in order to control and accelerate its own growth and to initiate metastases. To create metastases the tumor cells must first acquire the ability to detach from the main tumor and to adhere to, invade, and degrade the adjacent extracellular matrix. The cells must then be able to enter the lumen of the vessels where they home the distant tissues and organs by forming secondary tumors. The acquisition of this phenotype is related to the phenomenon of epithelial-to-mesenchymal transition. On the molecular level, this process is typified by a change in the expression of epithelial markers and by the enhancement of the expression of mesenchymal markers like vimentin that are responsible for cell migration and invasion. Metallothioneins have been shown to help protect against apoptosis. The expression of MT by tumor cells plays an important and complex role not only because of its pro-proliferative, anti-apoptotic activity, but also because it inhibits the immune response. The aim of the present study was to evaluate the immunoreactivity of vimentin and MT in the salivary gland adenocarcinoma and its stroma in order to observe the phenomenon of stromal remodeling. The tissue samples of salivary gland adenocarcinomas and their stromas and the palatine tonsils which constituted the reference group were obtained during routine surgical procedures. The immunoreactivity of vimentin, metalothionein, CD56, CD57 antigens was evaluated by the immunohistochemistry method in 30 tissue samples of parotid adenocarcinoma. The patient's consent was obtained in each case. A statistically significantly higher level of MT immunoreactivity was observed in the adenocarcinoma tissue slides than in either the stromal slides or the reference slides while no differences in MT immunoreactivity were detected when the stroma and reference tissue slides were compared. A statistically significantly higher vimentin immunoreactivity level was

  11. Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats.

    PubMed

    Wei, Xiao-Yan; Zhao, Yu; Wong-Riley, Margaret T T; Ju, Gong; Liu, Ying-Ying

    2012-09-01

    The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-BötC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-BötC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-BötC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.

  12. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma

    PubMed Central

    Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S.; McMurray, John S.; Gandhi, Varsha

    2016-01-01

    The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3–7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27–43%), but not in DKO MEFs or MEFs expressing respective Casp3–7 catalytic mutants (12–13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30–73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18–36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation. PMID:26658105

  13. Interleukin (IL)-8 immunoreactivity of injured axons and surrounding oligodendrocytes in traumatic head injury.

    PubMed

    Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

    2016-06-01

    Interleukin (IL)-8 has been suggested to be a positive regulator of myelination in the central nervous system, in addition to its principal role as a chemokine for neutrophils. Immunostaining for beta-amyloid precursor protein (AβPP) is an effective tool for detecting traumatic axonal injury, although AβPP immunoreactivity can also indicate axonal injury due to hypoxic causes. In this study, we examined IL-8 and AβPP immunoreactivity in sections of corpus callosum obtained from deceased patients with blunt head injury and from equivalent control tissue. AβPP immunoreactivity was detected in injured axons, such as axonal bulbs and varicose axons, in 24 of 44 head injury cases. These AβPP immunoreactive cases had survived for more than 3h. The AβPP immunostaining pattern can be classified into two types: traumatic (Pattern 1) and non-traumatic (Pattern 2) axonal injuries, which we described previously [Hayashi et al. Int. J. Legal Med. 129 (2015) 1085-1090]. Three of 44 control cases also showed AβPP immunoreactive injured axons as Pattern 2. In contrast, IL-8 immunoreactivity was detected in 7 AβPP immunoreactive and in 2 non-AβPP immunoreactive head injury cases, but was not detected in any of the 44 control cases, including the 3 AβPP immunoreactive control cases. The IL-8 immunoreactive cases had survived from 3 to 24 days, whereas those cases who survived less than 3 days (n=29) and who survived 90 days (n=1) were not IL-8 immunoreactive. Moreover, IL-8 was detected as Pattern 1 axons only. In addition, double immunofluorescence analysis showed that IL-8 is expressed by oligodendrocytes surrounding injured axons. In conclusion, our results suggest that immunohistochemical detection of IL-8 may be useful as a complementary diagnostic marker of traumatic axonal injury.

  14. Cholecystokinin octapeptide-like immunoreactivity: histochemical localization in rat brain.

    PubMed Central

    Innis, R B; Corrêa, F M; Uhl, G R; Schneider, B; Snyder, S H

    1979-01-01

    Cholecystokinin octapeptide-like (CCK-OP-like) immunoreactivity was localized in the rat brain by using the indirect immunofluorescence method. Specificity in immunohistochemical studies was demonstrated by the virtual elimination of staining with either preimmune sera or sera preadsorbed with CCK-OP and by the achievement of similar fluorescent patterns with two different primary anti-CCK-OP sera. CCK-OP-like fluorescence was localized in neuronal cell bodies, fibers, and varicose terminals. The most dense collections of CCK-OP cells occurred in the periaqueductal gray and in the dorsomedial hypothalamus. Substantial numbers of cells and fibers also were present in the medial/dorsal and perirhinal cortex; more limited groups of cells were found in the pyramidal layer of the hippocampus and in the dorsal raphe. Images PMID:284371

  15. Immunoreactive atrial natriuretic peptide in the guinea pig spleen

    SciTech Connect

    Vollmar, A.M.; Friedrich, A.; Schulz, R. )

    1989-01-01

    The presence of immunoreative ANP precursor-like material in the guinea pig spleen is suggested. This is based on the following experimental evidence: An acidic extract of guinea pig spleen analyzed by Sephadex G-50 gel filtration contained 4.6 pmol/g wet tissue immunoreactive atrial natriuretic peptide (IR-ANP), IR-ANP coeluting with 15 kDa synthetic ANP (2-126). Gel filtrated IR-ANP material was further submitted to reverse phase high performance liquid chromatography and monitored by radioimmunoassay employing two antisera. One antiserum recognizes the C-terminal of ANP (1-126), the second is directed against the N-terminal sequence. Both antisera revealed material eluting with synthetic ANP (2-126). Furthermore, immunohistochemical analysis suggests this ANP-like material to be localized mainly at the periphery of the white pulp of the spleen. These findings link ANP with the immune system.

  16. Parvalbumin-immunoreactive neurons in the human claustrum.

    PubMed

    Hinova-Palova, D V; Edelstein, L; Landzhov, B V; Braak, E; Malinova, L G; Minkov, M; Paloff, A; Ovtscharoff, W

    2014-09-01

    The morphology and distribution of parvalbumin-immunoreactive neurons (PV-ir) were studied in the human claustrum. PV-ir neurons were observed throughout the claustrum, with the highest numbers noted in the central (broadest) portion as compared with the dorsal and ventral aspects. Reaction product was evident in the neuronal perikarya, dendritic processes, and spines. In the majority of these labeled neurons, the cytoplasm was devoid of lipofuscin pigment. Cell bodies varied widely in both shape and size, ranging from oval and small, to multipolar and large. PV-ir neurons were classified into two groups, primarily based on dendritic morphology: spiny neurons with long and straight dendrites, and aspiny neurons with thin and curving dendritic processes. PV-ir fibers were seen throughout the neuropil, with many immuno-positive puncta noted.

  17. Melatonin Immunoreactivity in Malignant Small Intestinal Neuroendocrine Tumours

    PubMed Central

    Söderquist, Fanny; Janson, Eva Tiensuu; Rasmusson, Annica J.; Ali, Abir; Stridsberg, Mats; Cunningham, Janet L.

    2016-01-01

    Background/Aims Small intestinal neuroendocrine tumours (SI-NETs) are derived from enterochromaffin cells. After demonstrating melatonin in enterochromaffin cells, we hypothesized that SI-NETs may express and secrete melatonin, which may have an impact on clinical factors and treatment response. Methods Tumour tissue from 26 patients with SI-NETs, representing paired sections of primary tumour and metastasis, were immunohistochemically stained for melatonin and its receptors, MT1 and MT2. Plasma melatonin and immunoreactivity (IR) for melatonin, MT1 and MT2 in tumour cells were compared to other tumour markers and clinical parameters. Melatonin was measured at two time points in fasting morning plasma from 43 patients with SI-NETs. Results Melatonin IR was found in all SI-NETS. Melatonin IR intensity in primary tumours correlated inversely to proliferation index (p = 0.022) and patients reported less diarrhoea when melatonin IR was high (p = 0.012). MT1 IR was low or absent in tumours. MT2 expression was medium to high in primary tumours and generally reduced in metastases (p = 0.007). Plasma-melatonin ranged from 4.5 to 220.0 pg/L. Higher levels were associated with nausea at both time points (p = 0.027 and p = 0.006) and flush at the second sampling. In cases with disease stabilization or remission (n = 34), circulating melatonin levels were reduced in the second sample (p = 0.038). Conclusion Immunoreactive melatonin is present in SI-NETs. Circulating levels of melatonin in patients with SI-NETs are reduced after treatment. Our results are congruent with recent understanding of melatonin’s endocrine and paracrine functions and SI-NETs may provide a model for further studies of melatonin function. PMID:27736994

  18. Dissociation between changes in immunoreactive parathormone and its biological indices induced by cimetidine in primary hyperparathyroidism.

    PubMed

    van der Velden, P C; Fischer, H R; Hackeng, W H; Schopman, W; Koorevaar, G; den Ottolander, G J; Silberbusch, J

    1981-01-01

    In three out of four patients with primary hyperparathyroidism, 2 000 mg of cimetidine daily caused a reduction of immunoreactive parathormone (iPTH) when measured at 8.30 and 11.30 on days 16 and 17 on treatment. Serum Ca, PO4 and maximal tubular reabsorption of PO4 remained unchanged. Excretion of cAMP/100 ml GFR remained elevated to at least the same extent as before treatment. Two patients, in whom cimetidine treatment was continued for an additional 4 weeks, did not show further hormonal or biochemical changes compared with the evaluation on days 16 and 17. We conclude that reduction of iPTH is not accompanied by any change in biological activity of this hormone. The reason for this discrepancy remains unclear. PMID:6277160

  19. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    PubMed

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  20. Beacon-like immunoreactivity in the hypothalamus of Sprague-Dawley rats.

    PubMed

    Brailoiu, G Cristina; Dun, Siok L; Yang, Jun; Chang, Jaw Kang; Castellino, Sonya; Dun, Nae J

    2002-01-14

    Distribution of the novel peptide beacon in the hypothalamus of Sprague-Dawley rats was examined by immunohistochemical methods. Beacon-immunoreactive (irBC) neurons were found in the paraventricular, supraoptic, and accessory neurosecretory nuclei, and intensely labeled fibers in the median eminence and infundibulo-pituitary stalk. Scattered cells and/or fibers were noted in the suprachiasmatic nucleus, arcuate nucleus, retrochiasmatic area, lateral and medial preoptic area, as well as anterior and lateral hypothalamic area. The wide distribution of irBC in the hypothalamus of Sprague-Dawley rats suggests that the peptide may influence, in addition to a proposed role in feeding, a multitude of biological activities associated with the hypothalamic-pituitary axis.

  1. Neurotrophin-receptor immunoreactive neurons in mesopontine regions involved in the control of behavioral states.

    PubMed

    Yamuy, J; Sampogna, S; Chase, M H

    2000-06-01

    The microinjection of nerve growth factor (NGF) and neurotrophin-3 (NT-3) into the rostral pontine tegmentum of adult cats rapidly induces long-lasting episodes of rapid eye movement (REM) sleep [J. Yamuy, F.R. Morales, M.H. Chase, Induction of rapid eye movement sleep by the microinjection of nerve growth factor into the pontine reticular formation of the cat, Neuroscience 66 (1995) 9-13]. Because this effect may be mediated by neurotrophin receptors, we sought to determine the distribution of neurons that contain low- and high-affinity neurotrophin receptors in regions of the feline pons and mesencephalon which are involved in the generation of REM sleep as well as neuronal groups that are involved in the control of REM sleep-related patterns of physiological activity. Using antibodies directed against p75, trkA, trkB and trkC, immunolabeled neurons were present in the latero-dorsal and pedunculo-pontine tegmental nuclei, the peribrachial nuclei, medial and lateral pontine reticular formation, the raphe nuclei, and the locus coeruleus. Giant reticular cells and large neurons in the mesencephalic trigeminal nucleus were immunoreactive for p75 and all trk receptors. Neurons that were devoid of neurotrophin-receptor immunoreactivity were intermingled with immunostained neurons in all explored structures. Thus, both low- and high-affinity neurotrophin receptors are conspicuously present in neurons located in mesopontine regions of adult cats. These data underscore the importance of neurotrophin-induced trophic actions on mesopontine neurons. Furthermore, the results support the hypothesis that NGF and NT-3 may modulate the electrical activity of neurons in the rostral pontine tegmentum that are responsible for the generation of REM sleep by acting on one or more of the neurotrophin receptors. PMID:10825475

  2. Synapsin I-like immunoreactivity in nerve fibers associated with lingual taste buds of the rat.

    PubMed

    Finger, T E; Womble, M; Kinnamon, J C; Ueda, T

    1990-02-01

    Immunoreactivity to synapsin I, a neuronal phosphoprotein, was localized in free-floating tissue sections prepared from lingual tissue of rats. Many nerve fibers within the tissue exhibited clear immunoreactivity including motor endplates on striated muscle, autonomic fibers innervating blood vessels or glands, and sensory fibers innervating muscles or the lingual epithelium including taste buds. Numerous immunoreactive fibers occurred within each taste bud, with fewer, fine fibers being dispersed in the epithelium between taste buds. The majority of the intragemmal immunoreactive fibers extended throughout the taste buds most of the distance outward from the basal lamina toward the surface of the epithelium. Fine, perigemmal fibers reached nearly to the epithelial surface. Ultrastructural analysis of the immunoreactive sensory fibers revealed that synapsin I-immunoreactivity occurred diffusely throughout the cytoplasm, and heavily in association with microvesicles. The synaptic vesicles at the taste receptor cell-to-afferent fiber synapse were, however, not immunoreactive for synapsin I, although these vesicles fall into the size class shown to be immunoreactive in other systems. This absence of synapsin I may be a common property of vesicles in axonless short receptor cells. PMID:2108194

  3. Dopamine D1 and D2 receptor immunoreactivities in the arcuate-median eminence complex and their link to the tubero-infundibular dopamine neurons.

