Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

Energy Technology Data Exchange (ETDEWEB)

Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

2014-12-15

Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

Aggregated nanoplatelets: optical properties and optically induced deaggregation

International Nuclear Information System (INIS)

Jayabalan, J; Singh, Asha; Chari, Rama; Srivastava, Himanshu; Srivastava, A K; Mukhopadhyay, P K; Oak, S M

2008-01-01

A study of aggregation and laser-induced deaggregation of silver nanospheres and nanoplatelets in colloidal form is presented. Changes in the extinction spectrum caused by aggregation are explained using a two-particle approximation. In the case of platelets, controlled laser irradiation is shown to reverse the aggregation process.

Aggregated nanoplatelets: optical properties and optically induced deaggregation

Energy Technology Data Exchange (ETDEWEB)

Jayabalan, J; Singh, Asha; Chari, Rama [Laser Physics Application Division, Raja Ramanna Centre for Advanced Technology, Indore 452013 (India); Srivastava, Himanshu; Srivastava, A K [Indus Synchrotrons Utilization Division, Raja Ramanna Centre for Advanced Technology, Indore 452013 (India); Mukhopadhyay, P K; Oak, S M [Solid State Laser Division, Raja Ramanna Centre for Advanced Technology, Indore 452013 (India)], E-mail: jjaya@cat.ernet.in

2008-11-05

A study of aggregation and laser-induced deaggregation of silver nanospheres and nanoplatelets in colloidal form is presented. Changes in the extinction spectrum caused by aggregation are explained using a two-particle approximation. In the case of platelets, controlled laser irradiation is shown to reverse the aggregation process.

UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53Ser-15 phosphorylation

International Nuclear Information System (INIS)

Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader; Shahin, Allen; Patel, Mahendra; Galadari, Sehamuddin

2009-01-01

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser 15 . Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not prevent Dubca cell apoptosis, suggesting that p53 Ser-15 phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.

Inhibition of peptide aggregation by means of enzymatic phosphorylation

Directory of Open Access Journals (Sweden)

Kristin Folmert

2016-11-01

Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

Aggregate complexes of HIV-1 induced by multimeric antibodies.

Science.gov (United States)

Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

2014-10-02

Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

Science.gov (United States)

Zhang, Zhao; Zhang, Yun; Mou, Zheng; Chu, Shifeng; Chen, Xiaoyu; He, Wenbin; Guo, Xiaofeng; Yuan, Yuhe; Takahashi, Masami; Chen, Naihong

2014-01-01

Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402. PMID:24718602

Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells

DEFF Research Database (Denmark)

Birkelund, Svend; Johnsen, H; Christiansen, Gunna

1994-01-01

. By use of a monoclonal antibody against phosphotyrosine, we showed that three classes of proteins are tyrosine phosphorylated: a triple band of 68, 66, and 64 kDa, a 97-kDa band, and a 140-kDa band. The phosphorylation could be detected by immunoblotting from 15 min after infection of HeLa cells. We...... inactive. Attachment of EBs to host cells is medicated by a heparan sulfate-like glycosaminoglycan. Following attachment, the EB is internalized within a membrane-bound vesicle, and during the first 8 h of infection the vesicles are transported to a perinuclear location where they aggregate and fuse...

Fisetin stimulates autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors

OpenAIRE

Sunhyo Kim; Ki Ju Choi; Sun-Jung Cho; Sang-Moon Yun; Jae-Pil Jeon; Young Ho Koh; Jihyun Song; Gail V. W. Johnson; Chulman Jo

2016-01-01

The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer?s disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3?-induced tau aggregation model. However, there was no difference in activities of tau kinase...

Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

Science.gov (United States)

Korzeniewski, Bernard; Zoladz, Jerzy A

2003-08-15

Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial proteins and by an intensification of the parallel activation of ATP usage and ATP supply (increase in direct stimulation of oxidative phosphorylation complexes accompanying stimulation of ATP consumption) during exercise.

PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

Directory of Open Access Journals (Sweden)

Mao-lin HUANG

2014-09-01

Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei, E-mail: fchen@wayne.edu

2014-05-01

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

Aggregation in charged nanoparticles solutions induced by different interactions

Energy Technology Data Exchange (ETDEWEB)

Abbas, S.; Kumar, Sugam; Aswal, V. K., E-mail: vkaswal@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kohlbrecher, J. [Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen (Switzerland)

2016-05-23

Small-angle neutron scattering (SANS) has been used to study the aggregation of anionic silica nanoparticles as induced through different interactions. The nanoparticle aggregation is induced by addition of salt (NaCl), cationic protein (lysozyme) and non-ionic surfactant (C12E10) employing different kind of interactions. The results show that the interaction in presence of salt can be explained using DLVO theory whereas non-DLVO forces play important role for interaction of nanoparticles with protein and surfactant. The presence of salt screens the repulsion between charged nanoparticles giving rise to a net attraction in the DLVO potential. On the other hand, strong electrostatic attraction between nanoparticle and oppositely charged protein leads to protein-mediated nanoparticle aggregation. In case of non-ionic surfactant, the relatively long-range attractive depletion interaction is found to be responsible for the particle aggregation. Interestingly, the completely different interactions lead to similar kind of aggregate morphology. The nanoparticle aggregates formed are found to have mass fractal nature having a fractal dimension (~2.5) consistent with diffusion limited type of fractal morphology in all three cases.

GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

DEFF Research Database (Denmark)

Adelfinger, L; Turecek, R; Ivankova, K

2014-01-01

released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

Science.gov (United States)

Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

2017-12-01

Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

HSP27 phosphorylation modulates TRAIL-induced activation of Src-Akt/ERK signaling through interaction with β-arrestin2.

Science.gov (United States)

Qi, Shimei; Xin, Yinqiang; Qi, Zhilin; Xu, Yimiao; Diao, Ying; Lan, Lei; Luo, Lan; Yin, Zhimin

2014-03-01

Heat shock protein 27 (HSP27) regulates critical cellular functions such as development, differentiation, cell growth and apoptosis. A variety of stimuli induce the phosphorylation of HSP27, which affects its cellular functions. However, most previous studies focused on the role of HSP27 protein itself in apoptosis, the particular role of its phosphorylation state in signaling transduction remains largely unclear. In the present study, we reported that HSP27 phosphorylation modulated TRAIL-triggered pro-survival signaling transduction. In HeLa cells, suppression of HSP27 phosphorylation by specific inhibitor KRIBB3 or MAPKAPK2 (MK2) knockdown and by overexpression of non-phosphorylatable HSP27(3A) mutant demonstrated that hindered HSP27 phosphorylation enhanced the TRAIL-induced apoptosis. In addition, reduced HSP27 phosphorylation by KRIBB3 treatment or MK2 knockdown attenuated the TRAIL-induced activation of Akt and ERK survival signaling through suppressing the phosphorylation of Src. By overexpression of HSP27(15A) or HSP27(78/82A) phosphorylation mutant, we further showed that phosphorylation of HSP27 at serine 78/82 residues was essential to TRAIL-triggered Src-Akt/ERK signaling transduction. Co-immunoprecipitation and confocal microscopy showed that HSP27 interacted with Src and scaffolding protein β-arrestin2 in response of TRAIL stimulation and suppression of HSP27 phosphorylation apparently disrupted the TRAIL-induced interaction of HSP27 and Src or interaction of HSP27 and β-arrestin2. We further demonstrated that β-arrestin2 mediated HSP27 action on TRAIL-induced Src activation, which was achieved by recruiting signaling complex of HSP27/β-arrestin2/Src in response to TRAIL. Taken together, our study revealed that HSP27 phosphorylation modulates TRAIL-triggered activation of Src-Akt/ERK pro-survival signaling via interacting with β-arrestin2 in HeLa cells. Copyright © 2013 Elsevier Inc. All rights reserved.

SCGB3A2 Inhibits Acrolein-Induced Apoptosis through Decreased p53 Phosphorylation.

Science.gov (United States)

Kurotani, Reiko; Shima, Reika; Miyano, Yuki; Sakahara, Satoshi; Matsumoto, Yoshie; Shibata, Yoko; Abe, Hiroyuki; Kimura, Shioko

2015-04-28

Chronic obstructive pulmonary disease (COPD), a major global health problem with increasing morbidity and mortality rates, is anticipated to become the third leading cause of death worldwide by 2020. COPD arises from exposure to cigarette smoke. Acrolein, which is contained in cigarette smoke, is the most important risk factor for COPD. It causes lung injury through altering apoptosis and causes inflammation by augmenting p53 phosphorylation and producing reactive oxygen species (ROS). Secretoglobin (SCGB) 3A2, a secretory protein predominantly present in the epithelial cells of the lungs and trachea, is a cytokine-like small molecule having anti-inflammatory, antifibrotic, and growth factor activities. In this study, the effect of SCGB3A2 on acrolein-related apoptosis was investigated using the mouse fibroblast cell line MLg as the first step in determining the possible therapeutic value of SCGB3A2 in COPD. Acrolein increased the production of ROS and phosphorylation of p53 and induced apoptosis in MLg cells. While the extent of ROS production induced by acrolein was not affected by SCGB3A2, p53 phosphorylation was significantly decreased by SCGB3A2. These results demonstrate that SCGB3A2 inhibited acrolein-induced apoptosis through decreased p53 phosphorylation, not altered ROS levels.

SCGB3A2 Inhibits Acrolein-Induced Apoptosis through Decreased p53 Phosphorylation

International Nuclear Information System (INIS)

Kurotani, Reiko; Shima, Reika; Miyano, Yuki; Sakahara, Satoshi; Matsumoto, Yoshie; Shibata, Yoko; Abe, Hiroyuki; Kimura, Shioko

2015-01-01

Chronic obstructive pulmonary disease (COPD), a major global health problem with increasing morbidity and mortality rates, is anticipated to become the third leading cause of death worldwide by 2020. COPD arises from exposure to cigarette smoke. Acrolein, which is contained in cigarette smoke, is the most important risk factor for COPD. It causes lung injury through altering apoptosis and causes inflammation by augmenting p53 phosphorylation and producing reactive oxygen species (ROS). Secretoglobin (SCGB) 3A2, a secretory protein predominantly present in the epithelial cells of the lungs and trachea, is a cytokine-like small molecule having anti-inflammatory, antifibrotic, and growth factor activities. In this study, the effect of SCGB3A2 on acrolein-related apoptosis was investigated using the mouse fibroblast cell line MLg as the first step in determining the possible therapeutic value of SCGB3A2 in COPD. Acrolein increased the production of ROS and phosphorylation of p53 and induced apoptosis in MLg cells. While the extent of ROS production induced by acrolein was not affected by SCGB3A2, p53 phosphorylation was significantly decreased by SCGB3A2. These results demonstrate that SCGB3A2 inhibited acrolein-induced apoptosis through decreased p53 phosphorylation, not altered ROS levels

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence.

Science.gov (United States)

Byun, H-O; Jung, H-J; Kim, M-J; Yoon, G

2014-09-01

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.

Juvenile Hormone Prevents 20-Hydroxyecdysone-induced Metamorphosis by Regulating the Phosphorylation of a Newly Identified Broad Protein*

Science.gov (United States)

Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-01-01

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

Phosphorylation and disassembly of intermediate filaments in mitotic cells

International Nuclear Information System (INIS)

Chou, Yinghao; Rosevear, E.; Goldman, R.D.