    PubMed

    Romero-Fernandez, W; Borroto-Escuela, D O; Vargas-Barroso, V; Narváez, M; Di Palma, M; Agnati, L F; Larriva Sahd, J; Fuxe, K

    2014-07-18

    Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tubero-infundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region.  Dopamine D1 and D2 receptors may therefore directly and differentially

  4. Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

    PubMed Central

    Romero-Fernandez, W.; Borroto-Escuela, D.O.; Vargas-Barroso, V.; Narváez, M.; Di Palma, M.; Agnati, L.F.; Sahd, J. Larriva

    2014-01-01

    Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially

  5. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    SciTech Connect

    Tu, Yihui; Xue, Huaming; Francis, Wendy; Davies, Andrew P.; Pallister, Ian; Kanamarlapudi, Venkateswarlu; Xia, Zhidao

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  6. Human plasma C-peptide immunoreactivity: its correlation with immunoreactive insulin in diabetes, and chronic liver and renal diseases.

    PubMed

    Kajinuma, H; Kanazawa, Y; Sando, H; Hayashi, M; Kawazu, S; Kosaka, K

    1979-02-01

    The correlation between plasma C-peptide immunoreactivity (CPR) and immunoreactive insulin (IRI) was investigated during the oral glucose tolerance test in 20 normals, 127 diabetics, and 39 non-diabetics with chronic liver or renal disorders. When all subjects were included, the increment of CPR 30 minutes after glucose load (deltaCPR) correlated well with that of IRI (deltaIRI) (r = 0.66, p less than 0.001), but the return of CPR towards the basal level was delayed as compared with IRI. The positive correlation was also observed between the sum of 6 IRI and that of 6 CPR values during the glucose tolerance test in diabetics and controls (r = 0.53, p less than 0.001). deltaCPR/deltaBS (30 min.) was also well correlated with deltaIRI/deltaBS (30 min.), and was specifically low in diabetics. Insulin-treated maturity-onset diabetics showed low but considerable CPR responses while no CPR responses were observed in insulin-treated juvenile diabetics. In each plasma sample, CPR always exceeded IRI on the molar basis. At fasting CPR/IRI ratio was 15.6 +/- 1.7 (mean +/- SE) in normals and 14.9 +/- 1.3 approximately 16.9 +/- 1.0 in diabetics. In chronic liver diseases IRI response was augmented while CPR response was not different from that of controls, and the molar ratio of CPR/IRI was significantly low (9.5 +/- 1.1). On the contrary, it exceeded that of normals in chronic renal diseases (35.7 +/- 14.9). It is concluded that, first, the plasma CPR response appears to be a valuable indicator of pancreatic B-cell function, and second, it is, nevertheless, modified in chronic liver or renal disorders.

  7. Histamine-immunoreactive local neurons in the antennal lobes of the Hymenoptera

    PubMed Central

    Dacks, Andrew M.; Reisenman, Carolina E.; Paulk, Angelique C.; Nighorn, Alan J.

    2010-01-01

    Neural networks receive input which is transformed before being sent as output to higher centers of processing. These transformations are often mediated by local interneurons (LNs) that influence output based on activity across the network. In primary olfactory centers, the LNs that mediate these lateral interactions are extremely diverse. For instance, the antennal lobes (ALs) of bumble bees possess both GABA and histamine-immunoreactive (HA-ir) LNs, and both are neurotransmitters associated with fast forms of inhibition. Although the GABAergic network of the AL has been extensively studied, we sought to examine the anatomical features of the HA-ir LNs in relation to the other cellular elements of the bumble bee AL. As a population, HA-ir LNs densely innervate the glomerular core while sparsely arborizing in the outer glomerular rind, overlapping with the terminals of olfactory receptor neurons. Individual fills of HA-ir LNs revealed heavy arborization of the outer ring of a single “principal” glomerulus and sparse arborization in the core of other glomeruli. In contrast, projection neurons, and GABA-immunoreactive LNs project throughout the glomerular volume. To provide insight as to the selective pressures that resulted in the evolution of HA-ir LNs, we determined the phylogenetic distribution of HA-ir LNs in the AL. HA-ir LNs were present in all but the most basal hymenopteran examined, although there were significant morphological differences between major groups within the Hymenoptera. The ALs of other insect taxa examined lacked HA-ir LNs, suggesting that this population of LNs arose within the Hymenoptera and underwent extensive morphological modification. PMID:20533353

  8. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    NASA Astrophysics Data System (ADS)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

  9. Stability of polypeptide immunoreactants and polyvinyl alcohol as a blocking agent on polyester cloth during dry storage.

    PubMed

    Boyd, S; Yamazaki, H

    1995-08-01

    During dry storage at 32 degrees C for 70 days, polypeptide immunoreactants (rabbit IgG and rabbit antiperoxidase antibody) adsorbed onto polyester cloth demonstrated no loss of immunoreactivity. Polyvinyl alcohol of molecular weight 30,000-70,000 provided a stable blocking capacity on the immunoreactant-adsorbed cloth without affecting immunoreactivity. Cloth-based enzyme immunoassay systems are suitable for field testing and transportation in the absence of refrigeration.

  10. Immunoreactive trypsin in the adult respiratory distress syndrome.

    PubMed

    Deby-Dupont, G; Haas, M; Pincemail, J; Braun, M; Lamy, M; Deby, C; Franchimont, P

    1984-01-01

    With the purpose of studying the role of proteinases in the development of ARDS, plasma levels of immunoreactive trypsin (IRT) and amylase were measured in 43 intensive care patients at risk of developing ARDS (22 polytrauma, seven abdominal surgery, four burns, two DIC and eight pancreatitis). Twenty four of these 43 patients developed ARDS and 31 presented abnormal IRT values (above 70 micrograms/L). Twenty-one of these 31 patients had ARDS; a significant correlation thus appeared between ARDS and abnormal IRT values. In nine patients, IRT values were higher than 800 micrograms/L and remained high for 3 to 4 days. A statistically significant correlation also appeared between abnormal IRT and septic phenomena: 20 patients with high IRT values presented septic problems. When IRT values were high, amylase values were often also abnormal: 12 of 23 patients with high IRT had abnormal amylase levels (the eight patients with documented pancreatitis were excluded); no other clinical signs or symptoms of pancreatitis were present in these patients. IRT could be one of the mediators of ARDS in septic patients. It is not clear that the pancreas is the origin of IRT in all cases.

  11. Distribution of alarin immunoreactivity in the mouse brain.

    PubMed

    Eberhard, Nicole; Mayer, Christian; Santic, Radmila; Navio, Ruben Peco; Wagner, Andrea; Bauer, Hans Christian; Sperk, Guenther; Boehm, Ulrich; Kofler, Barbara

    2012-01-01

    Alarin is a 25 amino acid peptide that belongs to the galanin peptide family. It is derived from the galanin-like peptide gene by a splice variant, which excludes exon 3. Alarin was first identified in gangliocytes of neuroblastic tumors and later shown to have a vasoactive function in the skin. Recently, alarin was demonstrated to stimulate food intake as well as the hypothalamic-pituitary-gonadal axis in rodents, suggesting that it might be a neuromodulatory peptide in the brain. However, the individual neurons in the central nervous system that express alarin have not been identified. Here, we determined the distribution of alarin-like immunoreactivity (alarin-LI) in the adult murine brain. The specificity of the antibody against alarin was demonstrated by the absence of labeling after pre-absorption of the antiserum with synthetic alarin peptide and in transgenic mouse brains lacking neurons expressing the GALP gene. Alarin-LI was observed in different areas of the murine brain. A high intensity of alarin-LI was detected in the accessory olfactory bulb, the medial preoptic area, the amygdala, different nuclei of the hypothalamus such as the arcuate nucleus and the ventromedial hypothalamic nucleus, the trigeminal complex, the locus coeruleus, the ventral chochlear nucleus, the facial nucleus, and the epithelial layer of the plexus choroideus. The distinct expression pattern of alarin in the adult mouse brain suggests potential functions in reproduction and metabolism.

  12. Distribution of parvalbumin immunoreactivity in the rat septal area.

    PubMed

    Alonso, J R; Coveñas, R; Lara, J; Aijón, J

    1990-01-01

    The distribution of parvalbumin (PV)-containing neurons and processes in the septal area of the rat brain was studied using a monoclonal antibody and the avidin-biotin immunoperoxidase method. PV-immunoreactive neurons were mainly located in the medial septum/diagonal band complex and in the horizontal limb of the diagonal band of Broca, showing a high density of heavily immunostained neurons and fibers. Nonimmunoreactive cells surrounded by PV-positive cells and processes were observed in the same region, but no pericellular basket-like arrangements were found. On the contrary, the dorsal, intermediate, and ventral nuclei of the lateral septum were practically devoid of PV-positive neurons and processes. Thus, in these nuclei only a very low density of isolated neurons was labeled; these were specially scattered in the ventrolateral septal nucleus and in the dorsolateral septal nucleus just below the corpus callosum. Delicate PV-positive axonal plexuses were also observed in the dorsal and intermediate nuclei of the lateral septum. The immunopositive neurons displayed very different sizes and morphologies among the various septal nuclei and inside each of them, indicating that they do not belong to a single morphological class of neurons. Finally, the distribution of PV in the rat septal area is not directly related to cholinergic and GABAergic septal neurons.

  13. Immunoreactive inhibin concentration in blood tested under variable sampling conditions.

    PubMed

    Blaakaer, J; Micic, S; Høgdall, C K

    1996-06-01

    The stability of immunoreactive (i.r.) inhibin in blood samples drawn and handled under different conditions and at different time intervals were studied. Ten serum and plasma samples drawn in 1994 from healthy volunteers were compared to samples collected in 1986 from 10 healthy women admitted for laparoscopic sterilization and analysed 6 years later. All samples were drawn on the twelfth day of the menstrual cycle and handled under identical clinical conditions (22 degrees C). The concentrations in the 1986 samples were similar to the Se-i.r. inhibin levels from 1994. Different clotting temperatures, repetitive freezing and thawing or hemolysis had no effects on the i.r. inhibin values, whereas non-hemolysed samples left at room temperature (22 degrees C) for 3 days were significantly lower, which might be due to a statistical type 2 error. No differences in concentration between serum and plasma i.r. inhibin were demonstrated. In conclusion, i.r. inhibin is a very stable peptide hormone in both serum and plasma if drawn and handled under normal conditions.

  14. Beacon-like immunoreactivity in the hypothalamus of domestic chick.

    PubMed

    Esposito, V; de Girolamo, P; Gargiulo, G; Dun, N J

    2006-12-01

    Beacon-immunoreactive (B-ir) fibres and neurons in the hypothalamus of the domestic chick (Gallus domesticus) were studied using an immunohistochemical technique in order to verify the presence and elucidate the pattern of distribution of this novel peptide in an avian brain. B-ir neurons were seen in the n. supraopticus, pars ventralis and pars externus; n. magnocellularis preopticus, pars dorsalis, medialis and ventralis; n. preopticus periventricularis; n. suprachiasmaticus, pars medialis; n. ventrolateralis thalami. Only few B-ir cells were scattered in the most anterior part of the lateral hypothalamic area. B-ir fibres, appearing as thin punctuate structures, were seen mainly along the walls of the third ventricle and in the ventromedial hypothalamus. Labelled fibres and terminals were located in the external and internal zones of the anterior and posterior median eminence. In particular, fibre terminals were seen close to the capillary loops of the hypothalamo-hypophysial portal system. The anatomical data of the present study regarding the distribution of B-ir in the chick hypothalamus suggest that beacon may play a key role in the regulation of the neuroendocrine system by acting as a neuromodulator and/or neurotransmitter.

  15. The effects of 1 week of REM sleep deprivation on parvalbumin and calbindin immunoreactive neurons in central visual pathways of kittens.