1989-01-01

As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of P i per mol of protein in interphase to 1.9 mol of P i per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of P i per mol of protein to 1.5 mol of P i per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional 32 P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

Science.gov (United States)

Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

2014-11-01

The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

International Nuclear Information System (INIS)

Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto

2006-01-01

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to ∼200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

Science.gov (United States)

Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-09-19

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes

DEFF Research Database (Denmark)

Nielsen, M; Svejgaard, A; Skov, S

1994-01-01

that stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL-2 ligation, whereas...... an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL-2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that IL-2...... induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84-kDa protein in antigen-specific human T cell lines....

Cofilin phosphorylation is elevated after F-actin disassembly induced by Rac1 depletion.

Science.gov (United States)

Liu, Linna; Li, Jing; Zhang, Liwang; Zhang, Feng; Zhang, Rong; Chen, Xiang; Brakebusch, Cord; Wang, Zhipeng; Liu, Xinyou

2015-01-01

Cytoskeletal reorganization is essential to keratinocyte function. Rac1 regulates cytoskeletal reorganization through signaling pathways such as the cofilin cascade. Cofilin severs actin filaments after activation by dephosphorylation. Rac1 was knocked out in mouse keratinocytes and it was found that actin filaments disassembled. In the epidermis of mice in which Rac1 was knocked out only in keratinocytes, cofilin phosphorylation was aberrantly elevated, corresponding to repression of the phosphatase slingshot1 (SSH1). These effects were independent of the signaling pathways for p21-activated kinase/LIM kinase (Pak/LIMK), protein kinase C, or protein kinase D or generation of reactive oxygen species. Similarly, when actin polymerization was specifically inhibited or Rac1 was knocked down, cofilin phosphorylation was enhanced and SSH1 was repressed. Repression of SSH1 partially blocked actin depolymerization induced by Rac1 depletion. Therefore, aberrant cofilin phosphorylation that induces actin polymerization might be a consequence of actin disassembly induced by the absence of Rac1. © 2015 International Union of Biochemistry and Molecular Biology.

Ultrasensitive detection of target analyte-induced aggregation of gold nanoparticles using laser-induced nanoparticle Rayleigh scattering.

Science.gov (United States)

Lin, Jia-Hui; Tseng, Wei-Lung

2015-01-01

Detection of salt- and analyte-induced aggregation of gold nanoparticles (AuNPs) mostly relies on costly and bulky analytical instruments. To response this drawback, a portable, miniaturized, sensitive, and cost-effective detection technique is urgently required for rapid field detection and monitoring of target analyte via the use of AuNP-based sensor. This study combined a miniaturized spectrometer with a 532-nm laser to develop a laser-induced Rayleigh scattering technique, allowing the sensitive and selective detection of Rayleigh scattering from the aggregated AuNPs. Three AuNP-based sensing systems, including salt-, thiol- and metal ion-induced aggregation of the AuNPs, were performed to examine the sensitivity of laser-induced Rayleigh scattering technique. Salt-, thiol-, and metal ion-promoted NP aggregation were exemplified by the use of aptamer-adsorbed, fluorosurfactant-stabilized, and gallic acid-capped AuNPs for probing K(+), S-adenosylhomocysteine hydrolase-induced hydrolysis of S-adenosylhomocysteine, and Pb(2+), in sequence. Compared to the reported methods for monitoring the aggregated AuNPs, the proposed system provided distinct advantages of sensitivity. Laser-induced Rayleigh scattering technique was improved to be convenient, cheap, and portable by replacing a diode laser and a miniaturized spectrometer with a laser pointer and a smart-phone. Using this smart-phone-based detection platform, we can determine whether or not the Pb(2+) concentration exceed the maximum allowable level of Pb(2+) in drinking water. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein kinase C {alpha} activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

DEFF Research Database (Denmark)

Thomassen, Martin; Rose, Adam John; Jensen, Thomas Elbenhardt

2011-01-01

Exercise induced phosphorylation of FXYD1 is a potential important regulator of Na(+), K(+) pump activity. It was investigated if skeletal muscle contractions induce phosphorylation of FXYD1 and if Protein Kinase C a (PKCa) activity is a prerequisite for this possible mechanism. In part 1, human...... muscle biopsies were obtained at rest, after 30 s of high intensity exercise (166±31% of VO(2max)) and after a subsequent 20 min of moderate intensity exercise (79±8% of VO(2max)). In general, FXYD1 phosphorylation was increased compared to rest both after 30 s (P...

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

Science.gov (United States)

Zhang, Jun-Xia; Qu, Xin-Liang; Chu, Peng; Xie, Du-Jiang; Zhu, Lin-Lin; Chao, Yue-Lin; Li, Li; Zhang, Jun-Jie; Chen, Shao-Liang

2018-05-01

Uncoupled endothelial nitric oxide synthase (eNOS) produces O 2 - instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O 2 - production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O 2 - burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O 2 - releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Histone phosphorylation during radiation-induced mitotic delay in synchronous plasmodia of Physarum polycephalum

International Nuclear Information System (INIS)

Brewer, E.N.; Oleinick, N.L.

1980-01-01

Using the nearly perfect synchrony of the mitotic stages in Physarum plasmodia, and making use of 32 P as a tracer, studies were made to define the time course of histone phosphorylation during the late G2 and prophase and the alterations in that time course accompanying radiation-induced mitotic delay. Histone H1 was phosphorylated throughout the last 2-3 hours of the mitotic cycle coincident with the early stages of chromosome condensation. H1 phosphorylation appeared to be reduced in irradiated plasmodia. It is postulated that a longer time period, i.e. the mitotic delay, may be required to obtain the same eventual level of H1-phosphate. In normal cultures, nucleosome core histones were phosphorylated late in G2 and prophase, the peak corresponding closely with the γ-transition point. In irradiated plasmodia, phosphorylation of the core histones had an extended time course similar to H1. (U.K.)

Inflammation Induces TDP-43 Mislocalization and Aggregation.

Directory of Open Access Journals (Sweden)

Ana Sofia Correia

Full Text Available TAR DNA-binding protein 43 (TDP-43 is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS and frontotemporal lobar degeneration (FTLD. Here we report that lipopolysaccharide (LPS-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43(A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

International Nuclear Information System (INIS)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

2010-01-01

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Energy Technology Data Exchange (ETDEWEB)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates); Galadari, Sehamuddin, E-mail: sehamuddin@uaeu.ac.ae [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)

2010-04-09

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α1A-adrenergic receptors.

Science.gov (United States)

Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Romero-Ávila, M Teresa; Alfonzo-Méndez, Marco A; Pupo, André S; García-Sáinz, J Adolfo

2017-12-01

In LNCaP cells that stably express α 1A -adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α 1A -Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α 1A -adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α 1A -AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined. Copyright © 2017 Elsevier B.V. All rights reserved.

Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

OpenAIRE

Korzeniewski, Bernard; Zoladz, Jerzy A

2003-01-01

Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial protein...

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

Directory of Open Access Journals (Sweden)

Kanae Ando

2016-03-01

Full Text Available Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer's disease (AD. β-amyloid (Aβ lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.

Patterns of gravity induced aggregate migration during casting of fluid concretes

DEFF Research Database (Denmark)

Spangenberg, Jon; Roussel, N.; Hattel, Jesper Henri

2012-01-01

In this paper, aggregate migration patterns during fluid concrete castings are studied through experiments, dimensionless approach and numerical modeling. The experimental results obtained on two beams show that gravity induced migration is primarily affecting the coarsest aggregates resulting in...

Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts

International Nuclear Information System (INIS)

Xinbo Cheng; Nge Cheong; Ya Wang; Iliakis, George

1996-01-01

Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or γ-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21 cip1/waf1/sdi1 remains inducible. The results demonstrate a growth-stage and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells

Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

Directory of Open Access Journals (Sweden)

Huzoor Akbar

Full Text Available Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation.We utilized the Mx-cre;Cdc42(lox/lox inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+ mice. Platelets isolated from Cdc42(-/-, as compared to Cdc42(+/+, mice exhibited (a diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b inhibition of filopodia formation on immobilized CRP or fibrinogen, (c inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/- mice compared with Cdc42(+/+ mice.Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

Patterns of gravity induced aggregate migration during casting of fluid concretes

Energy Technology Data Exchange (ETDEWEB)

Spangenberg, J. [Department of Mechanical Engineering, Technical University of Denmark (DTU) (Denmark); Roussel, N., E-mail: Nicolas.roussel@lcpc.fr [Universite Paris Est, Laboratoire Central des Ponts et Chaussees (LCPC) (France); Hattel, J.H. [Department of Mechanical Engineering, Technical University of Denmark (DTU) (Denmark); Sarmiento, E.V.; Zirgulis, G. [Department of Structural Engineering, Norwegian University of Science and Technology (NTNU) (Norway); Geiker, M.R. [Department of Structural Engineering, Norwegian University of Science and Technology (NTNU) (Norway); Department of Civil Engineering, Technical University of Denmark (DTU) (Denmark)

2012-12-15

In this paper, aggregate migration patterns during fluid concrete castings are studied through experiments, dimensionless approach and numerical modeling. The experimental results obtained on two beams show that gravity induced migration is primarily affecting the coarsest aggregates resulting in a decrease of coarse aggregates volume fraction with the horizontal distance from the pouring point and in a puzzling vertical multi-layer structure. The origin of this multi layer structure is discussed and analyzed with the help of numerical simulations of free surface flow. Our results suggest that it finds its origin in the non Newtonian nature of fresh concrete and that increasing casting rate shall decrease the magnitude of gravity induced particle migration.

Patterns of gravity induced aggregate migration during casting of fluid concretes

International Nuclear Information System (INIS)

Spangenberg, J.; Roussel, N.; Hattel, J.H.; Sarmiento, E.V.; Zirgulis, G.; Geiker, M.R.

2012-01-01

In this paper, aggregate migration patterns during fluid concrete castings are studied through experiments, dimensionless approach and numerical modeling. The experimental results obtained on two beams show that gravity induced migration is primarily affecting the coarsest aggregates resulting in a decrease of coarse aggregates volume fraction with the horizontal distance from the pouring point and in a puzzling vertical multi-layer structure. The origin of this multi layer structure is discussed and analyzed with the help of numerical simulations of free surface flow. Our results suggest that it finds its origin in the non Newtonian nature of fresh concrete and that increasing casting rate shall decrease the magnitude of gravity induced particle migration.

Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis

Science.gov (United States)

Worsley, M.A.; Allen, C.E.; Billinton, A.; King, A.E.; Boissonade, F.M.

2014-01-01

Background Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase are transiently phosphorylated (activated) in the spinal cord and trigeminal nucleus by acute noxious stimuli. Acute stimulation of dental pulp induces short-lived ERK activation in trigeminal subnucleus caudalis (Vc), and p38 inhibition attenuates short-term sensitization in Vc induced by acute pulpal stimulation. We have developed a model to study central changes following chronic inflammation of dental pulp that induces long-term sensitization. Here, we examine the effects of chronic inflammation and acute stimulation on the expression of phosphorylated ERK (pERK), phosphorylated p38 (pp38) and Fos in Vc. Results Chronic inflammation alone induced bilateral expression of pERK and pp38 in Vc, but did not induce Fos expression. Stimulation of both non-inflamed and inflamed pulps significantly increased pERK and pp38 bilaterally; expression was greatest in inflamed, stimulated animals, and was similar following 10-min and 60-min stimulation. Stimulation for 60 min, but not 10 min, induced Fos in ipsilateral Vc; Fos expression was significantly greater in inflamed, stimulated animals. pERK was present in both neurons and astrocytes; pp38 was present in neurons and other non-neuronal, non-astrocytic cell types. Conclusions This study provides the first demonstration that chronic inflammation of tooth pulp induces persistent bilateral activation of ERK and p38 within Vc, and that this activation is further increased by acute stimulation. This altered activity in intracellular signaling is likely to be linked to the sensitization that is seen in our animal model and in patients with pulpitis. Our data indicate that pERK and pp38 are more accurate markers of central change than Fos expression. In our model, localization of pERK and pp38 within specific cell types differs from that seen following acute stimulation. This may indicate specific roles for different cell types in

Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3ζ protein

International Nuclear Information System (INIS)

Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

2009-01-01

Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3ζ. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3ζ is ∼3-folds higher than that between unphosphorylated 4R-tau and 14-3-3ζ. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3ζ to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3ζ. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3ζ exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3ζ suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate

International Nuclear Information System (INIS)

Pek, S.B.; Usami, Masaru; Bilir, N.; Fischer-Bovenkerk, C.; Ueda, Tetsufumi

1990-01-01

The authors have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, they considered the possibility that 3-phosphoglycerate may act as a coupling factor, and they searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [γ- 32 P]ATP and appropriate test substances and were subjected to NaDodSO 4 /PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion

The Platelet Aggregation-Inducing Factor Aggrus/Podoplanin Promotes Pulmonary Metastasis

Science.gov (United States)

Kunita, Akiko; Kashima, Takeshi G.; Morishita, Yasuyuki; Fukayama, Masashi; Kato, Yukinari; Tsuruo, Takashi; Fujita, Naoya

2007-01-01

Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor growth, metastasis, and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus-expressing cells, which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus (Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis. PMID:17392172

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

Science.gov (United States)

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

International Nuclear Information System (INIS)

Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

1987-01-01

Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of [ 3 H]serotonin, or alter the dose-responsive binding of 125 I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF

Eicosapentaenoic acid abolishes inhibition of insulin-induced mTOR phosphorylation by LPS via PTP1B downregulation in skeletal muscle.

Science.gov (United States)

Wei, Hong-Kui; Deng, Zhao; Jiang, Shu-Zhong; Song, Tong-Xing; Zhou, Yuan-Fei; Peng, Jian; Tao, Ya-Xiong

2017-01-05

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) increase insulin signaling in skeletal muscle. In the current study, we investigated the effect of eicosapentaenoic acid (EPA) on insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes. We showed that EPA did not affect basal and insulin-induced mTOR phosphorylation in myotubes. However, EPA abolished lipopolysaccharide (LPS) -induced deficiency in insulin signaling (P  0.05). In myotubes, LPS stimulated PTP1B expression via NF-κB and activation protein-1 (AP1). Pre-incubation of 50 μM EPA prevented the LPS-induced activation of AP1 and NF-κΒ as well as PTP1B expression (P < 0.05). Interestingly, incubation of peroxisome proliferator-activated receptor γ (PPARγ) antagonist (GW9662) prior to EPA treatment, the effect of EPA on insulin-induced mTOR phosphorylation was blocked. Accordingly, EPA did not inhibit the LPS-induced activation of AP1 or NF-κΒ as well as PTP1B expression when incubation of GW9662 prior to EPA treatment. The in vivo study showed that EPA prevented LPS-induced PTPT1B expression and a decrease in insulin-induced mTOR phosphorylation in muscle of mice. In summary, EPA abolished LPS inhibition of insulin-induced mTOR phosphorylation in myotubes, and one of the key mechanisms was to inhibit AP1 and NF-κB activation and PTP1B transcription. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

International Nuclear Information System (INIS)

Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

2016-01-01

XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells

Lead induced changes in phosphorylation of PSII proteins in low light grown pea plants.

Science.gov (United States)

Wioleta, Wasilewska; Anna, Drożak; Ilona, Bacławska; Kamila, Kąkol; Elżbieta, Romanowska

2015-02-01

Light-intensity and redox-state induced thylakoid proteins phosphorylation involved in structural changes and in regulation of protein turnover. The presence of heavy metal ions triggers a wide range of cellular responses including changes in plant growth and photosynthesis. Plants have evolved a number of mechanisms to protect photosynthetic apparatus. We have characterized the effect of lead on PSII protein phosphorylation in pea (Pisum sativum L.) plants grown in low light conditions. Pb ions affected only slightly photochemical efficiency of PSII and had no effect on organization of thylakoid complexes. Lead activated strongly phosphorylation of PSII core D1 protein and dephosphorylation of this protein did not proceed in far red light. D1 protein was also not degraded in this conditions. However, phosphorylation of LHCII proteins was not affected by lead. These results indicate that Pb(2+) stimulate the phosphorylation of PSII core proteins and by disturbing the disassembly of supercomplexes play a role in PSII repair mechanism. LHCII phosphorylation could control the distribution of energy between the photosystems in low light conditions. This demonstrates that plants may respond to heavy metals by induction different pathways responsible for protein protection under stress conditions.

High-speed centrifugation induces aggregation of extracellular vesicles.

Science.gov (United States)

Linares, Romain; Tan, Sisareuth; Gounou, Céline; Arraud, Nicolas; Brisson, Alain R

2015-01-01

Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

High-speed centrifugation induces aggregation of extracellular vesicles

Directory of Open Access Journals (Sweden)

Romain Linares

2015-12-01

Full Text Available Plasma and other body fluids contain cell-derived extracellular vesicles (EVs, which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

OpenAIRE

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-01-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity...

Pressure effects on the structure, kinetic, and thermodynamic properties of heat-induced aggregation of protein studied by FT-IR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Taniguchi, Y [Applied Chemistry Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Okuno, A [Research Department 3, Central Research, Bridgestone Co. Kodaira, Tokyo 187-8531 (Japan); Kato, M, E-mail: taniguti@sk.ritsumei.ac.j [Pharmaceutical Sciences Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan)

2010-03-01

Pressure can retrain the heat-induced aggregation and dissociate the heat-induced aggregates. We observed the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by FT-IR spectroscopy. The results suggest the {alpha}-helical structure collapses at the beginning of heat-induced aggregation through the swollen structure, and then the rearrangement of structure to the intermolecular {beta}-sheet takes place through partially unfolded structure. We determined the activation volume for the heat-induced aggregation ({Delta}V'' = +93 ml/mol) and the partial molar volume difference between native state and heat-induced aggregates ({Delta}V=+32 ml/mol). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular {beta}-sheet is unfavorable under high pressure.

Disruption of δ-opioid receptor phosphorylation at threonine 161 attenuates morphine tolerance in rats with CFA-induced inflammatory hypersensitivity.

Science.gov (United States)

Chen, Hai-Jing; Xie, Wei-Yan; Hu, Fang; Zhang, Ying; Wang, Jun; Wang, Yun

2012-04-01

Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not µ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.

Degradation and aggregation of delta sleep-inducing peptide (DSIP) and two analogs in plasma and serum

International Nuclear Information System (INIS)

Graf, M.V.; Saegesser, B.; Schoenenberger, G.A.

1987-01-01

The biostability of DSIP (delta sleep-inducing peptide) and two analogs in blood was investigated in order to determine if rates of inactivation contribute to variable effects in vivo. Incubation of DSIP in human or rat blood led to release of products having retention times on a gel filtration column equivalent to Trp. Formation of products was dependent on temperature, time, and species. Incubation of 125 I-N-Tyr-DSIP and 125 I-N-Tyr-P-DSIP, a phosphorylated analog, revealed slower degradation and, in contrast to DSIP, produced complex formation. An excess of unlabeled material did not displace the radioactivity supporting the assumption of non-specific binding/aggregation. It was concluded that the rapid disappearance of injected DSIP in blood was due to degradation, whereas complex formation together with slower degradation resulted in longer persistence of apparently intact analogs. Whether this could explain the sometimes stronger and more consistent effects of DSIP-analogs remains to be examined

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

Science.gov (United States)

Liu, Jingwen; Cai, Weicong; Fang, Xian; Wang, Xueting; Li, Guiling

2018-04-01

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy.

Science.gov (United States)

Huang, Chih-Yang; Lee, Fa-Lun; Peng, Shu-Fen; Lin, Kuan-Ho; Chen, Ray-Jade; Ho, Tsung-Jung; Tsai, Fu-Jen; Padma, Vijaya V; Kuo, Wei-Wen; Huang, Chih-Yang

2018-02-01

Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Inhibition of extracellular signal-regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)-induced cardiomyocyte hypertrophy and apoptosis by blocking insulin-like growth factor II receptor (IGF-IIR) signaling. However, the detailed mechanism by which ANG II induces ERK-mediated IGF-IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF-IIR expression via the angiotensin II type I receptor (AT 1 R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3-induced IGF-IIR protein stability by downregulating the E3 ubiquitin ligase of IGF-IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II-induced HSF1 S303 phosphorylation, resulting in IGF-IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG II-induced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126 degradation, which stabilizes IGF-IIR protein expression and eventually results in cardiac hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among hypertensive patients. © 2017 Wiley Periodicals, Inc.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

International Nuclear Information System (INIS)

Choi, Cheol-Hee; Lee, Byung-Hoon; Ahn, Sang-Gun; Oh, Seon-Hee

2012-01-01

Highlights: ► MG132 induces the phosphorylation of GSK3β Ser9 and, to a lesser extent, of GSK3β Thr390 . ► MG132 induces dephosphorylation of p70S6K Thr389 and phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 dephosphorylates GSK3β Ser9 and phosphorylates GSK3β Thr390 . ► Inactivation of p38 phosphorylates p70S6K Thr389 and increases the phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser 9 and, to a lesser extent, Thr 390 , the dephosphorylation of p70S6K at Thr 389 , and the phosphorylation of p70S6K at Thr 421 and Ser 424 . The specific p38 inhibitor SB203080 reduced the p-GSK3β Ser9 and autophagy through the phosphorylation of p70S6K Thr389 ; however, it augmented the levels of p-ERK, p-GSK3β Thr390 , and p-70S6K Thr421/Ser424 induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK Ser9 /p70S6K Thr380 and ERK/GSK3β Thr390 /p70S6K Thr421/Ser424 kinase signaling, which is involved in cell survival and cell death.

Genistein suppresses adhesion-induced protein tyrosine phosphorylation and invasion of B16-BL6 melanoma cells.

Science.gov (United States)

Yan, C; Han, R

1998-07-03

Protein tyrosine phosphorylation occurs as one of the earlier events in cancer cell-extracellular matrix (ECM) interaction. With immunoblot analysis and immunofluorescence microscopy, genistein was found to suppress the tyrosine phosphorylation of proteins located at the cell periphery, including a 125 kDa protein, when B16-BL6 melanoma cells attached to and interacted with ECM. When accompanied by the suppression of adhesion-induced protein tyrosine phosphorylation, the invasive potential of B16-BL6 cells through reconstituted basement membrane was decreased significantly. However, neither adhesive capability nor cell growth was significantly affected by genistein. Therefore, the interruption of cancer cell-ECM interaction by suppression of protein tyrosine phosphorylation may contribute to invasion prevention of genistein.