    PubMed

    Hogan, D; Roffwarg, H P; Shaffery, J P

    2001-12-01

    Many maturational processes in the brain are at high levels prenatally as well as neonatally before eye-opening, when extrinsic sensory stimulation is limited. During these periods of rapid brain development, a large percentage of time is spent in rapid eye movement (REM) sleep, a state characterized by high levels of endogenously produced brain activity. The abundance of REM sleep in early life and its ensuing decline to lower levels in adulthood strongly suggest that REM sleep constitutes an integral part of the activity-dependent processes that enable normal physiological and structural brain development. We examined the effect of REM sleep deprivation during the critical period for visual development on the development of two calcium-binding proteins that are associated with developmental synaptic plasticity and are found in the lateral geniculate nucleus (LGN) and visual cortex. In this study, REM sleep deprivation was carried out utilizing a computer-controlled, cage-shaking apparatus that successfully suppressed REM sleep. Body weight data suggested that this method of REM sleep deprivation produced less stress than the classical multiple-platform-over-water method. In REM sleep-deprived animals with normal binocular vision, the number of parvalbumin-immunoreactive (PV) neurons in LGN was found to be lower compared with control animals but was not affected in visual cortex. The pattern of calbindin-immunoreactivity (CaB) was unchanged at either site after REM sleep deprivation. Parvalbumin-immunoreactivity develops later than calbindin-immunoreactivity in the LGN, and the REM sleep deprivation that we applied from postnatal day 42-49 delayed this essential step in the development of the kitten's visual system. These data suggest that in early postnatal brain development, REM sleep facilitates the usual time course of the expression of PV-immunoreactivity in LGN neurons.

  16. FMRFamide-like immunoreactivity in the ventral ganglion of the fly Sarcophaga bullata: metamorphic changes.

    PubMed

    Sivasubramanian, P

    1991-01-01

    1. Localization of FMRFamide-like immunoreactivity was examined in the ventral ganglion of the fly Sarcophaga bullata using the indirect immunofluorescent method. 2. There are six large cells in the thoracic ganglion which are highly immunoreactive at all stages of development. 3. During metamorphosis the thoracic FLI neurons shift their position from ventrolateral to mid-ventral position and their axons terminate and elaborate a highly immunoreactive dorsal neural sheath. 4. It is suggested that the dorsal neural sheath may function as a neurohaemal organ from which FMRFamide-like substances may be released into the haemolymph to act as neurohormones.

  17. Functional consequences of caspase activation in cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Communal, Catherine; Sumandea, Marius; de Tombe, Pieter; Narula, Jagat; Solaro, R. John; Hajjar, Roger J.

    2002-04-01

    Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac myofilament including -actin, -actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of -actin and -actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using -adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca2+-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca2+ relationship and myofibrillar ATPase activity

  18. Presence of ACTH and beta-endorphin immunoreactive molecules in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) and their possible role in phagocytosis.

    PubMed

    Ottaviani, E; Petraglia, F; Montagnani, G; Cossarizza, A; Monti, D; Franceschi, C

    1990-01-01

    The presence of ACTH and beta-endorphin immunoreactive molecules in the cell-free hemolymph and in the hemocytes of the freshwater snail Planorbarius corneus were demonstrated by immunocytochemistry and RIA tests. Only spreading phagocytic hemocytes were positive, in contrast with other hemocytes devoid of phagocytic activity, i.e., round hemocytes. These data were confirmed by flow cytometry. Another cell type with marked phagocytic activity, i.e., digestive cells of digestive gland, were also positive to anti-ACTH. Corticotropin-releasing factor immunoreactive molecules were found in the cell-free hemolymph and hemocytes, by RIA. Our data suggest that cells with phagocytic activity, the oldest immune response, may represent a suitable model to unravel the tangled web of the common ancestor of the immune and the neuroendocrine systems.

  19. Target areas innervated by PACAP-immunoreactive retinal ganglion cells.

    PubMed

    Hannibal, Jens; Fahrenkrug, Jan

    2004-04-01

    The retinohypothalamic tract (RHT) originates from a subset of retinal ganglion cells (RGCs). The cells of the RHT co-store the neurotransmitters PACAP and glutamate, which in a complex interplay mediate light information to the circadian clock located in the suprachiasmatic nuclei (SCN). These ganglion cells are intrinsically photosensitive probably due to expression of melanopsin, a putative photoreceptor involved in light entrainment. In the present study we examined PACAP-containing retinal projections to the brain using intravitreal injection of the anterograde tracer cholera toxin subunit B (ChB) and double immunostaining for PACAP and ChB. Our results show that the PACAP-containing nerve fibres not only constituted the major projections to the SCN and the intergeniculate leaflet of the thalamus but also had a large terminal field in the olivary pretectal nucleus. The contralateral projection dominated except for the SCN, which showed bilateral innervation. PACAP-containing retinal fibres were also found in the ventrolateral preoptic nucleus, the anterior and lateral hypothalamic area, the subparaventricular zone, the ventral part of the lateral geniculate nucleus and the nucleus of the optic tract. Retinal projections not previously described in the rat also contained PACAP. These new projections were found in the lateral posterior nucleus, the posterior limitans nucleus, the dorsal part of the anterior pretectal nucleus and the posterior and medial pretectal nuclei. Only a few PACAP-containing retinal fibres were found in the superior colliculus. Areas innervated by PACAP-immunoreactive fibres also expressed the PACAP-specific PAC1 receptor as shown by in situ hybridization histochemistry. The findings suggest that PACAP plays a role as neurotransmitter in non-imaging photoperception to target areas in the brain regulating circadian timing, masking, regulation of sleep-wake cycle and pupillary reflex.

  20. Comparison of immunoreactive serum trypsinogen and lipase in Cystic Fibrosis

    SciTech Connect

    Lloyd-Still, J.D.; Weiss, S.; Wessel, H.; Fong, L.; Conway, J.J.

    1984-01-01

    The incidence of Cystic Fibrosis (CF) is 1 in 2,000. Early detection and treatment of CF may necessitate newborn screening with a reliable and cost-effective test. Serum immunoreactive trypsinogen (IRT) an enzyme produced by the pancreas, is detectable by radioimmunoassay (RIA) techniques. Recently, it has been shown that IRT is elevated in CF infants for the first few months of life and levels become subnormal as pancreatic insufficiency progresses. Other enzymes produced by the pancreas, such as lipase, are also elevated during this time. The author's earlier work confirmed previous reports of elevated IRT levels in CF infants. The development of a new RIA for lipase (nuclipase) has enabled comparison of these 2 pancreatic enzymes in C.F. Serum IRT and lipase determinations were performed on 2 groups of CF patients; infants under 1 year of age, and children between 1 and 18 years of age. Control populations of the same age groups were included. The results showed that both trypsin (161 +- 92 ng/ml, range 20 to 400) and lipase (167 +- 151 ng/ml, range 29 to 500) are elevated in CF in the majority of infants. Control infants had values of IRT ranging from 20 to 29.5 ng/ml and lipase values ranging from 23 to 34 ng/ml. IRT becomes subnormal in most CF patients by 8 years of age as pancreatic function insufficiency increases. Lipase levels and IRT levels correlate well in infancy, but IRT is a more sensitive indicator of pancreatic insufficiency in older patients with CF.

  1. Stomatin immunoreactivity in ciliated cells of the human airway epithelium.

    PubMed

    Fricke, Britta; Stewart, Gordon W; Treharne, Kathryn J; Mehta, Anil; Knöpfle, Gisela; Friedrichs, Nicolaus; Müller, Klaus-Michael; von Düring, Monika

    2003-07-01

    Stomatin is a widely distributed 32kD membrane protein of unknown function. In biochemical studies it is associated with cholesterol+sphingomyelin-rich 'rafts' in the cytomembrane. Genetic studies in C. elegans, supported by microscopic studies in mammalian tissue and co-expression studies in oocytes, suggest a functional link with the DEG/ENaC (degenerin/epithelial Na+ channel) superfamily of monovalent ion channels. Since ENaC channels play a prominent role in the physiology of the respiratory epithelium, we have studied the immunolocalization of stomatin in mature and developing human airway epithelium by means of Western blot analysis, immunocytochemistry, and immunoelectron microscopy. Stomatin immunoreactivity (stomatin-IR) was found in the ciliated cells of the conductive airway epithelium in a distinct distribution pattern with the strongest signal along the cilia. Immunogold labelling revealed immunogold particles at the basal bodies, along the cilia, and at the membrane of the microvilli. The presence of stomatin-IR paralleled the stages of ciliogenesis in airway development, and its appearance preceded the elongation of the axoneme and the cilial outgrowth. Due to its presence in the different cellular locations in the ciliated cell, we suggest that stomatin is involved in various cellular functions. From its ultrastructural position, stomatin could be a candidate for a membrane-associated mechanotransducer with a role in the control of ciliary motility. Stomatin as a raft protein might be a microtubule associated protein moving along the outer surface of the microtubules to its terminal site of action in the cilia. Stomatin-IR in microvilli supports the hypothesis of a co-localization with beta- and gamma- ENaC and, in conclusion, their potential functional interaction to control the composition of periciliary mucus electrolytes. PMID:12759749

  2. Fos immunoreactivity in the rat forebrain induced by electrical stimulation of the dorsolateral periaqueductal gray matter.

    PubMed

    Lim, Lee Wei; Temel, Yasin; Visser-Vandewalle, Veerle; Blokland, Arjan; Steinbusch, Harry

    2009-10-01

    Electrical stimulation of the dorsolateral periaqueductal gray (dlPAG) matter induces panic- or fear-like responses with intense emotional distress and severe anxiety. In this study, we evoked panic-like behaviour by dlPAG stimulation and evaluated the effect on neuronal activation in different brain regions. The number of c-Fos immunoreactive (c-Fos-ir) cells was measured semi-quantitatively through series of stained rat brain sections. Our results demonstrate strong neural activation in the medial prefrontal cortex, orbital cortex, anterior olfactory nuclei, secondary motor cortex, and the somatosensory cortex. Moderate increases in the number of c-Fos-ir cells were detected in various regions, including the hypothalamus, amygdala, and striatum. Additionally, there was mild expression of c-Fos-ir cells in the hippocampus, thalamus, and habenula regions. In conclusion, we have shown that deep brain stimulation of the dlPAG produced a distinctive pattern of neuronal activation across forebrain regions as compared to the sham and control animals.

  3. Distribution of hypocretin (orexin) immunoreactivity in the feline pons and medulla.

    PubMed

    Zhang, Jian Hua; Sampogna, Sharon; Morales, Francisco R; Chase, Michael H

    2004-01-01

    The distribution of hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) immunoreactivities in the cat brainstem was examined using immunohistochemical techniques. Hcrt-1- and hcrt-2-positive fibers with varicosities were detected in almost all brainstem regions. However, no hcrt-1- or hcrt-2-immunoreactive neuronal somata were observed in the cat brainstem. Both hcrt-1- and hcrt-2-labeled fibers exhibited different densities in distinct regions of the brainstem. In most brainstem regions, the intensity of hcrt-1 immunoreactivity was higher than that of hcrt-2 immunoreactivity. The highest densities of hcrt-1- and hcrt-2-positive fibers were found in the nucleus raphe dorsalis (RD), the laterodorsal tegmental nucleus (LDT) and the locus coeruleus (LC), suggesting an important role for these peptides in functions related to sleep-wake behavior. PMID:14672810

  4. Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates.

    PubMed

    Mizuno, J; Takeda, N

    1988-01-01

    1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

  5. Relation of smoking to immunoreactive endothelin in the bronchiolar epithelial cells.

    PubMed Central

    Shokeir, M. O.; Paré, P.; Wright, J. L.

    1994-01-01

    BACKGROUND--Endothelin is a potent bronchoconstrictor which appears to be important in asthma. To ascertain whether cigarette smoking is associated with any alteration in the proportion of bronchiolar epithelial cells which express endothelin immunoreactivity, the airways in the lungs of non-smokers and smokers were analysed. Since an increase in immunoreactivity has been found in the bronchial epithelial cells of asthmatic subjects, cigarette smokers with and without evidence of airway hyperresponsiveness were also selected. METHODS--A point counting method which examined the proportion of endothelin immunoreactive epithelial cells in membranous and respiratory bronchioles was used. RESULTS--Neither smoking itself nor evidence of airway hyperresponsiveness altered the percentage of endothelin immunoreactive epithelial cells in the membraneous and respiratory bronchioles. CONCLUSIONS--Cigarette smoke does not induce endothelin production in bronchiolar epithelial cells, and the airway hyperresponsiveness seen in some patients with lung disease induced by cigarette smoking is not related to exaggerated endothelin production in epithelial cells. PMID:8091324

  6. GABA and serotonin immunoreactivity during postembryonic brain development in the beetle Tenebrio molitor.

    PubMed

    Wegerhoff, R

    1999-05-01

    Analysis of the serotonin immunoreactive neurons in the central brain of the beetle Tenebrio molitor during postembryonic development shows that the basic structural characteristics of larval brain resemble those of the adult. Most, if not all, serotonin immunoreactive central brain neurons persist with metamorphosis. Their fate can be followed during development. GABA immunoreactivity occurs in about 360 neurons assembled in ten different clusters of somata in the larval midbrain. During metamorphosis no additional clusters are formed. However, the number of immunoreactive neurons increases to 450. Their morphological analysis is restricted to location of the somata and the distribution of arborizations within neuropil areas. Metamorphic transition of glomerular sub-units in the antennal lobes as well as ellipsoid body development can be followed by GABA immunohistochemistry. Furthermore, the study of these transitions proved useful in displaying changes during metamorphic pattern formation induced by sublethal application of the pyrethroid insecticide fenvalerate.

  7. Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system

    NASA Technical Reports Server (NTRS)

    D'Amelio, F.; Eng, L. F.; Gibbs, M. A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  8. Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity.

    PubMed

    Martinez-Guijarro, F J; Soriano, E; Del Rio, J A; Lopez-Garcia, C

    1991-10-01

    Zinc-positive boutons, originating in the medial cortex of lizards, exhibit glutamate immunoreactivity. This finding supports the presumed homology between lizard zinc-positive boutons and the hippocampal mossy fibres of mammals, which are also glutamate-immunoreactive and zinc-positive. Zinc-positive boutons of lizards contain a chelatable pool of zinc located in the hippocampal mossy fibres of mammals. These synaptic systems also contain glutamate, which indicates a possible simultaneous action of zinc and glutamate during synaptic transmission.

  9. Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

    PubMed

    Barakat, I; Droz, B

    1987-07-01

    Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

  10. Neurotensin-like immunoreactivities in human plasma: feeding responses and metabolism.

    PubMed

    Holst Pedersen, J; Fahrenkrug, J

    1986-01-01

    Feeding responses and day and night levels of plasma concentration of neurotensin (NT) and NT-fragments were studied in healthy subjects. Plasma levels were measured by three radioimmunoassays recognizing intact NT in addition to C- and N-terminal immunoreactivity. The metabolism of NT was studied following intravenous administration. In 106 subjects fasting levels of intact NT (median 18 pmol/l), C-terminal (median 30 pmol/l) and N-terminal immunoreactivity (median 95 pmol/l) were unrelated to sex or age. Postprandially plasma levels in seven subjects measured with all assays increased by a factor 1-3. Following a mixed meal the increase was biphasic, whereas the response to dairy cream was monophasic. Repetitive measurements during 24 hours showed that levels of N-terminal immunoreactivity fluctuated in a manner related to meal ingestion and were elevated throughout the daytime, whereas intact NT and C-terminal immunoreactivity changed little. Following intravenous infusion of 2.4 pmol/kg/min NT in 5 subjects the chromatographic pattern was similar to that seen postprandially. The plasma half life of intact NT and C-terminal immunoreactivity was 1.5 and 1.2 min, whereas that of N-terminal immunoreactivity was 10.0 min. The differences in circulating levels could be explained by these differences in metabolism, but the physiological significance remains to be elucidated.

  11. Immunoreactivity of lactic acid-treated mare's milk after simulated digestion.

    PubMed

    Fotschki, Joanna; Szyc, Anna; Wróblewska, Barbara

    2015-02-01

    The similarity of mare's milk to breast milk makes it an interesting substrate for the creation of dairy beverages. The aim of this study was to determine the immunoreactivity of the digested mare's milk products carried out by lactic acid fermentation with Lactobacillus casei LCY, Streptococcus thermophilus MK10 and Bifidobacterium animalis Bi30. Simulation of digestion with saliva, pepsin and pancreatin/bile salts was carried out. The immunoreactivity of the milk proteins was assessed by competitive ELISA. The separation of proteins was studied using a tricine SDS-PAGE method. It has been demonstrated that lactic acid fermentation significantly decreases the immunoreactivity of β-lactoglobulin, β-casein, κ-casein and bovine serum albumin. The level of reduction was connected to the type of bacterial strain. The simulated digestion processes caused the decline of immunoreactivity, and the decreases obtained in the experiment were as follows: lactoferrin: 95%, β-lactoglobulin: 94%, β-casein: 93%, α-lactalbumin: 82%, α-casein: 82%, bovine serum albumin: 76% and κ-casein: 37%. The results of the study indicated that microbial fermentation with tested strains is a valuable method for reducing the immunoreactivity of mare's milk proteins. However, further studies with other bacterial strains are needed to gain a higher level of elimination or total reduction of mare's milk immunoreactivity to possibly introduce fermented mare's milk into the diet of patients with immune-mediated digestive problems.

  12. Attenuation by butalbital of capsaicin-induced c-fos-like immunoreactivity in trigeminal nucleus caudalis.

    PubMed

    Cutrer, F M; Mitsikostas, D D; Ayata, G; Sanchez del Rio, M

    1999-01-01

    We examined the effects of butalbital (30, 100, and 1000 micrograms/kg) on the number of cells expressing c-fos-like immunoreactivity (c-fos-LI), a marker of neuronal activation, within lamina I, IIo of the trigeminal nucleus caudalis and the nucleus of the solitary tract 2 hours after the intracisternal injection of capsaicin (0.1 mL; 15.25 mg/mL) or vehicle in urethane-anesthetized guinea pigs (N = 45). Robust c-fos-LI was observed within nuclei of cells in the trigeminal nucleus caudalis after capsaicin (329 +/- 35). Butalbital dose-dependently reduced the number of labeled cells to a maximum of 66% (1000 micrograms/kg intraperitoneally [i.p.], P < .01) in lamina I, IIo but not within area postrema, medial reticular nucleus, or the nucleus of the solitary tract. Pretreatment with bicuculline (30 micrograms/kg i.p.) blocked the effect of butalbital, thereby suggesting the importance of the GABAA receptor to activation involved in the transmission of nociceptive information. Our studies suggest the possibility that GABAA receptors might provide an important therapeutic target in migraine and related headache disorders.

  13. Regulation of gamma-secretase activating protein by the 5Lipoxygenase: in vitro and in vivo evidence

    PubMed Central

    Chu, Jin; Li, Jian-Guo; Hoffman, Nicholas E.; Stough, Alexandra M.; Madesh, Muniswamy; Praticò, Domenico

    2015-01-01

    The formation of Aβ is directly controlled by the γ-secretase complex and its activator, γ-secretase activating protein (GSAP). GSAP derives from a C-terminal fragment of a larger precursor protein via a caspase-3 mediated cleavage. However, the mechanism regulating this process remains unknown. Here we provide in vitro experimental evidence that 5-Lipoxygenase (5LO) is as an endogenous regulator for GSAP formation, but not for other known γ-secretase modulators, by directly and specifically activating caspase-3. These results were confirmed in vivo by using transgenic mouse models of Alzheimer’s disease in which 5LO level and activity were modulated genetically or pharmacologically. Taken together, our findings demonstrate that GSAP cleavage via caspase-3 is regulated and depend upon the availability of 5LO further establishing this protein as an attractive and viable therapeutic target for Alzheimer’s disease. PMID:26076991

  14. Cytoprotective Activity of Glycyrrhizae radix Extract Against Arsenite-induced Cytotoxicity

    PubMed Central

    Kim, Sang Chan; Park, Sook Jahr; Lee, Jong Rok; Seo, Jung Cheol; Yang, Chae Ha

    2008-01-01

    Licorice, Glycyrrhizae radix, is one of the herbal medicines in East Asia that has been commonly used for treating various diseases, including stomach disorders. This study investigated the effect of licorice on arsenite (As)-induced cytotoxicity in H4IIE cells, a rat hepatocyte-derived cell line. Cell viability was significantly diminished in As-treated H4IIE cells in a time and concentration-dependent manner. Furthermore, results from flow cytometric assay and DNA laddering in H4IIE cells showed that As treatment induced apoptotic cell death by activating caspase-3. Licorice (0.1 and 1.0 mg ml−1) treatment significantly inhibited cell death and the activity of caspase-3 in response to As exposure. These results demonstrate that licorice induced a cytoprotective effect against As-induced cell death by inhibition of caspase-3. PMID:18604262

  15. Seasonal changes in hypothalamic gonadotropin-releasing hormone-I immunoreactivity in relation with testicular volume in adult male free-living European starlings (Sturnus vulgaris).

    PubMed

    Pintér, O; Péczely, P

    2010-09-01

    Birds from the temperate and cold zones show annual sexual activity accompanied by gonadal changes and fluctuation in their brain gonadotropin-releasing hormone (GnRH) levels. However, most of the studies were done on captive birds where the constant environment can profoundly modify periodical changes. Therefore our aim was to reveal annual variations of hypothalamic and gonadal changes in male, free-living European starlings (Sturnus vulgaris) captured directly from their natural environment. We analyzed hypothalamic GnRH-I immunoreactivity and testes volume. Four key time points of the active reproductive cycle and the photorefractory phase were studied. GnRH-I immunoreactivity was analyzed in the preoptic area (POA) and the median eminence (ME). Photorefractory birds (August) with regressed gonads had the lowest level of GnRH-I immunoreactivity compared to other birds from the active reproductive phases. These results suggest that parallel with the gonadal volume GnRH-I undergoes seasonal changes in adult male free-living European starlings. PMID:20724271

  16. GFAP immunoreactivity within the rat nucleus ambiguus after laryngeal nerve injury

    PubMed Central

    Berdugo-Vega, G; Arias-Gil, G; Rodriguez-Niedenführ, M; Davies, D C; Vázquez, T; Pascual-Font, A

    2014-01-01

    Changes that occur in astroglial populations of the nucleus ambiguus after recurrent (RLN) or superior (SLN) laryngeal nerve injury have hitherto not been fully characterised. In the present study, rat RLN and SLN were lesioned. After 3, 7, 14, 28 or 56 days of survival, the nucleus ambiguus was investigated by means of glial fibrillary acidic protein (GFAP) immunofluorescence or a combination of GFAP immunofluorescence and the application of retrograde tracers. GFAP immunoreactivity was significantly increased 3 days after RLN resection and it remained significantly elevated until after 28 days post injury (dpi). By 56 dpi it had returned to basal levels. In contrast, following RLN transection with repair, GFAP immunoreactivity was significantly elevated at 7 dpi and remained significantly elevated until 14 dpi. It had returned to basal levels by 28 dpi. Topographical analysis of the distribution of GFAP immunoreactivity revealed that after RLN injury, GFAP immunoreactivity was increased beyond the area of the nucleus ambiguus within which RLN motor neuron somata were located. GFAP immunoreactivity was also observed in the vicinity of neuronal somata that project into the uninjured SLN. Similarly, lesion of the SLN resulted in increased GFAP immunoreactivity around the neuronal somata projecting into it and also in the vicinity of the motor neuron somata projecting into the RLN. The increase in GFAP immunoreactivity outside of the region containing the motor neurons projecting into the injured nerve, may reflect the onset of a regenerative process attempting to compensate for impairment of one of the laryngeal nerves and may occur because of the dual innervation of the posterior cricoarytenoid muscle. This dual innervation of a very specialised muscle could provide a useful model system for studying the molecular mechanisms underlying axonal regeneration process and the results of the current study could provide the basis for studies into functional regeneration

  17. Decreased orexin (hypocretin) immunoreactivity in the hypothalamus and pontine nuclei in sudden infant death syndrome.

    PubMed

    Hunt, Nicholas J; Waters, Karen A; Rodriguez, Michael L; Machaalani, Rita

    2015-08-01

    Infants at risk of sudden infant death syndrome (SIDS) have been shown to have dysfunctional sleep and poor arousal thresholds. In animal studies, both these attributes have been linked to impaired signalling of the neuropeptide orexin. This study examined the immunoreactivity of orexin (OxA and OxB) in the tuberal hypothalamus (n = 27) and the pons (n = 15) of infants (1-10 months) who died from SIDS compared to age-matched non-SIDS infants. The percentage of orexin immunoreactive neurons and the total number of neurons were quantified in the dorsomedial, perifornical and lateral hypothalamus at three levels of the tuberal hypothalamus. In the pons, the area of orexin immunoreactive fibres were quantified in the locus coeruleus (LC), dorsal raphe (DR), laterodorsal tegmental (LDT), medial parabrachial, dorsal tegmental (DTg) and pontine nuclei (Pn) using automated methods. OxA and OxB were co-expressed in all hypothalamic and pontine nuclei examined. In SIDS infants, orexin immunoreactivity was decreased by up to 21 % within each of the three levels of the hypothalamus compared to non-SIDS (p ≤ 0.050). In the pons, a 40-50 % decrease in OxA occurred in the all pontine nuclei, while a similar decrease in OxB immunoreactivity was observed in the LC, LDT, DTg and Pn (p ≤ 0.025). No correlations were found between the decreased orexin immunoreactivity and previously identified risk factors for SIDS, including prone sleeping position and cigarette smoke exposure. This finding of reduced orexin immunoreactivity in SIDS infants may be associated with sleep dysfunction and impaired arousal.

  18. Decreased orexin (hypocretin) immunoreactivity in the hypothalamus and pontine nuclei in sudden infant death syndrome.