Biological Significance of the Suppression of Oxidative Phosphorylation in Induced Pluripotent Stem Cells

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Cheng Zhang

2017-11-01

Full Text Available We discovered that induced pluripotent stem cell (iPSC clones generated from aged tissue donors (A-iPSCs fail to suppress oxidative phosphorylation. Compared to embryonic stem cells (ESCs and iPSCs generated from young donors (Y-iPSCs, A-iPSCs show poor expression of the pluripotent stem cell-specific glucose transporter 3 (GLUT3 and impaired glucose uptake, making them unable to support the high glucose demands of glycolysis. Persistent oxidative phosphorylation in A-iPSCs generates higher levels of reactive oxygen species (ROS, which leads to excessive elevation of glutathione (a ROS-scavenging metabolite and a blunted DNA damage response. These phenotypes were recapitulated in Y-iPSCs by inhibiting pyruvate dehydrogenase kinase (PDK or supplying citrate to activate oxidative phosphorylation. In addition, oxidative phosphorylation in A-iPSC clones depletes citrate, a nuclear source of acetyl group donors for histone acetylation; this consequently alters histone acetylation status. Expression of GLUT3 in A-iPSCs recovers the metabolic defect, DNA damage response, and histone acetylation status.

Al cation induces aggregation of serum proteins.

Science.gov (United States)

Chanphai, P; Kreplak, L; Tajmir-Riahi, H A

2017-07-15

Al cation is known to induce protein fibrillation and causes several neurodegenerative disorders. We report the spectroscopic, thermodynamic analysis and AFM imaging for the Al cation binding process with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in Al-protein interactions with more hydrophobic b-LG forming stronger Al-protein complexes. Thermodynamic parameters ΔS, ΔH and ΔG showed Al-protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der Waals and H-bonding interactions prevail in HSA and BSA adducts. AFM clearly indicated that aluminum cations are able to force BSA and b-LG into larger or more robust aggregates than HSA, with HSA 4±0.2 (SE, n=801) proteins per aggregate, for BSA 17±2 (SE, n=148), and for b-LG 12±3 (SE, n=151). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced major alterations of protein conformations with the order of perturbations b-LG>BSA>HSA. Copyright © 2017 Elsevier B.V. All rights reserved.

Towards meso -Ester BODIPYs with Aggregation-Induced Emission Properties: The Effect of Substitution Positions

KAUST Repository

Chua, Ming Hui

2015-06-17

Three meso-ester boron dipyrromethene (BODIPY) dyes have been synthesized and functionalized with aggregation-induced emission (AIE)-active tetraphenylethene or triphenylethene moieties. It was found that functionalizing at the different positions of the BODIPY core resulted in the final dye having different emission properties in response to aggregation: from aggregation-induced quenching (ACQ) to being AIE active. X-ray crystallographic analysis was thus performed to provide an explanation for these differences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards meso -Ester BODIPYs with Aggregation-Induced Emission Properties: The Effect of Substitution Positions

KAUST Repository

Chua, Ming Hui; Ni, Yong; Garai, Monalisa; Zheng, Bin; Huang, Kuo-Wei; Xu, Qing Hua; Xu, Jianwei; Wu, Jishan

2015-01-01

Three meso-ester boron dipyrromethene (BODIPY) dyes have been synthesized and functionalized with aggregation-induced emission (AIE)-active tetraphenylethene or triphenylethene moieties. It was found that functionalizing at the different positions of the BODIPY core resulted in the final dye having different emission properties in response to aggregation: from aggregation-induced quenching (ACQ) to being AIE active. X-ray crystallographic analysis was thus performed to provide an explanation for these differences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

Energy Technology Data Exchange (ETDEWEB)

Pan, Xiao [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Whitten, Douglas A. [Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Wu, Ming [Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Chan, Christina [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Wilkerson, Curtis G. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Pestka, James J., E-mail: pestka@msu.edu [Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824 (United States)

2013-04-15

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

International Nuclear Information System (INIS)

Pan, Xiao; Whitten, Douglas A.; Wu, Ming; Chan, Christina; Wilkerson, Curtis G.; Pestka, James J.

2013-01-01

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

International Nuclear Information System (INIS)

Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

1989-01-01

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation

Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

International Nuclear Information System (INIS)

Tian Mei; Pan Yan; Liu Jianxiang; Ruan Jianlei; Su Xu

2011-01-01

Objective: To investigate 60 Co γ-ray induced damage in lymphocytes and the relationship between doses of 60 Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM. Methods: Cells were irradiated with 60 Co γ-rays in the range of 0-8 Gy. The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively. The micronucleus(MN)was analyzed by CB method to evaluate DNA damage. Results: FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses, and the fitting curve was shown as Y=3.96+11.29D-0.45D 2 . The level of phosphorylated ATM (p-ATM) was not changed significantly by using the same method. Western blot showed that p-ATM protein expression was significantly increased after irradiation compared with sham, irradiated group. The MN assay which represented DNA damage was sensitive to different doses. Conclusions: γ-ray irradiation could induce the phosphorylation of H2AX and ATM, which may play an important role in indicating DNA damage. Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage. (authors)

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

Directory of Open Access Journals (Sweden)

Fang Rao

Full Text Available Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ. We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg or increased (120, 180, 240 mmHg hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg. The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs. These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

Science.gov (United States)

Rao, Fang; Yang, Ren-Qiang; Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

Science.gov (United States)

Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

2016-07-01

Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

International Nuclear Information System (INIS)

Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

1986-01-01

Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [ 32 P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

Decrease of GSK3β phosphorylation in the rat nucleus accumbens core enhances cocaine-induced hyper-locomotor activity.

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Kim, Wha Y; Jang, Ju K; Lee, Jung W; Jang, Hyunduk; Kim, Jeong-Hoon

2013-06-01

Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant-induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants-induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine-induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N-terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine-induced hyper-locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine-induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co-activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors. © 2013 International Society for Neurochemistry.

Glycogen phosphorylation and Lafora disease.

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Roach, Peter J

2015-12-01

Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

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Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

2009-06-09

CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

Paradoxical Acceleration of Dithiothreitol-Induced Aggregation of Insulin in the Presence of a Chaperone

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Boris Kurganov

2010-11-01

Full Text Available The kinetics of dithiothreitol (DTT-induced aggregation of human recombinant insulin and the effect of α-crystallin, a representative of the family of small heat shock proteins, on the aggregation process have been studied using dynamic light scattering technique. Analysis of the distribution of the particles by size measured in the course of aggregation showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with a hydrodynamic radius (Rh of about 90 nm. When studying the effect of α-crystallin on the rate of DTT-induced aggregation of insulin, it was demonstrated that low concentrations of α-crystallin dramatically accelerated the aggregation process, whereas high concentrations of α-crystallin suppressed insulin aggregation. In the present study, at the molar stoichiometric ratio (insulin:α-crystallin less than 1:0.5, a pronounced accelerating effect of α-crystallin was observed; whereas a ratio exceeding the value of 1:0.6 caused suppression of insulin aggregation. The mechanisms underlying the dual effect of α-crystallin have been proposed. It is assumed that heterogeneous nucleation occurring on the surface of the α-crystallin particle plays the key role in the paradoxical acceleration of insulin aggregation by α-crystallin that may provide an alternative biologically significant pathway of the aggregation process.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

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Choi, Cheol-Hee [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Department of Pharmacology, College of Medicine, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Multiscreening Center for Drug Development, Seoul National University, Seoul 151-742 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: oshccw@hanmail.net [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of)

2012-02-24

Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

17-AAG induces cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy.

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Riedel, Michael; Goldbaum, Olaf; Schwarz, Lisa; Schmitt, Sebastian; Richter-Landsberg, Christiane

2010-01-18

The accumulation and aggregation of alpha-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. alpha-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved. In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human alpha-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of alpha-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of alpha-synuclein aggregates in OLN-A53T cells. Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation

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Zhou, Yan [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); College of Food Safety, Guizhou Medical University, Guiyang 550025 (China); Ruan, Zheng, E-mail: ruanzheng@ncu.edu.cn [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Zhou, Lili; Shu, Xugang [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Sun, Xiaohong [College of Food Safety, Guizhou Medical University, Guiyang 550025 (China); Mi, Shumei; Yang, Yuhui [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Yin, Yulong, E-mail: yinyulong@isa.ac.cn [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125 (China)

2016-01-22

Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. - Highlights: • Dietary supplementation with chlorogenic acid (CGA) improved endotoxin-induced liver injury. • Chlorogenic acid enhances ATP increase and shifts energy metabolism, which is correlated with up-regulation AMPK and PGC-1α. • The possible mechanism of CGA on mitochondrial biogenesis was correlated with up-regulation AMPK and PGC-1α.

Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation

International Nuclear Information System (INIS)

Zhou, Yan; Ruan, Zheng; Zhou, Lili; Shu, Xugang; Sun, Xiaohong; Mi, Shumei; Yang, Yuhui; Yin, Yulong

2016-01-01

Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. - Highlights: • Dietary supplementation with chlorogenic acid (CGA) improved endotoxin-induced liver injury. • Chlorogenic acid enhances ATP increase and shifts energy metabolism, which is correlated with up-regulation AMPK and PGC-1α. • The possible mechanism of CGA on mitochondrial biogenesis was correlated with up-regulation AMPK and PGC-1α.

The Effects of Insulin-Induced Hypoglycaemia on Tyrosine Hydroxylase Phosphorylation in Rat Brain and Adrenal Gland.

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Senthilkumaran, Manjula; Johnson, Michaela E; Bobrovskaya, Larisa

2016-07-01

In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.

Tau Phosphorylation by GSK3 in Different Conditions

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Jesús Avila

2012-01-01

Full Text Available Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau.

Aggregation-induced emission: phenomenon, mechanism and applications.

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Hong, Yuning; Lam, Jacky W Y; Tang, Ben Zhong

2009-08-07

It is textbook knowledge that chromophore aggregation generally quenches light emission. In this feature article, we give an account on how we observed an opposite phenomenon termed aggregation-induced emission (AIE) and identified the restriction of intramolecular rotation as a main cause for the AIE effect. Based on the mechanistic understanding, we developed a series of new fluorescent and phosphorescent AIE systems with emission colours covering the entire visible spectral region and luminescence quantum yields up to unity. We explored high-tech applications of the AIE luminogens as, for example, fluorescence sensors (for explosive, ion, pH, temperature, viscosity, pressure, etc.), biological probes (for protein, DNA, RNA, sugar, phospholipid, etc.), immunoassay markers, PAGE visualization agents, polarized light emitters, monitors for layer-by-layer assembly, reporters for micelle formation, multistimuli-responsive nanomaterials, and active layers in the fabrication of organic light-emitting diodes.

Novel direct factor Xa inhibitory compounds from Tenebrio molitor with anti-platelet aggregation activity.