    PubMed

    Hunt, Nicholas J; Waters, Karen A; Rodriguez, Michael L; Machaalani, Rita

    2015-08-01

    Infants at risk of sudden infant death syndrome (SIDS) have been shown to have dysfunctional sleep and poor arousal thresholds. In animal studies, both these attributes have been linked to impaired signalling of the neuropeptide orexin. This study examined the immunoreactivity of orexin (OxA and OxB) in the tuberal hypothalamus (n = 27) and the pons (n = 15) of infants (1-10 months) who died from SIDS compared to age-matched non-SIDS infants. The percentage of orexin immunoreactive neurons and the total number of neurons were quantified in the dorsomedial, perifornical and lateral hypothalamus at three levels of the tuberal hypothalamus. In the pons, the area of orexin immunoreactive fibres were quantified in the locus coeruleus (LC), dorsal raphe (DR), laterodorsal tegmental (LDT), medial parabrachial, dorsal tegmental (DTg) and pontine nuclei (Pn) using automated methods. OxA and OxB were co-expressed in all hypothalamic and pontine nuclei examined. In SIDS infants, orexin immunoreactivity was decreased by up to 21 % within each of the three levels of the hypothalamus compared to non-SIDS (p ≤ 0.050). In the pons, a 40-50 % decrease in OxA occurred in the all pontine nuclei, while a similar decrease in OxB immunoreactivity was observed in the LC, LDT, DTg and Pn (p ≤ 0.025). No correlations were found between the decreased orexin immunoreactivity and previously identified risk factors for SIDS, including prone sleeping position and cigarette smoke exposure. This finding of reduced orexin immunoreactivity in SIDS infants may be associated with sleep dysfunction and impaired arousal. PMID:25953524

  19. Differential effects of experimentally induced chronic pancreatitis on neuropeptide immunoreactivities in the feline pancreas.

    PubMed

    De Giorgio, R; Sternini, C; Widdison, A L; Alvarez, C; Brecha, N C; Reber, H A; Go, V L

    1993-11-01

    The distribution and concentration of calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and gastrin-releasing peptide (GRP) immunoreactivities in the pancreas of cats with experimentally induced chronic pancreatitis and of age- and sex-matched controls were investigated. By narrowing the main pancreatic duct between the head and the body to approximately 25% of its normal diameter, we induced within 5 weeks chronic pancreatitis restricted to the body and tail. In control animals, peptide immunoreactive nerves were distributed to the islets, acini, and ducts; the latter were predominantly innervated by fibers immunoreactive for NPY, VIP, or CGRP. The vasculature received an abundant supply of NPY-, CGRP-, and, to a lesser extent, SP-containing axons. Within intrapancreatic ganglia, peptide immunoreactivities were identified in fibers and ganglion cells, with the exception of CGRP and SP immunostaining, which could be visualized only in fibers. In animals with chronic pancreatitis, the innervation pattern of each peptidergic system was comparable to that described in controls. However, there was a remarkable increase in the density and staining intensity of VIP and NPY immunoreactive fibers in the exocrine parenchyma and fibrous septa of the body and tail, where chronic pancreatitis developed. Fibers immunoreactive for CGRP and SP also were moderately denser than in controls, whereas those containing GRP immunoreactivity did not show any detectable changes. In addition, a marked increase of the immunostaining for VIP and, to a much lesser extent, for NPY and GRP, was observed in neurites supplying the head of the pancreas, which appeared devoid of histologically detectable pathological alterations. Radioimmunoassay analysis confirmed the immunohistochemical observations. The increased density of distinct peptidergic nerves in the pancreas with induced chronic pancreatitis might be the result of

  20. Acid-sensing ion channel immunoreactivities in the cephalic neuromasts of adult zebrafish.

    PubMed

    Abbate, F; Madrigrano, M; Scopitteri, T; Levanti, M; Cobo, J L; Germanà, A; Vega, J A; Laurà, R

    2016-09-01

    The neuromasts are the morphofunctional unit of the lateral line system serving as mechanosensors for water flow and movement. The mechanisms underlying the detection of the mechanical stimuli in the vertebrate mechanosensory cells remain poorly understood at the molecular level, and no information is available on neuromasts. Mechanotransduction is the conversion of a mechanical stimulus into an electrical signal via activation of ion channels. The acid-sensing ion channels (ASICs) are presumably involved in mechanosensation, and therefore are expected to be expressed in the mechanoreceptors. Here we used immunohistochemistry to investigate the occurrence and distribution of ASICs in the cephalic neuromasts of the adult zebrafish. Specific immunoreactivity for ASIC1 and ASIC4 was detected in the hair cells while ASIC2 was restricted to the nerves supplying neuromasts. Moreover, supporting and mantle cells; i.e., the non-sensory cells of the neuromasts, also displayed ASIC4. For the first time, these results demonstrate the presence of the putative mechanoproteins ASIC1, ASIC2 and ASIC4 in neuromasts, suggesting a role for these ion channels in mechanosensation.

  1. Ontogeny of Fos protein-like immunoreactivity in the suprachiasmatic nucleus.

    PubMed

    Joyce, M P; Barr, G A

    1995-09-01

    This study examined the normal development of neuronal activity in the suprachiasmatic nuclei (SCN) of rats between age 3-60 days, using Fos protein-like immunoreactivity (Fos-LI) as a marker. At age 3 days, Fos-positive nuclei are sparsely distributed throughout the SCN. Between age 3-10 days, the density of labeled nuclei increases significantly. Fos-LI labeling is maximal at 10 days. Between age 10-14 days, the number of labeled nuclei decreases and remains relatively constant thereafter, although the intensity of the reaction product diminishes as the animal matures. By age 60 days, the number of Fos-LI labeled nuclei in the SCN is substantially decreased and is essentially the same as in the 3-day-old rat. The appearance of Fos-LI nuclei in the SCN during development appears to reflect the development of visual system afferents to the nucleus as well as the development of intrinsic SCN synaptology. PMID:8525462

  2. Acid-sensing ion channel immunoreactivities in the cephalic neuromasts of adult zebrafish.

    PubMed

    Abbate, F; Madrigrano, M; Scopitteri, T; Levanti, M; Cobo, J L; Germanà, A; Vega, J A; Laurà, R

    2016-09-01

    The neuromasts are the morphofunctional unit of the lateral line system serving as mechanosensors for water flow and movement. The mechanisms underlying the detection of the mechanical stimuli in the vertebrate mechanosensory cells remain poorly understood at the molecular level, and no information is available on neuromasts. Mechanotransduction is the conversion of a mechanical stimulus into an electrical signal via activation of ion channels. The acid-sensing ion channels (ASICs) are presumably involved in mechanosensation, and therefore are expected to be expressed in the mechanoreceptors. Here we used immunohistochemistry to investigate the occurrence and distribution of ASICs in the cephalic neuromasts of the adult zebrafish. Specific immunoreactivity for ASIC1 and ASIC4 was detected in the hair cells while ASIC2 was restricted to the nerves supplying neuromasts. Moreover, supporting and mantle cells; i.e., the non-sensory cells of the neuromasts, also displayed ASIC4. For the first time, these results demonstrate the presence of the putative mechanoproteins ASIC1, ASIC2 and ASIC4 in neuromasts, suggesting a role for these ion channels in mechanosensation. PMID:27443821

  3. Subfornical organ disconnection and Fos-like immunoreactivity in hypothalamic nuclei after intragastric hypertonic saline.

    PubMed

    Starbuck, Elizabeth M; Fitts, Douglas A

    2002-10-01

    The subfornical organ (SFO) may act as a sodium- or osmoreceptor that drives hypothalamic and other nuclei to secrete vasopressin and to elicit drinking. However, in response to mild doses of hypertonic saline, Fos-like immunoreactivity (Fos-ir) is absent in the SFO whereas it is well expressed in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. This suggests that the hypothalamus may be activated in advance of the SFO. In this study, the fibers connecting the SFO and hypothalamus were disconnected by a wire knife cut so that Fos-ir could be examined in both the SFO and hypothalamus after an intragastric (ig) load of 0.5% of body weight of 0.6 M NaCl. Compared with Fos-ir in isotonic-loaded rats, Fos-ir after the hypertonic load was not significantly elevated in the SFO or median preoptic nucleus in sham-cut or knife-cut rats and was only slightly elevated in the OVLT in sham-cut rats. However, the hypertonic load in sham-cut rats greatly elevated Fos-ir in the SON and in the entire PVN, but this expression was reduced significantly by 30-50% in knife-cut rats. Thus, the connectivity between SFO and the hypothalamus is critical for the full expression of Fos-ir in the hypothalamus during moderate ig hypertonic saline loading even when the SFO itself does not yet express Fos-ir. PMID:12270498

  4. Nitric oxide production and nitric oxide synthase immunoreactivity in Naegleria fowleri.

    PubMed

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A; Moreno-Fierros, Leticia; Jarillo-Luna, Adriana; Carrasco-Yepez, Marisela; Miliar-García, Angel; Campos-Rodríguez, Rafael

    2007-07-01

    Free-living ameba Naegleria fowleri produces an acute and fatal infectious disease called primary amebic meningoencephalitis (PAM), whose pathophysiological mechanism is largely unknown. The aim of this study was to investigate the role of nitric oxide (NO) in PAM. Although NO has a cytotoxic effect on various parasites, it is produced by others as part of the pathology, as is the case with Entamoeba histolytica. To test for the production of NO, we analyzed whether antibodies against mammalian NO synthase isoforms (neuronal, inducible, and endothelial) presented immunoreactivity to N. fowleri proteins. We found that the trophozoites produced NO in vitro. The Western blot results, which showed N. fowleri trophozoites, contained proteins that share epitopes with the three described mammalian NOS, but have relative molecular weights different than those described in the literature, suggesting that N. fowleri may contain undescribed NOS isoforms. Moreover, we found that trophozoites reacted to the NOS2 antibody, in amebic cultures as well as in the mouse brain infected with N. fowleri, suggesting that nitric oxide may participate in the pathogenesis of PAM. Further research aimed at determining whether N. fowleri contains active novel NOS isoforms could lead to the design of new therapies against this parasite.

  5. Analysis of the brain ACTH-immunoreactive peptide spectrum in inbred mice

    SciTech Connect

    Fedoseev, Yu.L.; Blednov, Yu.A.; Seredenin, S.B.

    1987-01-01

    Mice of the BALB/c (C) and C57BL/6 (B6) strains, characterized by high and low emotionality respectively in open field tests, have been shown to differ considerably in both the initial level and the time course of changes in the plasma ACTH concentration after exposure to stress in an open field and after administration of a benzodiazepine tranquilizer. The ACTH concentration in the pituitary gland of animals of these lines also differs. The ACTH molecule is known to contain regions with neurotropic activity. It can therefore be postulated that differences in the level of this hormone and the products of its bioconversion in the brain are an essential factor in the mechanisms of formation of the hereditary features of emotional behavior. In this first stage of this investigation, represented in this paper and undertaken to test this hypothesis, spectra of ACTH-immunoreactive peptides were studied in chromatographic fractions of an acid brain extract as well as in the blood plasma of mice belonging to B6 and C lines and their hybrids. The peptides were determined by radioimmunoassay.

  6. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1.

    PubMed

    Citron, Bruce A; Ameenuddin, Syed; Uchida, K; Suo, William Z; SantaCruz, Karen; Festoff, Barry W

    2016-07-15

    Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies. PMID:27138068

  7. Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

    PubMed

    Nie, C; Luo, Y; Zhao, X; Luo, N; Tong, A; Liu, X; Yuan, Z; Wang, C; Wei, Y

    2014-10-30

    The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

  8. Study of the temperature dependent immuno-reaction kinetics for the bio-functionalized magnetic nanoparticle assay of bio-markers of colorectal cancer

    NASA Astrophysics Data System (ADS)

    Yang, S. Y.; Chang, J. F.; Chen, T. C.; Yang, C. C.; Ho, C. S.

    2014-01-01

    By conjugating antibodies on magnetic nanoparticles, target antigens can be quantitatively detected by measuring the magnetic signals of the magnetic nanoparticles due to their association with target antigens. This method of detection is called magnetically labeled immunoassay. The assay characteristics of magnetically labeled immunoassay have been reported widely. However, the immuno-reaction kinetics of magnetically labeled immunoassay has not been studied. In this work, the reaction rates between magnetic nanoparticles and target antigens are measured at various temperatures. It is found that the temperature dependent reaction rate obeys Arrhenius's equation, which shows the collision frequency and activation energy for the immuno-reaction between antibody-functionalized magnetic nanoparticles and target antigens. The carcinoembryonic antigen, which is a regular blood bio-marker for in-vitro diagnosis of colorectal cancer, is used as a target antigen for the example.

  9. High frequency stimulation of the subthalamic nucleus increases c-fos immunoreactivity in the dorsal raphe nucleus and afferent brain regions.

    PubMed

    Tan, Sonny K H; Janssen, Marcus L F; Jahanshahi, Ali; Chouliaras, Leonidas; Visser-Vandewalle, Veerle; Lim, Lee Wei; Steinbusch, Harry W M; Sharp, Trevor; Temel, Yasin

    2011-10-01

    High frequency stimulation (HFS) of the subthalamic nucleus (STN) is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced Parkinson's disease, but this treatment can elicit disabling mood changes. Our recent experiments show that in rats, HFS of the STN both inhibits the firing of 5-HT (5-hydroxytryptamine; serotonin) neurons in the dorsal raphe nucleus (DRN) and elicits 5-HT-dependent behavioral effects. The neural circuitry underpinning these effects is unknown. Here we investigated in the dopamine-denervated rat the effect of bilateral HFS of the STN on markers of neuronal activity in the DRN as well as DRN input regions. Controls were sham-stimulated rats. HFS of the STN elicited changes in two 5-HT-sensitive behavioral tests. Specifically, HFS increased immobility in the forced swim test and increased interaction in a social interaction task. HFS of the STN at the same stimulation parameters, increased c-fos immunoreactivity in the DRN, and decreased cytochrome C oxidase activity in this region. The increase in c-fos immunoreactivity occurred in DRN neurons immunopositive for the GABA marker parvalbumin. HFS of the STN also increased the number of c-fos immunoreactive cells in the lateral habenula nucleus, medial prefrontal cortex but not significantly in the substantia nigra. Collectively, these findings support a role for circuitry involving DRN GABA neurons, as well as DRN afferents from the lateral habenula nucleus and medial prefrontal cortex, in the mood effects of HFS of the STN.

  10. Distribution of APGWamide-immunoreactivity in the brain and reproductive organs of adult pygmy squid, Idiosepius pygmaeus.

    PubMed

    Sirinupong, P; Suwanjarat, J; van Minnen, J

    2011-12-01

    The neuropeptide APGWamide is involved in the control of the reproductive behavior in molluscs. Using immunocytochemistry, we investigated the distribution of APGWa-immunoreactive neurons in the brain and reproductive organs of adult male and female specimen of Idiosepius pygmaeus. The study showed that the APGWamide-immunoreactive neurons and fibers are localized in the dorsal basal and vertical lobes of the supraesophageal mass, the palliovisceral lobe of the posterior subesophageal mass and olfactory lobe of the optic tract in male brains, with the highest number of APGWamide-immunoreactive neurons in the palliovisceral and olfactory lobes. In females, only the palliovisceral and olfactory lobes contained APGWa-immunoreactive neurons. The number of APGWamide-immunoreactive neurons in male I. pygmaeus brain is significantly higher than in females. Furthermore, APGWamide-immunoreactive fibers are localized exclusively in male reproductive organs and mantle muscles. Together these data suggest a role for APGW-amide in the control of male reproduction.

  11. Mapping of alpha-melanocyte-stimulating hormone-like immunoreactivity in the cat brainstem.

    PubMed

    Coveñas, R; de León, M; Narváez, J A; Aguirre, J A; Tramu, G

    2000-04-01

    The distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures was studied in the brainstem of the cat using an indirect immunoperoxidase technique. Immunoreactivity was observed in several brainstem nuclei of the cat in which no immunoreactivity had been previously reported. Immunoreactive fibres were observed in the following; the inferior central nucleus; the pontine gray nuclei; the Kölliker-Fuse nucleus; the motor trigeminal nucleus, the anteroventral cochlear nucleus; the abducens nucleus; the retrofacial nucleus; the superior, lateral, inferior, and medial vestibular nuclei; the lateral nucleus of the superior olive; the external cuneate nucleus; the nucleus of the trapezoid body; the postpyramidal nucleus of the raphe; the medial accessory inferior olive; the dorsal accessory nucleus of the inferior olive; the nucleus ambiguus; the principal nucleus of the inferior olive; the preolivary nucleus; the nucleus ruber; the substantia nigra; and in the area postrema. Our results point to a more widespread distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures in the cat brainstem than that reported in previous studies carried out in the same region of the cat, rat and humans.

  12. Occurrence of gustducin-immunoreactive cells in von Ebner's glands of guinea pigs.

    PubMed

    Ibira, Yuki; Yokosuka, Hiroyuki; Haga-Tsujimura, Maiko; Yoshie, Sumio

    2013-11-01

    An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner's glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP3R-3), phospholipase Cβ2 (PLCβ2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception. PMID:23604549

  13. Radioimmunoassay for human pancreatic ribonuclease and measurement of serum immunoreactive pancreatic ribonuclease in patients with malignant tumors

    SciTech Connect

    Kurihara, M.; Ogawa, M.; Ohta, T.; Kurokawa, E.; Kitahara, T.; Murata, A.; Matsuda, K.; Kosaki, G.; Watanabe, T.; Wada, H.

    1984-05-01

    A method for radioimmunoassay of human pancreatic RNase was developed. The method is sensitive, reproducible, and specific. Almost no cross-reactivity exists between human pancreatic and liver RNases. A good correlation was observed between the serum concentration of pancreatic RNase as measured by radioimmunoassay and its enzymatic activity using polycytidylic acid as substrate. The concentration of serum pancreatic RNase correlates well with age, blood urea nitrogen, and albumin contents but does not correlate with serum amylase activity. Using the data of 52 patients with malignant tumors except pancreatic cancer, serum RNase level could be expressed by a multiple regression equation: Immunoreactive RNase content in pancreatic cancer was elevated in patients with complications from renal failure. Serum pancreatic RNase contents in patients with pancreatic cancer measured by radioimmunoassay agreed well with the values calculated using the equation derived from the data of patients with other malignant tumors.

  14. Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates.

    PubMed

    Mizuno, J; Takeda, N

    1988-01-01

    1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

  15. Effects of heat and high-pressure treatments on the solubility and immunoreactivity of almond proteins.

    PubMed

    Zhang, Yan; Zhang, Jieqiong; Sheng, Wei; Wang, Shuo; Fu, Tong-Jen

    2016-05-15

    The effects of dry and moist heat, autoclave sterilization and high-pressure treatment on the biochemical characteristics and immunological properties of almond proteins were investigated. Changes in the solubility and immunoreactivity of almond proteins extracted from treated almond flour were evaluated using a total protein assay, indirect competitive inhibition enzyme-linked immunosorbent assay (IC-ELISA), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Almond proteins were stable during dry-heat treatment at temperatures below 250°C. Dry heat at 400°C, boiling, autoclave sterilization and high-pressure treatment in the presence of water at ⩾ 500 MPa greatly reduced the solubility and immunoreactivity of almond proteins. SDS-PAGE revealed that the protein profiles of almond flour samples treated under these conditions also changed significantly. The synergistic effects of heat, pressure and the presence of water contributed to significant changes in solubility and immunoreactivity of almond proteins.

  16. [Changes in neuropeptide Y and substance P immunoreactive nerve fibres and immunocompetent cells in hepatitis].

    PubMed

    Fehér, Erzsébet

    2015-11-22

    Neuropeptide Y and substance P were thought to play a role in the function of immune cells and in amplification or elimination of the inflammatory processes. In hepatitis the number of both neuropeptide Y and substance P immunoreactive nerve fibres are increased, where the increase of neoropeptide Y is significant. A large number of lymphocytes and mast cells are also stained for neuropeptide Y and substance P. Very close associations (less than 1 µm) were observed between neuropeptide Y immunoreactive nerve fibres and immune cells stained also with neuropeptide Y. Some immune cells were also found to be immunoreactive for tumor necrosis factor-α and NF-κB. Some of the SP IR immunocells were also stained for TNF-α and nuclear factor kappaB. Based on these data it is hypothesized that neuropeptid Y and substance P released from nerve fibres and immune cells play a role in inflammation and elimination of inflammation in hepatitis.

  17. Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

    SciTech Connect

    Hashimoto, K.; Hattori, T.; Murakami, K.; Suemaru, S.; Kawada, Y.; Kageyama, J.; Ota, Z.

    1985-02-18

    The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of /sup 125/I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neurointermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.

  18. Effects of heat and high-pressure treatments on the solubility and immunoreactivity of almond proteins.

    PubMed

    Zhang, Yan; Zhang, Jieqiong; Sheng, Wei; Wang, Shuo; Fu, Tong-Jen

    2016-05-15

    The effects of dry and moist heat, autoclave sterilization and high-pressure treatment on the biochemical characteristics and immunological properties of almond proteins were investigated. Changes in the solubility and immunoreactivity of almond proteins extracted from treated almond flour were evaluated using a total protein assay, indirect competitive inhibition enzyme-linked immunosorbent assay (IC-ELISA), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Almond proteins were stable during dry-heat treatment at temperatures below 250°C. Dry heat at 400°C, boiling, autoclave sterilization and high-pressure treatment in the presence of water at ⩾ 500 MPa greatly reduced the solubility and immunoreactivity of almond proteins. SDS-PAGE revealed that the protein profiles of almond flour samples treated under these conditions also changed significantly. The synergistic effects of heat, pressure and the presence of water contributed to significant changes in solubility and immunoreactivity of almond proteins. PMID:26776044

  19. Somatostatin-immunoreactive nerve cell bodies and fibers in the medulla oblongata et spinalis.

    PubMed

    Forssmann, W G; Burnweit, C; Shehab, T; Triepel, J

    1979-10-01

    Complete serial sectioning of the medulla oblongata in monkey, cat, guinea pig, and japanese dancing mouse and incubation for somatostatin-immunoreaction was carried out. Numerous regions of the medulla oblongata such as the nucleus reticularis gigantocellularis, nucleus cuneatus et gracillis, nucleus raphe magnus, nucleus tractus solitarius, nucleus vestibularis, and parts of the oliva contain dense networks of somatostatin-immunoreactive nerve fibers. Cell bodies were seen in the nucleus reticularis medullae oblongatae. In the spinal cord the sections from each segment were analyzed, showing the highest concentrations of somatostatinergic fibers in the substantia gelantinosa of the columna dorsalis. Cell bodies were seen in the zona intermedia centralis, especially in the upper cervical segments. Many positive fibers were also seen in the entire zona intermedia and the columna ventralis. Especially prominent was the immunoreactivity in the zona intermediolateralis of the thoracic segments and the columna ventralis of the lower lumbar and sacral segments.

  20. Pre- and postoperative changes in serum myelin basic protein immunoreactivity in neurosurgical patients.

    PubMed

    Hoyle, N R; Seeldrayers, P A; Moussa, A H; Paul, E A; Thomas, D G

    1984-07-01

    In 44 patients undergoing neurosurgical procedures for intracranial tumors, subarachnoid hemorrhage, or spinal and peripheral nerve lesions, serum myelin basic protein (MBP) immunoreactivity was measured preoperatively and serially in the first 10 postoperative days. The double-antibody radioimmunoassay method was used, with a detection limit of 2.5 ng/ml in serum. Clinical evaluation was carried out at admission and on successive days during the period of neurosurgical management; outcome was assessed later. In the early postoperative phase, there was a fall in MBP immunoreactivity in all groups of patients. In the groups with intracranial tumor and subarachnoid hemorrhage, there was a subsequent rise in MBP immunoreactivity before the end of the 10-day period, which was not found in the group with spinal and peripheral nerve lesions.

  1. Existence of subtypes of gustducin-immunoreactive cells in the vallate taste bud of guinea pigs.

    PubMed

    Ohkubo, Yasuhiro; Yokosuka, Hiroyuki; Kumakura, Masahiko; Yoshie, Sumio

    2007-12-01

    Vallate taste buds in the guinea-pig tongue were immunohistochemically investigated with regard to the colocalization of gustducin with calbindin-D28K (=spot 35 protein) and type III inositol triphosphate receptor (IP(3)R-3) in order to characterize gustducin-immunoreactive cells. Individual taste bud cells ranged from totally immunopositive to totally immunonegative for these three molecules. Among the immunoreactive cells, gustducin-immunoreactive cells were divided into two cell populations: one immunopositive and the other immunonegative for calbindin-D28K. Applying our previous data to the present results, the former cells should belong to Type III cells designated by electron microscopy. This finding provides new evidence regarding the taste bud types of cells expressing gustducin in the guinea pig. PMID:18431029

  2. An optimized activity-based probe for the study of caspase-6 activation

    PubMed Central

    Edgington, Laura E.; van Raam, Bram J.; Verdoes, Martijn; Wierschem, Christoph; Salvesen, Guy S.; Bogyo, Matthew

    2012-01-01

    Summary While significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when the executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspases-3/-6 /-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe AB50. Using this probe we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the pro-enzyme. We also demonstrate that caspase-6 activation does not require active caspases-3/-7 suggesting that it may auto-activate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions. PMID:22444589

  3. OXYTOCIN-IMMUNOREACTIVE INNERVATION OF IDENTIFIED NEURONS IN THE RAT DORSAL VAGAL COMPLEX

    PubMed Central

    Llewellyn-Smith, Ida J.; Kellett, Daniel O.; Jordan, David; Browning, Kirsteen N.; Travagli, R. Alberto

    2011-01-01

    Background Oxytocin (OXT) has been implicated in reproduction and social interactions as well as in the control of digestion and blood pressure. OXT-immunoreactive axons occur in the dorsal vagal complex (DVC; nucleus tractus solitarius, NTS, dorsal motor nucleus of the vagus, DMV, and area postrema, AP), which contains neurons that regulate autonomic homeostasis. The aim of the present work was to provide a systematic investigation of the OXT-immunoreactive innervation of DVC neurons involved in the control of gastrointestinal (GI) function. Methods We studied DMV neurons identified by 1) prior injection of retrograde tracers in the stomach, ileum or cervical vagus or 2) induction of c-fos expression by glucoprivation with 2-deoxyglucose. Another subgroup of DMV neurons was identified electrophysiologically by stimulation of the cervical vagus and then juxtacellularly labelled with biotinamide. We used two- or three-color immunoperoxidase labelling for studies at the light microscopic level. Results Close appositions from OXT-immunoreactive varicosities were found on the cell bodies, dendrites and axons of DMV neurons that projected to the GI tract and that responded to 2-deoxyglucose as well as juxtacellularly-labelled DMV neurons. Double staining for OXT and choline acetyltransferase revealed that OXT innervation was heavier in the caudal and lateral DMV than in other regions. OXT-immunoreactive varicosities also closely apposed a small subset of tyrosine hydroxylase-immunoreactive NTS and DMV neurons. Conclusions and inferences Our results provide the first anatomical evidence for direct OXT-immunoreactive innervation of GI-related neurons in the DVC. PMID:22188490

  4. Anion exchanger immunoreactivity in human salivary glands in health and Sjögren's syndrome.

    PubMed Central

    Vázquez, J. J.; Vázquez, M.; Idoate, M. A.; Montuenga, L.; Martínez-Ansó, E.; Castillo, J. E.; García, N.; Medina, J. F.; Prieto, J.