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Lee, Wonhwa; Kim, Mi-Ae; Park, InWha; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup

2017-11-01

Tenebrio molitor is an edible insect that has antimicrobial, anticancer, and antihypertensive effects. The aim of this study was to identify the unreported bioactive compounds from T. molitor larvae with inhibitory activities against factor Xa (FXa) and platelet aggregation. Isolated compounds were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. A diketopiperazine (1, cyclo( L -Pro- L -Tyr)) and a phenylethanoid (2, N-acetyltyramine) were isolated and inhibited the catalytic activity of FXa in a mixed inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619. They inhibited ADP- and U46619-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) and the expression of P-selectin and PAC-1 in platelets. They also improved the production of nitric oxide and inhibited the oversecretion of endothelin-1 compared to that of the ADP- or U46619-treated group. In an animal model of arterial and pulmonary thrombosis, the isolated compounds showed enhanced antithrombotic effects. They also elicited anticoagulant effects in mice. Compounds 1-2 inhibited ADP-, collagen-, or U46619-induced platelet aggregation and showed similar anti-thrombotic efficacy to rivaroxaban, a positive control. Therefore, 1-2 could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphorylated α-Synuclein-Copper Complex Formation in the Pathogenesis of Parkinson’s Disease

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Juan Antonio Castillo-Gonzalez

2017-01-01

Full Text Available Parkinson’s disease is the second most important neurodegenerative disorder worldwide. It is characterized by the presence of Lewy bodies, which are mainly composed of α-synuclein and ubiquitin-bound proteins. Both the ubiquitin proteasome system (UPS and autophagy-lysosomal pathway (ALS are altered in Parkinson’s disease, leading to aggregation of proteins, particularly α-synuclein. Interestingly, it has been observed that copper promotes the protein aggregation process. Additionally, phosphorylation of α-synuclein along with copper also affects the protein aggregation process. The interrelation among α-synuclein phosphorylation and its capability to interact with copper, with the subsequent disruption of the protein degradation systems in the neurodegenerative process of Parkinson’s disease, will be analyzed in detail in this review.

Aspirin-Mediated Acetylation Protects Against Multiple Neurodegenerative Pathologies by Impeding Protein Aggregation.

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Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Kakraba, Samuel; Alla, Ramani; Mehta, Jawahar L; Shmookler Reis, Robert J

2017-12-10

Many progressive neurological disorders, including Alzheimer's disease (AD), Huntington's disease, and Parkinson's disease (PD), are characterized by accumulation of insoluble protein aggregates. In prospective trials, the cyclooxygenase inhibitor aspirin (acetylsalicylic acid) reduced the risk of AD and PD, as well as cardiovascular events and many late-onset cancers. Considering the role played by protein hyperphosphorylation in aggregation and neurodegenerative diseases, and aspirin's known ability to donate acetyl groups, we asked whether aspirin might reduce both phosphorylation and aggregation by acetylating protein targets. Aspirin was substantially more effective than salicylate in reducing or delaying aggregation in human neuroblastoma cells grown in vitro, and in Caenorhabditis elegans models of human neurodegenerative diseases in vivo. Aspirin acetylates many proteins, while reducing phosphorylation, suggesting that acetylation may oppose phosphorylation. Surprisingly, acetylated proteins were largely excluded from compact aggregates. Molecular-dynamic simulations indicate that acetylation of amyloid peptide energetically disfavors its association into dimers and octamers, and oligomers that do form are less compact and stable than those comprising unacetylated peptides. Hyperphosphorylation predisposes certain proteins to aggregate (e.g., tau, α-synuclein, and transactive response DNA-binding protein 43 [TDP-43]), and it is a critical pathogenic marker in both cardiovascular and neurodegenerative diseases. We present novel evidence that acetylated proteins are underrepresented in protein aggregates, and that aggregation varies inversely with acetylation propensity after diverse genetic and pharmacologic interventions. These results are consistent with the hypothesis that aspirin inhibits protein aggregation and the ensuing toxicity of aggregates through its acetyl-donating activity. This mechanism may contribute to the neuro-protective, cardio

C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease.

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Kleinknecht, Alexandra; Popova, Blagovesta; Lázaro, Diana F; Pinho, Raquel; Valerius, Oliver; Outeiro, Tiago F; Braus, Gerhard H

2016-06-01

Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by �

GPNMB ameliorates mutant TDP-43-induced motor neuron cell death.

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Nagahara, Yuki; Shimazawa, Masamitsu; Ohuchi, Kazuki; Ito, Junko; Takahashi, Hitoshi; Tsuruma, Kazuhiro; Kakita, Akiyoshi; Hara, Hideaki

2017-08-01

Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP-43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP-43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)-positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba1)-positive microglia. GPNMB aggregates were localized in the microtubule-associated protein 2 (MAP-2)-positive neuron and neurofilament H non-phosphorylated (SMI-32)-positive neuron, and these were co-localized with TDP-43 aggregates in the spinal cord of ALS patients. Mock or TDP-43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP-43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP-43 plasmids and serum-free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP-43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP-43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Sorbitol crystallization-induced aggregation in frozen mAb formulations.

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Piedmonte, Deirdre Murphy; Hair, Alison; Baker, Priti; Brych, Lejla; Nagapudi, Karthik; Lin, Hong; Cao, Wenjin; Hershenson, Susan; Ratnaswamy, Gayathri

2015-02-01

Sorbitol crystallization-induced aggregation of mAbs in the frozen state was evaluated. The effect of protein aggregation resulting from sorbitol crystallization was measured as a function of formulation variables such as protein concentration and pH. Long-term studies were performed on both IgG1 and IgG2 mAbs over the protein concentration range of 0.1-120 mg/mL. Protein aggregation was measured by size-exclusion HPLC (SE-HPLC) and further characterized by capillary-electrophoresis SDS. Sorbitol crystallization was monitored and characterized by subambient differential scanning calorimetry and X-ray diffraction. Aggregation due to sorbitol crystallization is inversely proportional to both protein concentration and formulation pH. At high protein concentrations, sorbitol crystallization was suppressed, and minimal aggregation by SE-HPLC resulted, presumably because of self-stabilization of the mAbs. The glass transition temperature (Tg ') and fragility index measurements were made to assess the influence of molecular mobility on the crystallization of sorbitol. Tg ' increased with increasing protein concentration for both mAbs. The fragility index decreased with increasing protein concentration, suggesting that it is increasingly difficult for sorbitol to crystallize at high protein concentrations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

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Mills, E N; Huang, L; Noel, T R; Gunning, A P; Morris, V J

2001-06-11

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

Halogenated salicylaldehyde azines: The heavy atom effect on aggregation-induced emission enhancement properties

International Nuclear Information System (INIS)

Chen, Xiao-tong; Tong, Ai-jun

2014-01-01

This study investigates the heavy-atom effect (HAE) on aggregation-induced emission enhancement (AIEE) properties of salicylaldehyde azines. For this purpose, a series of halogenated salicylaldehyde azine derivatives, namely, chloro-salicylaldehyde azine (1), bromo-salicylaldehyde azine (2) and iodo-salicylaldehyde azine (3) are synthesized. 1 and 2 display typical AIEE characteristics of salicylaldehyde azine compounds; whereas for the iodo-substituent in 3, is found to be effective “external” heavy atom quenchers to salicylaldehyde azine fluorescence in aggregated state. Based on its weak fluorescence in aggregated state and relative strong fluorescence in dispersed state, 3 can also be applied as a turn-on fluorescence probe for egg albumin detection attributed to hydrophobic interaction. -- Highlights: • This study investigates the heavy-atom effect (HAE) on aggregation-induced emission enhancement (AIEE) properties of salicylaldehyde azines. • Chloro- and bromo-salicylaldehyde display typical AIEE properties of salicylaldehyde azine, whereas the iodo-substitute quenches AIEE in aggregated state. • Iodo-salicylaldehyde can be applied as a turn-on fluorescence probe for egg albumin detection attributed to hydrophobic interaction

SP600125 Induces Src and Type I IGF Receptor Phosphorylation Independent of JNK

Directory of Open Access Journals (Sweden)

Qingbin Kong

2014-09-01

Full Text Available c-Jun N-terminal kinases (JNK are members of the mitogen-activated protein kinase (MAPK family that have important roles in signal transduction. The small molecule SP600125 is widely used in biochemical studies as a JNK inhibitor. However, recent studies indicate that SP600125 may also act independent of JNK. Here, we report that SP600125 can induce Src, type I insulin-like growth factor receptor (IGF-IR, Akt and Erk1/2 phosphorylation. Notably, these effects are independent of its inhibition of JNK. Inhibition of Src abrogates the stimulation of IGF-IR, Akt and Erk1/2 phosphorylation. IGF-IR knockdown blunts the induction of both Akt and Erk1/2 phosphorylation by SP600125. Moreover, combination of SP600125 and the Src inhibitor saracatinib synergistically inhibits cell proliferation. We conclude that SP600125 can activate Src-IGF-IR-Akt/Erk1/2 signaling pathways independent of JNK.

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

Science.gov (United States)

Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

1999-01-01

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

Rubrene analogues with the aggregation-induced emission enhancement behaviour

DEFF Research Database (Denmark)

Zhang, Xiaoxu; Sørensen, Jakob Kryger; Fu, Xiaowei

2014-01-01

In the light of the principle of aggregation-induced emission enhancement (AIEE), the rubrene analogue with orange light-emitting properties is designed and synthesized by substituting the phenyl side groups of rubrene with thienyl groups. To the best of our knowledge, this is the first report on...

Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis

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Michael I. Carr

2016-09-01

Full Text Available ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2S394A knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2S394A mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2S394A mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies.

CELLS OVEREXPRESSING HSP27 SHOW ACCELERATED RECOVERY FROM HEAT-INDUCED NUCLEAR-PROTEIN AGGREGATION

NARCIS (Netherlands)

KAMPINGA, HH; BRUNSTING, JF; STEGE, GJJ; KONINGS, AWT; LANDRY, J

1994-01-01

Protein denaturation/aggregation upon cell exposure to heat shock is a likely cause of cell death. in the nucleus, protein aggregation has often been correlated to inhibition of nuclear located processes and heat-induced cell killing. in Chinese hamster 023 cells made thermotolerant by a prior

Changes in tau phosphorylation levels in the hippocampus and frontal cortex following chronic stress

Energy Technology Data Exchange (ETDEWEB)

Yang, C.; Guo, X. [Wuhan University, Renmin Hospital, Department of Psychiatry, Wuhan, China, Department of Psychiatry, Renmin Hospital, Wuhan University, Wuhan (China); Wang, G.H. [Wuhan University, Renmin Hospital, Department of Psychiatry, Wuhan, China, Department of Psychiatry, Renmin Hospital, Wuhan University, Wuhan (China); Wuhan University, Institute of Neuropsychiatry, Wuhan, China, Institute of Neuropsychiatry, Wuhan University, Wuhan (China); Wang, H.L.; Liu, Z.C.; Liu, H.; Zhu, Z.X.; Li, Y. [Wuhan University, Renmin Hospital, Department of Psychiatry, Wuhan, China, Department of Psychiatry, Renmin Hospital, Wuhan University, Wuhan (China)

2014-03-03

Studies have indicated that early-life or early-onset depression is associated with a 2- to 4-fold increased risk of developing Alzheimers disease (AD). In AD, aggregation of an abnormally phosphorylated form of the tau protein may be a key pathological event. Tau is known to play a major role in promoting microtubule assembly and stabilization, and in maintaining the normal morphology of neurons. Several studies have reported that stress may induce tau phosphorylation. The main aim of the present study was to investigate possible alterations in the tau protein in the hippocampus and frontal cortex of 32 male Sprague-Dawley rats exposed to chronic unpredictable mild stress (CUMS) and then re-exposed to CUMS to mimic depression and the recurrence of depression, respectively, in humans. We evaluated the effects of CUMS, fluoxetine, and CUMS re-exposure on tau and phospho-tau. Our results showed that a single exposure to CUMS caused a significant reduction in sucrose preference, indicating a state of anhedonia. The change in behavior was accompanied by specific alterations in phospho-tau protein levels, but fluoxetine treatment reversed the CUMS-induced impairments. Moreover, changes in sucrose preference and phospho-tau were more pronounced in rats re-exposed to CUMS than in those subjected to a single exposure. Our results suggest that changes in tau phosphorylation may contribute to the link between depression and AD.