    1995-01-01

    Salivary gland ducts play a relevant role in saliva secretion through transport processes. Na(+)-independent chloride-bicarbonate anion exchangers (AE) may be involved in these processes by generating ion fluxes into the salivary secretion. In Sjögren's syndrome, a disorder with gland dysfunction, there might be an impaired expression of AE proteins. Here we study AE immunoreactivities in human salivary glands, both in health and in Sjögren's syndrome. Immunohistochemistry was carried out on salivary glands from normal subjects and patients with Sjögren's syndrome, using two monoclonal antibodies against AE1 and AE2. Normal salivary glands showed AE2 immunoreactivity, which was restricted to the epithelium of the ducts, with no staining at the acini. A strong positivity was seen in the basolateral portion of the striated ducts, while interlobular duct cells showed a discrete positivity at their apical pole. In salivary glands from most of the patients with Sjögren's syndrome, AE2 immunoreactivity was absent in the ducts as well as in the acini. In both normal and diseased salivary glands, AE1 immunoreactivity was only located at the erythrocyte membrane. The recently reported AE0 was discarded because no AE0 message was found in salivary glands by reverse transcription polymerase chain reaction. In conclusion, AE2 immunoreactivity is observed in the ducts of normal salivary glands, particularly in the striated ducts. AE2 immunoreactivity is virtually absent in salivary glands from patients with Sjögren's syndrome, which may reflect either a loss of AE2 after inflammatory atrophy, or a primary defect occurring in the disease. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:7778681

  5. On the localization of Thy-1-like immunoreactivity in the rodent and human nervous system.

    PubMed

    Seiger, A; Almqvist, P; Granholm, A C; Olson, L

    1986-01-01

    The neuronal localisation of the surface glycoprotein Thy-1 was studied using the adult mouse iris whole-mount preparation. Polyclonal antibodies to Thy-1 and indirect immunohistochemical techniques were used on fixed tissues. In the adult intact mouse iris a plexus of delicate bundles and fibres was found in both the dilator and sphincter regions. Ovoid negative spots along the bundles were numerous, probably indicating the location of supportive cells. The ciliary body contained strongly immunoreactive bundles oriented in radial and circular patterns. Numerous Thy-1-positive mast cells were found in the irides. All Thy-1-immunoreactive fibres disappeared in intraocular iris transplants after 4 days, leaving only the Thy-1-positive mast cells. A Thy-1-positive fibre plexus reappeared in intraocular iris transplants after 4 weeks, strongly indicating that Thy-1-immunoreactive fibres in adult mouse irides are associated with the nerve fibres and not with their supportive tissue. Distribution of Thy-1-like immunoreactivity in the developing human nervous system is presented for the first time, and its temporal changes are followed from the eighth gestational week to adulthood. At eight weeks the spinal cord and lower brain stem seemed to show virtually no immunoreactivity. At 10 and 31 weeks gestational age immunoreactivity was found preferentially in white matter areas with a granular appearance, becoming more densely aggregated at the later stage. Two months postnatally the internal capsule was strongly positive in an otherwise negative neuropil, only to disappear completely in the adult brain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2875228

  6. Pharmacological activity in growth inhibition and apoptosis of cultured human leiomyomal cells of tropical plant Scutellaria barbata D. Don (Lamiaceae).

    PubMed

    Lee, Tae-Kyun; Lee, Yun-Jeong; Kim, Dong-Il; Kim, Hyung-Min; Chang, Young-Chae; Kim, Cheorl-Ho

    2006-01-01

    Scutellaria barbata D. Don (Lamiaceae) (SB), which is known in traditional Korean medicine, has been used as an anti-inflammatory and antitumor agent. Since uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium, we aimed to determine the growth inhibition and the induction of apoptotic cell death brought about by the herb SB in two different leiomyomal cells, named LM-1 and LM-2, and to clarify the mechanism of this apoptosis. Water-soluble ingredients of SB, and the leiomyomal cell lines of LM-1 and LM-2, were used in vitro. Growth inhibition, induction of cell death, morphological features, the presence of DNA ladders, increases in Caspase 3-like activity, the effects of a Caspase 3 inhibitor on apoptotic cell death, and the release of Cytochrome C by SB were analyzed. SB inhibited the growth and decreased the viability of the leiomyomal cells. The viability of normal myomatrial smooth muscle cells (SMC) in the presence of low concentrations of SB was higher than those of leiomyomal cells. Apoptotic bodies and DNA ladders were observed to be induced in leiomyomal cells of LM-1 and LM-2 by SB. The synthetic tetrapeptide Caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited the apoptotic cell death of leiomyomal cells induced by SB. The Caspase 3-like activity in leiomyomal cells LM-1 and LM-2 increased after the addition of SB. Cytochrome C was released from mitochondria into the cytosol 8h after the addition of SB, and reached a peak at 16h. The peak of Cytochrome C release was earlier than that of Caspase 3-like activity. We concluded that SB inhibited the growth of the leiomyomal cells and induced apoptosis. The apoptosis of leiomyomal cells induced by SB was associated with the release of Cytochrome C from the mitochondria, followed by an increase in Caspase 3-like activity.

  7. Mapping brain Fos immunoreactivity in response to water deprivation and partial rehydration: Influence of sodium intake.

    PubMed

    Dalmasso, Carolina; Antunes-Rodrigues, José; Vivas, Laura; De Luca, Laurival A

    2015-11-01

    Water deprivation (WD) followed by water intake to satiety, produces satiation of thirst and partial rehydration (PR). Thus, WD-PR is a natural method to differentiate thirst from sodium appetite. WD-PR also produces Fos immunoreactivity (Fos-ir) in interconnected areas of a brain circuit postulated to subserve sodium appetite. In the present work, we evaluated the effect of sodium intake on Fos-ir produced by WD-PR in brain areas operationally defined according to the literature as either facilitatory or inhibitory to sodium intake. Isotonic NaCl was available for ingestion in a sodium appetite test performed immediately after a single episode of WD-PR. Sodium intake decreased Fos-ir in facilitatory areas such as the lamina terminalis (particularly subfornical organ and median preoptic nucleus), central amygdala and hypothalamic parvocellular paraventricular nucleus in the forebrain. Sodium intake also decreased Fos-ir in inhibitory areas such as the area postrema, lateral parabrachial nucleus and nucleus of the solitary tract in the hindbrain. In contrast, sodium intake further increased Fos-ir that was activated by water deprivation in the dorsal raphe nucleus, another inhibitory area localized in the hindbrain. WD-PR increased Fos-ir in the core and shell of the nucleus accumbens. Sodium intake reduced Fos-ir in both parts of the accumbens. In summary, sodium intake following WD-PR reduced Fos-ir in most facilitatory and inhibitory areas, but increased Fos-ir in another inhibitory area. It also reduced Fos-ir in a reward area (accumbens). The results suggest a functional link between sodium intake and the activity of the hindbrain-forebrain circuitry subserving reward and sodium appetite in response to water deprivation.

  8. Transient expression of choline acetyltransferase-like immunoreactivity in Purkinje cells of the developing rat cerebellum.

    PubMed

    Gould, E; Butcher, L L

    1987-08-01

    The expression of choline acetyltransferase (ChAT)-like immunoreactivity was studied immunohistochemically in the cerebelli of developing rats. Brains were examined from the day of birth (postnatal day 1: P1) until adulthood. From P4 through P21, several Purkinje cells in the uvula, nodule, and flocculus of the cerebellum demonstrated ChAT-like immunoreactivity. After P23, no ChAT-positive neurons were observed in any region of the cerebellum. This finding paralleled the transient expression of acetylcholinesterase in Purkinje cells of these same cerebellar areas during development.

  9. Immunohistochemical localization of corticotropin-releasing factor- and arginine vasotocin-like immunoreactivities in the brain and pituitary of the American bullfrog (Rana catesbeiana) during development and metamorphosis.

    PubMed

    Carr, J A; Norris, D O

    1990-05-01

    Immunoperoxidase staining for corticotropin-releasing factor (CRF) in the median eminence was sparce or absent from premetamorphic tadpoles, but increased dramatically by late prometamorphosis. Quantitative photometry revealed that CRF-like immunostaining material in the median eminence was most dense in metamorphic climax tadpoles. Arginine vasotocin (AVT)-like immunostaining material was visualized in perikarya of the magnocellular nucleus, with extensive fiber staining seen in the medial basal and infundibular hypothalamus as well as in the median eminence and pars nervosa of the pituitary. AVT-like immunoreactive perikarya were virtually absent in premetamorphic tadpoles, but their number increased greatly by Taylor-Kollros stage XII and continued to increase after this stage. Quantitative photometry revealed that AVT-like immunoreactivity in the pars nervosa increased greatly at Taylor-Kollros stage XII and remained intense after this stage. AVT-like immunoreactivity did not appear in the median eminence until Taylor-Kollros stage XVI. Localization of AVT-like immunoperoxidase staining around portal vessels in the median eminence suggests an anatomical mechanism for delivery of AVT to anterior pituitary corticotropes. These results indicate that both CRF and vasotocinergic neuronal systems develop just before the activation of interrenal steroidogenesis which occurs during the later stages of metamorphosis in this species.

  10. Quantitative changes of GABA-immunoreactive cells in the hindlimb representation of the rat somatosensory cortex after 14-day hindlimb unloading by tail suspension

    NASA Technical Reports Server (NTRS)

    D'Amelio, F.; Fox, R. A.; Wu, L. C.; Daunton, N. G.

    1996-01-01

    The present study was aimed at evaluating quantitatively gamma-aminobutyric acid (GABA) immunoreactivity in the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension. A reduction in the number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-containing terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system of hindlimb-suspended animals, it is suggested that the unloading due to hindlimb suspension alters afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the reduction in immunoreactivity of local circuit GABAergic neurons and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.

  11. Phospho-histone H3 (pHH3) immuno-reactivity as a prognostic marker in non-functioning pituitary adenomas.

    PubMed

    Hightower, Erica; Cabanillas, Maria E; Fuller, Greg N; McCutcheon, Ian E; Hess, Kenneth R; Shah, Komal; Waguespack, Steven G; Corley, Lynda J; Devin, Jessica K

    2012-12-01

    Nonfunctioning pituitary adenomas (NFPA) are typically benign neoplasms that can cause significant morbidity through local mass effects. MIB-1/Ki-67 and p53 immuno-reactivity are used to predict aggressive behavior but have known limitations. No marker to date is widely used to reliably predict tumor progression. Phospho-histone H3 (pHH3) is a protein phosphorylated during chromatin condensation in mitosis, and thus anti-pHH3 immunocytochemistry is able to assess mitotic activity. Study objectives were to determine the relationship among pHH3, MIB-1/Ki-67, and p53 in NFPA, and to evaluate the relationship between these indices and time to progression (TTP). Seventy-six patients with NFPA operated on by a single neurosurgeon at University of Texas M. D. Anderson Cancer Center from 1992 to 2006 were identified from a database and met all criteria for inclusion in this clinicopathology study. PHH3, MIB-1/Ki-67, and p53 immuno-reactivity was evaluated in each case. Retrospective review was used to determine TTP. With 282 person-years of follow-up, 19 progression events were observed. A correlation was found between MIB-1/Ki-67 and p53 immuno-reactivity (r = 0.25, p = 0.031). PHH3 did not correlate with either. When markers were dichotomized at the median, only MIB-1/Ki-67 correlated with TPP (log rank p = 0.018). Rank correlation analysis confirmed a significant inverse correlation between both MIB-1/Ki-67 (Dxy = -0.33, p = 0.036) and p53 (Dxy = -0.40, 0.016) immuno-reactivity and TTP. Our results support previous data suggesting that MIB-1/Ki-67 and p53 have clinical utility as prognostic markers for tumor progression. PHH3 did not prove to be associated with TTP in this retrospective study limited by few progression events. PMID:22120760

  12. Decreased calcineurin immunoreactivity in the postmortem brain of a patient with schizophrenia who had been prescribed the calcineurin inhibitor, tacrolimus, for leukemia

    PubMed Central

    Wada, Akira; Kunii, Yasuto; Matsumoto, Jyunya; Hino, Mizuki; Nagaoka, Atsuko; Niwa, Shin-ichi; Yabe, Hirooki

    2016-01-01

    N immunore-activity in human brain was stronger than the effect of schizophrenia. PMID:27462157

  13. Effects of food deprivation on goal-directed behavior, spontaneous locomotion, and c-Fos immunoreactivity in the amygdala.