Changes in tau phosphorylation levels in the hippocampus and frontal cortex following chronic stress

International Nuclear Information System (INIS)

Yang, C.; Guo, X.; Wang, G.H.; Wang, H.L.; Liu, Z.C.; Liu, H.; Zhu, Z.X.; Li, Y.

2014-01-01

Studies have indicated that early-life or early-onset depression is associated with a 2- to 4-fold increased risk of developing Alzheimers disease (AD). In AD, aggregation of an abnormally phosphorylated form of the tau protein may be a key pathological event. Tau is known to play a major role in promoting microtubule assembly and stabilization, and in maintaining the normal morphology of neurons. Several studies have reported that stress may induce tau phosphorylation. The main aim of the present study was to investigate possible alterations in the tau protein in the hippocampus and frontal cortex of 32 male Sprague-Dawley rats exposed to chronic unpredictable mild stress (CUMS) and then re-exposed to CUMS to mimic depression and the recurrence of depression, respectively, in humans. We evaluated the effects of CUMS, fluoxetine, and CUMS re-exposure on tau and phospho-tau. Our results showed that a single exposure to CUMS caused a significant reduction in sucrose preference, indicating a state of anhedonia. The change in behavior was accompanied by specific alterations in phospho-tau protein levels, but fluoxetine treatment reversed the CUMS-induced impairments. Moreover, changes in sucrose preference and phospho-tau were more pronounced in rats re-exposed to CUMS than in those subjected to a single exposure. Our results suggest that changes in tau phosphorylation may contribute to the link between depression and AD

Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

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Rui Li

2016-03-01

Full Text Available For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7, BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

Science.gov (United States)

Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

2015-11-13

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells

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Jeon, Bo Kyung; Kwon, Kihwan; Kang, Jihee Lee; Choi, Youn-Hee

2015-01-01

Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress. PMID:26234813

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation.

Directory of Open Access Journals (Sweden)

Jens T Stieler

Full Text Available Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD. Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with

Factors influencing radiation-induced impairment of rat liver mitochondrial oxidative phosphorylation

International Nuclear Information System (INIS)

Alexander, K.C.; Aiyar, A.S.; Sreenivasan, A.

1975-01-01

The influence of some experimental conditions on the radiation-induced impairment of oxidative phosphorylation in rat liver mitochondria has been studied. Shielding of the liver during whole body irradiation of the animal does not significantly alter the decreased efficiency of phosphorylation. There exists a great disparity in the in vivo and in vitro radiation doses required for the manifestation of damage to liver mitochondria. While these observations point to the abscopal nature of the radiation effects, direct involvement of the adrenals has been ruled out by studies with adrenalectomised rats. Prior administration of the well known radio-protective agents, serotonin or 2-aminoethyl isothiouronium bromide hydrobromide, is effective in preventing the derangement of mitochondrial function following radioexposure. The hypocholesterolemic drug ethyl-α-p-chlorophenoxy isobutyrate, which is known to influence hepatic mitochondrial turnover, does not afford any significant protection against either mitochondrial damage or the mortality of the animals due to whole body irradiation. (author)

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

Science.gov (United States)

Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

2017-08-01

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Curcumin Attenuates Amyloid-β Aggregate Toxicity and Modulates Amyloid-β Aggregation Pathway.

Science.gov (United States)

Thapa, Arjun; Jett, Stephen D; Chi, Eva Y

2016-01-20

The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.

Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications.

Science.gov (United States)

Yang, Tao-Tao; Cao, Na; Zhang, Hai-Hui; Wei, Jian-Bo; Song, Xiao-Xia; Yi, Dong-Min; Chao, Shuai-Heng; Zhang, Li-Da; Kong, Ling-Fei; Han, Shuang-Yin; Yang, Yu-Xiu; Ding, Song-Ze

2018-04-15

Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either 13 C-urea breath test or immunohistochemistry staining. In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

International Nuclear Information System (INIS)

Mori, Jun; Takahashi-Yanaga, Fumi; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki

2005-01-01

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G 0 /G 1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3β (GSK-3β). Depletion of endogenous GSK-3β by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3β and found that DIF-1 dephosphorylated GSK-3β on Ser 9 and induced the nuclear translocation of GSK-3β, suggesting that DIF-1 activated GSK-3β. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr 286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3β-mediated phosphorylation of Thr 286

Cotinine improves visual recognition memory and decreases cortical Tau phosphorylation in the Tg6799 mice.

Science.gov (United States)

Grizzell, J Alex; Patel, Sagar; Barreto, George E; Echeverria, Valentina

2017-08-01

Alzheimer's disease (AD) is associated with the progressive aggregation of hyperphosphorylated forms of the microtubule associated protein Tau in the central nervous system. Cotinine, the main metabolite of nicotine, reduced working memory deficits, synaptic loss, and amyloid β peptide aggregation into oligomers and plaques as well as inhibited the cerebral Tau kinase, glycogen synthase 3β (GSK3β) in the transgenic (Tg)6799 (5XFAD) mice. In this study, the effect of cotinine on visual recognition memory and cortical Tau phosphorylation at the GSK3β sites Serine (Ser)-396/Ser-404 and phospho-CREB were investigated in the Tg6799 and non-transgenic (NT) littermate mice. Tg mice showed short-term visual recognition memory impairment in the novel object recognition test, and higher levels of Tau phosphorylation when compared to NT mice. Cotinine significantly improved visual recognition memory performance increased CREB phosphorylation and reduced cortical Tau phosphorylation. Potential mechanisms underlying theses beneficial effects are discussed. Copyright © 2017. Published by Elsevier Inc.

Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of β-endorphin in a mouse pituitary cell line

International Nuclear Information System (INIS)

Fagarasan, M.O.; Axelrod, J.; Bishop, J.F.; Rinaudo, M.S.

1990-01-01

Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates β-endorphin release and potentiates the secretion induced by many secretagogues. Desensitization of protein kinase C (PKC) by pretreatment with phorbol ester [phorbol 12-tetradecanoate 13-acetate (TPA)] for 8 hr abolished the secretion induced by TPA as well as the enhancement of TPA-induced β-endorphin release produced by IL-1. Desensitization of PKC only partly abolished the potentiating effects of IL-1 on corticotropin-releasing factor-induced β-endorphin secretion. In contrast, IL-1-induced β-endorphin release was independent of PKC. The authors observed that treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-, 20-, and 60-kDa proteins within minutes, presumably by early activation of protein kinases. Prolonged treatment with TPA, which was shown to desensitize an 87-kDa protein (a substrate for PKC), had no effect on IL-1-induced phosphorylation of 20-, 60-, and 87-kDa proteins, indicating that the phosphorylation of these proteins does not involve PKC. IL-1 does not generate cAMP in AtT-20 cells, suggesting that a cAMP-dependent protein kinase is also not involved. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of β-endorphin

The effect of carbohydrates and lipids on the radiation-induced aggregation of proteins

International Nuclear Information System (INIS)

Delincee, H.; Jakubick, V.

1977-01-01

Myoglobin, ovalbumin and serum albumin have been irradiated in aqueous solution in the presence of varying amounts of carbohydrates and lipids, simulating a model food. Gel chromatography revealed the induction of protein aggregates, the formation of which depended strongly on protein concentration. The addition of carbohydrates (trehalose, starch) greatly reduced the amount of radiation-induced aggregates, whereas the addition of lipids (sunflower oil) had practically no effect on aggregate formation. However, if both carbohydrates and lipids were added, the decrease in aggregation caused by the carbohydrate addition was counteracted by the addition of the lipid; as increasing amounts of lipid were added, the effect of carbohydrate addition became smaller. (author)

Enzyme-induced aggregation of whey proteins with Bacillus licheniformis protease

NARCIS (Netherlands)

Creusot, N.P.

2006-01-01

Whey proteins are commonly used as ingredient in food. In relation with the gelation properties of whey proteins, this thesis deals with understanding the mechanism of peptide-induced aggregation of whey protein hydrolysates made with Bacillus licheniformis protease (BLP). The results show that BLP

Neurofilament phosphorylation and disruption: A possible mechanism of chronic aluminium toxicity in Wistar rats

International Nuclear Information System (INIS)

Kaur, Amarpreet; Joshi, Kusum; Minz, Ranjana Walker; Gill, Kiran Dip

2006-01-01

The present study was designed to investigate the possible effects of chronic aluminium exposure on neurofilament phosphorylation and its subsequent disruption in various regions of the rat brain. An intra-gastric dose of aluminium (10 mg/kg bw for 12 weeks) resulted in a marked enhancement of Ca 2+ /CaM dependent protein kinase activity as compared to cAMP dependent protein kinase. The levels of phosphoprotein phosphatase were found to be significantly depleted only in the cerebral cortex. After in vitro phosphorylation using [ 32 γ-P] ATP, various proteins were resolved on one-dimensional 8% SDS-PAGE, stained with Coomassie Blue and autoradiographed. The amount of 32 P-incorporated was quantified using ADOPE PHOTOSHOP (7.0). The 200 kDa neurofilament protein was identified using immunoblotting. Finally, the extent of phosphorylation induced neurofilamentous damage was assessed using immunocytochemical studies. The cytoskeletal proteins were found to be aggregated and disrupted in all the three neuronal regions following 12 weeks of aluminium treatment. This study lends further support to the possible role of aluminium as a potent neurotoxic agent and in the etiopathogenisis of various neurodegenerative diseases

Methylglyoxal-induced modification causes aggregation of myoglobin

Science.gov (United States)

Banerjee, Sauradipta; Maity, Subhajit; Chakraborti, Abhay Sankar

2016-02-01

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200 μM) on the monomeric heme protein myoglobin (Mb) (100 μM) in a time-dependent manner (7 to 18 days incubation at 25 °C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased β-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7 days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14 days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18 days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

International Nuclear Information System (INIS)

Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

1987-01-01

In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase [A-kinase], from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from 32 P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the 32 P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase

Inhibitory Effect of Waste Glass Powder on ASR Expansion Induced by Waste Glass Aggregate

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Shuhua Liu

2015-10-01

Full Text Available Detailed research is carried out to ascertain the inhibitory effect of waste glass powder (WGP on alkali-silica reaction (ASR expansion induced by waste glass aggregate in this paper. The alkali reactivity of waste glass aggregate is examined by two methods in accordance with the China Test Code SL352-2006. The potential of WGP to control the ASR expansion is determined in terms of mean diameter, specific surface area, content of WGP and curing temperature. Two mathematical models are developed to estimate the inhibitory efficiency of WGP. These studies show that there is ASR risk with an ASR expansion rate over 0.2% when the sand contains more than 30% glass aggregate. However, WGP can effectively control the ASR expansion and inhibit the expansion rate induced by the glass aggregate to be under 0.1%. The two mathematical models have good simulation results, which can be used to evaluate the inhibitory effect of WGP on ASR risk.