    PubMed

    Moscarello, J M; Ben-Shahar, O; Ettenberg, A

    2009-01-30

    Previous work in our laboratory has shown that food deprivation and food presentation produce different patterns of neuronal activity (as measured by c-Fos immunoreactivity) in the medial prefrontal cortex and nucleus accumbens of rats. Since the amygdala has been implicated in both motivational and reinforcement processes and has neuronal connections to both the prefrontal cortex and nucleus accumbens, it was of interest to assess amygdaloid c-Fos immunoreactivity during similar manipulations of food deprivation and presentation. In the current study, c-Fos counts in both basolateral and central amygdalar nuclei were observed to increase in rats 12- and 36-h food deprived (relative to 0-h controls)-an effect reversed by the presentation of either a small or large meal (2.5 or 20g of food). In another experiment, rats working on a progressive ratio schedule of reinforcement exhibited elevated break-points as a function of food deprivation, a result consistent with the view that the feeding manipulations increased the subjects' level of motivation. In contrast, food deprivation reduced the spontaneous locomotor activity of rats, presumably as a result of an inherent energy-conservation strategy when no food is readily available. These data suggest that the state of food deprivation is associated with: (a) enhanced behavioral output only when food is attainable (increased goal-directed behavior, but decreased spontaneous activity), and (b) increased synaptic engagement in neuronal circuits involved in affective valuation and related decision-making (increased c-Fos counts in the amygdala). PMID:18706934

  14. Effect of low-intensity treadmill exercise on behavioural measures and hippocampal parvalbumin immunoreactivity in the rat.

    PubMed

    Nguyen, Jason C D; Killcross, A Simon; Jenkins, Trisha A

    2013-11-01

    Exercise has been demonstrated to have positive effects on both the body and brain. The present study aimed to determine the behavioural and morphological consequence of low-intensity running. Rats were exercised on a treadmill for a total of 30 days, 30 min/day. Social interaction, locomotor activity and behaviour on an elevated plus maze were assessed post-treatment. Exercised animals demonstrated more passive interaction and less time not interacting than control animals that were not exercised. Conversely, locomotor and anxiety measures showed no effect of exercise. Analysis of brains demonstrated an increase in expression of parvalbumin immunoreactive neurons in the hippocampus localised to the CA1 and CA2/3 regions. These results demonstrate that low-intensity exercise leads to changes in social behaviour as well as neuroplastic morphological changes within the hippocampus.

  15. Kinetic and thermodynamic parameters for thermal denaturation of ovine milk lactoferrin determined by its loss of immunoreactivity.

    PubMed

    Navarro, F; Harouna, S; Calvo, M; Pérez, M D; Sánchez, L

    2015-07-01

    Lactoferrin is a protein with important biological functions that can be obtained from milk and by-products derived from the dairy industry, such as whey. Although bovine lactoferrin has been extensively studied, ovine lactoferrin is not quite as well known. In the present study, the effect of several heat treatments in 3 different media, over a temperature range from 66 to 75°C, has been studied on lactoferrin isolated from sheep milk. Denaturation of lactoferrin was determined by measuring its immunoreactivity with specific polyclonal antibodies. Kinetic and thermodynamic parameters obtained indicate that lactoferrin denatures by heat more rapidly in whey than in phosphate buffer or milk. The value of activation energy found for the denaturation process of lactoferrin when treated in whey is higher (390kJ/mol) than that obtained in milk (194kJ/mol) or phosphate buffer (179kJ/mol). This indicates that a great amount of energy is necessary to start denaturation of ovine lactoferrin, probably due to the interaction of this protein with other whey proteins. The changes in the hydrophobicity of lactoferrin after heat treatments were determined by fluorescence measurement using acrylamide. The decrease in the hydrophobicity constant was very small for the treatments from 66 to 75°C, up to 20min, which indicates that lactoferrin conformation did not experienced a great change. The results obtained in this study permit the prediction of behavior of ovine lactoferrin under several heat treatments and show that high-temperature, short-time pasteurization (72°C, 15 s) does not cause loss of its immunoreactivity and, consequently, would not affect its conformation and biological activity. PMID:25958286

  16. Kinetic and thermodynamic parameters for thermal denaturation of ovine milk lactoferrin determined by its loss of immunoreactivity.

    PubMed

    Navarro, F; Harouna, S; Calvo, M; Pérez, M D; Sánchez, L

    2015-07-01

    Lactoferrin is a protein with important biological functions that can be obtained from milk and by-products derived from the dairy industry, such as whey. Although bovine lactoferrin has been extensively studied, ovine lactoferrin is not quite as well known. In the present study, the effect of several heat treatments in 3 different media, over a temperature range from 66 to 75°C, has been studied on lactoferrin isolated from sheep milk. Denaturation of lactoferrin was determined by measuring its immunoreactivity with specific polyclonal antibodies. Kinetic and thermodynamic parameters obtained indicate that lactoferrin denatures by heat more rapidly in whey than in phosphate buffer or milk. The value of activation energy found for the denaturation process of lactoferrin when treated in whey is higher (390kJ/mol) than that obtained in milk (194kJ/mol) or phosphate buffer (179kJ/mol). This indicates that a great amount of energy is necessary to start denaturation of ovine lactoferrin, probably due to the interaction of this protein with other whey proteins. The changes in the hydrophobicity of lactoferrin after heat treatments were determined by fluorescence measurement using acrylamide. The decrease in the hydrophobicity constant was very small for the treatments from 66 to 75°C, up to 20min, which indicates that lactoferrin conformation did not experienced a great change. The results obtained in this study permit the prediction of behavior of ovine lactoferrin under several heat treatments and show that high-temperature, short-time pasteurization (72°C, 15 s) does not cause loss of its immunoreactivity and, consequently, would not affect its conformation and biological activity.

  17. Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats.

    PubMed

    Poulsen, S S; Thulesen, J; Hartmann, B; Kissow, H L; Nexø, E; Thim, L

    2003-09-15

    Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.

  18. Fos-like immunoreactivity and thirst following hyperosmotic loading in rats with subdiaphragmatic vagotomy.

    PubMed

    Starbuck, Elizabeth M; Wilson, Wendy L; Fitts, Douglas A

    2002-03-29

    If receptors in the gut relay information about increases in local osmolality to the brain via the vagus nerve, then vagotomy should diminish this signaling and reduce both thirst and brain Fos-like immunoreactivity (Fos-ir). Water intake in response to hypertonic saline (i.p. or i.g., 1 M NaCl, 1% BW; i.g., 0.6 M NaCl, 0.5% BW) was reduced during 120 min in rats with subdiaphragmatic vagotomy (VGX) compared to sham-VGX rats. Brain Fos-ir was examined in response to both i.g. loads. After the smaller load, VGX greatly reduced Fos-ir in the supraoptic nucleus (SON) and the magnocellular and parvocellular areas of the paraventricular nucleus (PVN). Fos-ir in the subfornical organ (SFO) and nucleus of the solitary tract (NTS) was not affected. After the larger load, VGX significantly reduced Fos-ir in the parvocellular PVN and in the NTS, but not in the other regions. Thus, decreased water intake by VGX rats was accompanied by decreased Fos-ir in the parvocellular PVN after the same treatments, indicating a role for the abdominal vagus in thirst in response to signaling from gut osmoreceptors. The decreased water intake in the VGX group was not reflected as a decrease in Fos-ir in the SFO. Absorption of the larger i.g. load may have activated Fos-ir through more rapidly increasing systemic osmolality, thereby obscuring a role for the vagus at this dose in the SON and magnocellular PVN. PMID:11897101

  19. Presence of kisspeptin-like immunoreactivity in human adrenal glands and adrenal tumors.

    PubMed

    Takahashi, Kazuhiro; Shoji, Itaru; Shibasaki, Akiko; Kato, Ichiro; Hiraishi, Keisuke; Yamamoto, Hajime; Kaneko, Kiriko; Murakami, Osamu; Morimoto, Ryo; Satoh, Fumitoshi; Ito, Sadayoshi; Totsune, Kazuhito

    2010-05-01

    Kisspeptins are neuropeptides which activate the hypothalamo-pituitary gonadal axis and are considered to play important physiological roles in the reproduction. Kisspeptins have also been reported to stimulate the aldosterone secretion from the adrenal cortex. However, the expression of kisspeptins in human adrenal glands and adrenal tumors has not been clarified yet. We, therefore, studied the presence of kisspeptin-like immunoreactivity (LI) in human adrenal glands and adrenal tumors (adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas) by radioimmunoassay and immunocytochemistry. Kisspeptin-LI was detected in all the tissues examined; normal portions of adrenal glands (3.0 +/- 2.3 pmol/g wet weight, n = 21, mean +/- SD), aldosterone-producing adenomas (4.6 +/- 3.3 pmol/g wet weight, n = 10), cortisol-producing adenomas (2.7 +/- 1.4 pmol/g wet weight, n = 14), adrenocortical carcinomas (1.7 +/- 0.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.8 +/- 0.8 pmol/g wet weight, n = 6). There was no significant difference in kisspeptin-LI levels among them. Immunocytochemistry showed positive kisspeptin-immunostaining in normal adrenal glands, with stronger immunostaining found in the medulla. Furthermore, positive kisspeptin-immunostaining was found in all types of adrenal tumors examined; adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas. The intensity of kisspeptin-immunostaining in these adrenal tumors was, however, not so strong as that in normal adrenal medulla. The present study has shown for the first time the presence of kisspeptin-LI in adrenal glands and adrenal tumors.

  20. Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

    PubMed Central

    Moss, C.

    1998-01-01

    Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested

  1. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease.

    PubMed

    Gray, Daniel C; Mahrus, Sami; Wells, James A

    2010-08-20

    Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.

  2. Influence of resting tension on immunoreactive atrial natriuretic peptide secretion by rat atria superfused in vitro

    SciTech Connect

    Schiebinger, R.J.; Linden, J.

    1986-07-01

    Atrial natriuretic peptide is a potent diuretic hormone secreted by the atria in response to volume expansion. We examined the effect of resting tension on atrial natriuretic peptide secretion by rat atria superfused in vitro. Left atria were hooked between an electrode and force transducer and superfused with medium 199. The atria were studied at a pacing frequency of 0 or 3 Hz. Atrial natriuretic peptide content of the superfusate was measured by radioimmunoassay. In nonpaced and paced atria, increasing resting tension three- to five-fold caused immunoreactive atrial natriuretic peptide secretion to increase by 35 +/- 5% (mean +/- SEM, n = 6, p less than 0.01) and 30 +/- 3% (n = 4, p less than 0.01), respectively. Lowering resting tension by 50% in nonpaced and paced atria lowered immunoreactive atrial natriuretic peptide secretion by 30 +/- 3% (n = 7, p less than 0.01) and 24 +/- 3% (n = 6, p less than 0.01), respectively. To exclude the possibility that release of norepinephrine or acetylcholine from endogenous nerve endings was mediating this effect, the atria were superfused with the combination of propranolol 0.1 microM, phentolamine 1.0 microM, and atropine 10 microM. These concentrations of the antagonists were 125-fold or higher than their Kd for binding to their respective receptors. The antagonists did not block the rise in immunoreactive atrial natriuretic peptide secretion; neither did they inhibit an established rise in immunoreactive atrial natriuretic peptide secretion induced by increasing the resting tension.

  3. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    PubMed

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

  4. Reduction of IgE immunoreactivity of whole peanut (Arachis hypogaea L.) after pulsed light illumination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pulsed light (PL), a novel food processing and preservation technology, has been shown in literature to reduce allergen levels on peanut, soybean, almond, and shrimp protein extracts. This study investigated how PL affected the immunoreactivity of whole peanut kernels at two sample-to-lamp distance...

  5. Distribution of presumptive chemosensory afferents with FMRFamide- or substance P-like immunoreactivity in decapod crustaceans.

    PubMed

    Schmidt, M

    1997-01-23

    In five species of decapod crustaceans--Cherax destructor (crayfish), Carcinus maenas (crab), Homarus americanus (clawed lobster), Eriocheir sinensis (crab), Macrobrachium rosenbergii (shrimp)--immunocytochemical stainings revealed the presence of sensory afferents with FMRFamide-like immunoreactivity in the central nervous system. These afferents were extremely thin, very numerous, and innervated all sensory neuropils except the optic and olfact