Rate laws of the self-induced aggregation kinetics of Brownian particles

Science.gov (United States)

Mondal, Shrabani; Sen, Monoj Kumar; Baura, Alendu; Bag, Bidhan Chandra

2016-03-01

In this paper we have studied the self induced aggregation kinetics of Brownian particles in the presence of both multiplicative and additive noises. In addition to the drift due to the self aggregation process, the environment may induce a drift term in the presence of a multiplicative noise. Then there would be an interplay between the two drift terms. It may account qualitatively the appearance of the different laws of aggregation process. At low strength of white multiplicative noise, the cluster number decreases as a Gaussian function of time. If the noise strength becomes appreciably large then the variation of cluster number with time is fitted well by the mono exponentially decaying function of time. For additive noise driven case, the decrease of cluster number can be described by the power law. But in case of multiplicative colored driven process, cluster number decays multi exponentially. However, we have explored how the rate constant (in the mono exponentially cluster number decaying case) depends on strength of interference of the noises and their intensity. We have also explored how the structure factor at long time depends on the strength of the cross correlation (CC) between the additive and the multiplicative noises.

Maintained activity of glycogen synthase kinase-3β despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

International Nuclear Information System (INIS)

Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

2010-01-01

Glycogen synthase kinase-3β (GSK3β) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3β. However, the inactive form of GSK3β which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3β substrates, such as β-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3β at serine-9 and other substrates including tau, β-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3β inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3β may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3β inhibitors could be a valuable drug candidate in AD.

Tyrosine phosphorylation of a 66KD soluble protein and augmentation of lectin induced mitogenesis by DMSO in human T lymphocytes

International Nuclear Information System (INIS)

Wedner, H.J.; Bass, G.

1986-01-01

The authors have demonstrated that induction of mitogenesis in human T lymphocytes is associated with the tyrosine phosphorylation of a 66KD soluble substrate-TPP 66. Since DMSO has been shown to be a non-specific stimulator of tyrosine protein kinases they have examined the effect of DMSO on both activation and tyrosine phosphorylation in human T cells. Human peripheral blood T lymphocytes were isolated by dextran sedimentation, Ficol/Paque centrifugation and nylon wool filtration. Phosphorylation was performed in cells incubated with [ 32 P] orthophosphate followed by DMSO for 30 min. TPP 66 was identified by 2-D PAGE, autoradiography, and HV electrophoresis of the hydrolyzed protein. Concentrations of DMSO from 1% to 50% induced the tyrosine phosphorylation of TPP 66 with maximal stimulation seen at 20%. DMSO alone did not activate the T cells (measured by [ 3 H] thymidine incorporation) when tested at high concentrations for 30 sec to 10 min. (longer incubations were markedly toxic) or low concentrations for 12 to 48 hrs. Low concentrations of DMSO 0.1%-0.5% did however, markedly augment [ 3 H] thymidine incorporation induced by PHA or Con A. These data suggest that tyrosine phosphorylation of TPP 66 alone may not constitute sufficient signal for the activation sequence to begin but the phosphorylation of this soluble substrate may be a critical factor in the propagation of the activation sequence

Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein

International Nuclear Information System (INIS)

Huzoor-Akbar; Anwer, K.

1986-01-01

This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 μM) blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in 32 P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

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Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Increased phosphorylation of histone H3 at serine 10 is involved in Epstein-Barr virus latent membrane protein-1-induced carcinogenesis of nasopharyngeal carcinoma

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Li, Binbin; Huang, Guoliang; Zhang, Xiangning; Li, Rong; Wang, Jian; Dong, Ziming; He, Zhiwei

2013-01-01

Increased histone H3 phosphorylation is an essential regulatory mechanism for neoplastic cell transformation. We aimed to explore the role of histone H3 phosphorylation at serine10 (p-H3Ser10) in Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1)-induced carcinogenesis of nasopharyngeal carcinoma (NPC). The expression of p-H3Ser10 was detected by the immunohistochemical analysis in NPC, chronic nasopharyngitis and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using the small interfering RNA (siRNA)-H3 and histone H3 mutant (S10A), the effect of histone H3 Ser10 motif on LMP1-induced CNE1 cell proliferation, transformation and activator protein-1 (AP-1) activation were evaluated by CCK-8, focus-forming and reporter gene assay respectively. Mitogen- and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation were detected by in vitro kinase assay and western blot. Using MSK1 inhibitor H89 or siRNA-MSK1, the regulatory role of MSK1 on histone H3 phosphorylation and AP-1 activation were analyzed. Immunohistochemical analysis revealed that the expression of p-H3Ser10 was significantly higher in the poorly differentiated NPC tissues than that in chronic nasopharyngitis (p <0.05) and normal nasopharynx tissues (p <0.001). Moreover, high level of p-H3Ser10 was positively correlated with the expression of LMP1 in NPC tissues (χ 2 =6.700, p =0.01; C=0.350) and cell lines. The knockdown and mutant (S10A) of histone H3 suppressed LMP1-induced CNE1 cell proliferation, foci formation and AP-1 activation. In addition, LMP1 could increase MSK1 kinase activity and phosphorylation. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA blocked LMP1-induced phosphorylation of histone H3 at Ser10 and AP-1 activation. EBV-LMP1 can induce phosphorylation of histone H3 at Ser10 via MSK1. Increased phosphorylation of histone H3 at Ser10 is likely a crucial regulatory mechanism involved in LMP1-induced carcinogenesis of

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

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Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Primary Blast-Induced Changes in Akt and GSK3β Phosphorylation in Rat Hippocampus

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Yushan Wang

2017-08-01

Full Text Available Traumatic brain injury (TBI due to blast from improvised explosive devices has been a leading cause of morbidity and mortality in recent conflicts in Iraq and Afghanistan. However, the mechanisms of primary blast-induced TBI are not well understood. The Akt signal transduction pathway has been implicated in various brain pathologies including TBI. In the present study, the effects of simulated primary blast waves on the phosphorylation status of Akt and its downstream effector kinase, glycogen synthase kinase 3β (GSK3β, in rat hippocampus, were investigated. Male Sprague-Dawley (SD rats (350–400 g were exposed to a single pulse shock wave (25 psi; ~7 ms duration and sacrificed 1 day, 1 week, or 6 weeks after exposure. Total and phosphorylated Akt, as well as phosphorylation of its downstream effector kinase GSK3β (at serine 9, were detected with western blot analysis and immunohistochemistry. Results showed that Akt phosphorylation at both serine 473 and threonine 308 was increased 1 day after blast on the ipsilateral side of the hippocampus, and this elevation persisted until at least 6 weeks postexposure. Similarly, phosphorylation of GSK3β at serine 9, which inhibits GSK3β activity, was also increased starting at 1 day and persisted until at least 6 weeks after primary blast on the ipsilateral side. In contrast, p-Akt was increased at 1 and 6 weeks on the contralateral side, while p-GSK3β was increased 1 day and 1 week after primary blast exposure. No significant changes in total protein levels of Akt and GSK were observed on either side of the hippocampus at any time points. Immunohistochemical results showed that increased p-Akt was mainly of neuronal origin in the CA1 region of the hippocampus and once phosphorylated, the majority was translocated to the dendritic and plasma membranes. Finally, electrophysiological data showed that evoked synaptic N-methyl-d-aspartate (NMDA receptor activity was

Temperature dependent rapid annealing effect induces amorphous aggregation of human serum albumin.

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Ishtikhar, Mohd; Ali, Mohd Sajid; Atta, Ayman M; Al-Lohedan, Hammad; Badr, Gamal; Khan, Rizwan Hasan

2016-01-01

This study represents an analysis of the thermal aggregation of human serum albumin (HSA) induced by novel rosin modified compounds. The aggregation process causes conformational alterations in the secondary and tertiary structures of the proteins. The conversion of globular protein to amorphous aggregates was carried out by spectroscopic, calorimetric and microscopic techniques to investigate the factors that are responsible for the structural, conformational and morphological alteration in the protein. Our outcome results show that the aggregation of HSA was dependent on the hydrophobicity, charge and temperature, because the formation of amorphous aggregates occurs in the presence of a novel cationic rosin compound, quaternary amine of rosin diethylaminoethyl ester (QRMAE), at 40°C and pH 7.4 (but at 25°C on similar pH value, there was no evidence of aggregate formation). In addition, the parent compound of QRMAE, i.e., abietic acid, and other derivatives such as nonionic rosin compounds [(RMPEG-750) and (RMA-MPEG-750)] do not shows the aggregating property. This work provides precise and necessary information that aid in the understanding the effects of rosin derivative compounds on HSA. This study also restrains important information for athletes, health providers, pharmaceutical companies, industries, and soft drink-processing companies. Copyright © 2015 Elsevier B.V. All rights reserved.

Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

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Hirose, H; Sakuma, N; Kaji, N; Nakayama, K; Inoue, K; Sekijima, M; Nojima, T; Miyakoshi, J

2007-02-01

An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.

Effects of the gelatin plasma substitutes Haemaccel, Plasmagel and Plasmion (Geloplasma) on collagen-, ADP- and adrenaline-induced aggregation of human platelets in vitro.

Science.gov (United States)

Stibbe, J; van der Plas, P M; Ong, G L; ten Hoor, F; Nauta, J; de Jong, D S; Krenning-Douma, E; Gomes, M

1981-01-01

The effect of some gelatin plasma substitutes (Haemaccel, plasmagel and Plasmion (Geloplasma), which are widely used in Europe) on collagen-, ADP- and adrenaline-induced platelet aggregation in human PRP in vitro was studied under controlled conditions (pH, electrolyte composition). Haemaccel inhibited these aggregations, both in citrated as well as in heparinised PRP, whereas they were enhanced by both Plasmagel and Plasmion as compared to the appropriate control. Increasing teh concentration of the inducer overcame the inhibition by Haemaccel. Haemaccel inhibited, while Plasmion enhanced 14C-serotonin release induced by collagen, ADP or adrenaline. Also in the presence of indomethacin (90 muM) Haemaccel inhibited aggregation induced by high concentrations of collagen and the primary aggregation induced by ADP and adrenaline, while Plasmion enhanced these aggregations induced by ADP and adrenaline, while Plasmion enhanced these aggregations. The inhibition by Haemaccel was not caused by binding of Ca2+ to haemaccel.

Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

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Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

2010-03-01

Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

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Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

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Riedl, Julia; Preusser, Matthias; Nazari, Pegah Mir Seyed; Posch, Florian; Panzer, Simon; Marosi, Christine; Birner, Peter; Thaler, Johannes; Brostjan, Christine; Lötsch, Daniela; Berger, Walter; Hainfellner, Johannes A; Pabinger, Ingrid; Ay, Cihan

2017-03-30

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts ( P < .001) and higher D-dimer levels ( P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens ( P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P = 010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors. © 2017 by The American Society of Hematology.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

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Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

2017-09-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

International Nuclear Information System (INIS)

McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

2010-01-01

Research highlights: → cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. → Pka1 phosphorylation is further induced by physiological stresses. → Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. → Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1 + or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

Induced simplified neutrosophic correlated aggregation operators for multi-criteria group decision-making

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Şahin, Rıdvan; Zhang, Hong-yu

2018-03-01

Induced Choquet integral is a powerful tool to deal with imprecise or uncertain nature. This study proposes a combination process of the induced Choquet integral and neutrosophic information. We first give the operational properties of simplified neutrosophic numbers (SNNs). Then, we develop some new information aggregation operators, including an induced simplified neutrosophic correlated averaging (I-SNCA) operator and an induced simplified neutrosophic correlated geometric (I-SNCG) operator. These operators not only consider the importance of elements or their ordered positions, but also take into account the interactions phenomena among decision criteria or their ordered positions under multiple decision-makers. Moreover, we present a detailed analysis of I-SNCA and I-SNCG operators, including the properties of idempotency, commutativity and monotonicity, and study the relationships among the proposed operators and existing simplified neutrosophic aggregation operators. In order to handle the multi-criteria group decision-making (MCGDM) situations where the weights of criteria and decision-makers usually correlative and the criterion values are considered as SNNs, an approach is established based on I-SNCA operator. Finally, a numerical example is presented to demonstrate the proposed approach and to verify its effectiveness and practicality.

Andrographolide Attenuates LPS-Induced Cardiac Malfunctions Through Inhibition of IκB Phosphorylation and Apoptosis in Mice

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Jinlong Zhang

2015-11-01

Full Text Available Background/Aims: Cardiac malfunction is a common complication in sepsis and significantly increases the mortality of patients in septic shock. However, no studies have examined whether andrographolide (And reduces LPS-induced myocardial malfunction. Methods: Left ventricular systolic and diastolic functions were examined using echocardiography. TNF-a and IL-1ß protein levels were detected by an enzyme-linked immunosorbent assay (ELISA. NO oxidation products were determined using Griess reagent. Protein expression levels of inhibitors of NF-κBa (IκB and phospho-IκB were determined via Western blot. Oxidative injury was determined by measuring myocardial lipid peroxidation and superoxide dismutase activity. Cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL and cardiac caspase 3/7 activity. Results: And blunted LPS-induced myocardial malfunctions in mice. LPS induced TNF-a, IL-1ß, and NO production as well as I-κB phosphorylation. Cardiac apoptosis was attenuated via incubation with And, but the extent of oxidative injury remained unaffected. Conclusion: And prevents LPS-induced cardiac malfunctions in mice by inhibiting TNF-a, IL-1ß, and NO production, IκB phosphorylation, and cardiac apoptosis, indicating that And may be a potential agent for preventing myocardial malfunction during sepsis.

Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

Science.gov (United States)

Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

2013-10-01

In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

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Slavka eBekesova

2015-05-01

Full Text Available The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+ used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™ offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy.

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Woolf, N; Pearson, B E; Bondzie, P A; Meyer, R D; Lavaei, M; Belkina, A C; Chitalia, V; Rahimi, N

2017-09-18

Adhesion to extracellular matrix (ECM) is crucially important for survival of normal epithelial cells as detachment from ECM triggers specific apoptosis known as anoikis. As tumor cells lose the requirement for anchorage to ECM, they rely on cell-cell adhesion 'multicellular aggregation' for survival. Multicellular aggregation of tumor cells also significantly determines the sensitivity of tumor cells to the cytotoxic effects of chemotherapeutics. In this report, we demonstrate that expression of immunoglobulin containing and proline-rich receptor-1 (IGPR-1) is upregulated in human primary colon cancer. Our study demonstrates that IGPR-1 promotes tumor multicellular aggregation, and interfering with its adhesive function inhibits multicellular aggregation and, increases cell death. IGPR-1 supports colon carcinoma tumor xenograft growth in mouse, and inhibiting its activity by shRNA or blocking antibody inhibits tumor growth. More importantly, IGPR-1 regulates sensitivity of tumor cells to the chemotherapeutic agent, doxorubicin/adriamycin by a mechanism that involves doxorubicin-induced AKT activation and phosphorylation of IGPR-1 at Ser220. Our findings offer novel insight into IGPR-1's role in colorectal tumor growth, tumor chemosensitivity, and as a possible novel anti-cancer target.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

International Nuclear Information System (INIS)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

2015-01-01

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

The effect of magnetic field induced aggregates on ultrasound propagation in aqueous magnetic fluid

International Nuclear Information System (INIS)

Parekh, Kinnari; Upadhyay, R.V.

2017-01-01

Ultrasonic wave propagation in the aqueous magnetic fluid is investigated for different particle concentrations. The sound velocity decreases while acoustic impedance increases with increasing concentrations. The velocity anisotropy is observed upon application of magnetic field. The velocity anisotropy fits with Tarapov’s theory suggests the presence of aggregates in the system. We report that these aggregates are thermodynamically unstable and the length of aggregate changes continuously with increasing concentration and, or magnetic field and resulted in a decrease in effective magnetic moment. The Taketomi's theory fits well with the experimental data suggesting that the particle clusters are aligned in the direction of the magnetic field. The radius of cluster found to increase with increasing concentration, and then decreases whereas the elastic force constant increases and then becomes constant. The increase in cluster radius indicates elongation of aggregate length due to tip-to-tip interaction of aggregates whereas for higher concentration, the lateral alignment is more favorable than tip-to-tip alignment of aggregates which reduces the cluster radius making elastic force constant to raise. Optical images show that the chains are fluctuating and confirming the lateral alignment of chains at higher fields. - Highlights: • Magnetic field induced aggregates investigated using ultrasonic wave in aqueous magnetic fluid. • Velocity anisotropy induces upon applications of magnetic field. • Tarapov’s theory fit shows reduction in effective magnetic moment as concentration increases. • Taketomi's theory shows alignment of clusters in field direction. • Cluster radius increases and then decreases with increasing volume fractions. • Optical images show that fluctuating chains and lateral alignment of chains at higher fields.

The effect of magnetic field induced aggregates on ultrasound propagation in aqueous magnetic fluid

Energy Technology Data Exchange (ETDEWEB)

Parekh, Kinnari, E-mail: kinnariparekh.rnd@charusat.ac.in [Dr. KC Patel R& D Center, Charotar University of Science & Technology, Changa, 388421 Dist. Anand, Gujarat (India); Upadhyay, R.V. [PD Patel Institute of Applied Sciences, Charotar University of Science & Technology, Changa, 388421 Dist. Anand, Gujarat (India)

2017-06-01

Ultrasonic wave propagation in the aqueous magnetic fluid is investigated for different particle concentrations. The sound velocity decreases while acoustic impedance increases with increasing concentrations. The velocity anisotropy is observed upon application of magnetic field. The velocity anisotropy fits with Tarapov’s theory suggests the presence of aggregates in the system. We report that these aggregates are thermodynamically unstable and the length of aggregate changes continuously with increasing concentration and, or magnetic field and resulted in a decrease in effective magnetic moment. The Taketomi's theory fits well with the experimental data suggesting that the particle clusters are aligned in the direction of the magnetic field. The radius of cluster found to increase with increasing concentration, and then decreases whereas the elastic force constant increases and then becomes constant. The increase in cluster radius indicates elongation of aggregate length due to tip-to-tip interaction of aggregates whereas for higher concentration, the lateral alignment is more favorable than tip-to-tip alignment of aggregates which reduces the cluster radius making elastic force constant to raise. Optical images show that the chains are fluctuating and confirming the lateral alignment of chains at higher fields. - Highlights: • Magnetic field induced aggregates investigated using ultrasonic wave in aqueous magnetic fluid. • Velocity anisotropy induces upon applications of magnetic field. • Tarapov’s theory fit shows reduction in effective magnetic moment as concentration increases. • Taketomi's theory shows alignment of clusters in field direction. • Cluster radius increases and then decreases with increasing volume fractions. • Optical images show that fluctuating chains and lateral alignment of chains at higher fields.

Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells

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Marino, Maria; Acconcia, Filippo; Trentalance, Anna

2003-01-01

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1–S transition by the parallel stimulation of both PKC-α and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17β-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1–S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1–S phase transition. PMID:12808053

Human herpesvirus 6B induces phosphorylation of p53 in its regulatory domain by a CK2- and p38-independent pathway

DEFF Research Database (Denmark)

Øster, Bodil; Bundgaard, B; Hupp, T R

2008-01-01

Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although...

Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats.

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Choi, Miyeon; Lee, Seung Hoon; Park, Min Hyeop; Kim, Yong-Seok; Son, Hyeon

2017-08-05

Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine. Copyright © 2017 Elsevier Inc. All rights reserved.

PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

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Aiko Amagai

2012-01-01

Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

International Nuclear Information System (INIS)

Tseng, Ya-Shih; Lee, Jenq-Chang; Huang, Chi-Ying F; Liu, Hsiao-Sheng

2009-01-01

Overexpression of Aurora-A and mutant Ras (Ras V12 ) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras V12 and wild-type Aurora-A (designated WT) or Ras V12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. Overexpression of wild-type Aurora-A and mutation of Ras V12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras V12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras V12 transformants. Wild-type-Aurora-A enhances focus formation and aggregation of the Ras V12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway

PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

Science.gov (United States)

Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation

Science.gov (United States)

Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira

2018-01-01

Background Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. Methods We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. Results In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. Conclusions These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers. PMID:29629343

A high-fructose diet induces changes in pp185 phosphorylation in muscle and liver of rats

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M. Ueno

2000-12-01

Full Text Available Insulin stimulates the tyrosine kinase activity of its receptor resulting in the tyrosine phosphorylation of pp185, which contains insulin receptor substrates IRS-1 and IRS-2. These early steps in insulin action are essential for the metabolic effects of insulin. Feeding animals a high-fructose diet results in insulin resistance. However, the exact molecular mechanism underlying this effect is unknown. In the present study, we determined the levels and phosphorylation status of the insulin receptor and pp185 (IRS-1/2 in liver and muscle of rats submitted to a high-fructose diet evaluated by immunoblotting with specific antibodies. Feeding fructose (28 days induced a discrete insulin resistance, as demonstrated by the insulin tolerance test. Plasma glucose and serum insulin and cholesterol levels of the two groups of rats, fructose-fed and control, were similar, whereas plasma triacylglycerol concentration was significantly increased in the rats submitted to the fructose diet (P<0.05. There were no changes in insulin receptor concentration in the liver or muscle of either group. However, insulin-stimulated receptor autophosphorylation was reduced to 72 ± 4% (P<0.05 in the liver of high-fructose rats. The IRS-1 protein levels were similar in both liver and muscle of the two groups of rats. In contrast, there was a significant decrease in insulin-induced pp185 (IRS-1/2 phosphorylation, to 83 ± 5% (P<0.05 in liver and to 77 ± 4% (P<0.05 in muscle of the high-fructose rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance induced by high-fructose feeding.

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

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Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

2016-02-15

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

p53 Aggregates penetrate cells and induce the co-aggregation of intracellular p53.

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Karolyn J Forget

Full Text Available Prion diseases are unique pathologies in which the infectious particles are prions, a protein aggregate. The prion protein has many particular features, such as spontaneous aggregation, conformation transmission to other native PrP proteins and transmission from an individual to another. Protein aggregation is now frequently associated to many human diseases, for example Alzheimer's disease, Parkinson's disease or type 2 diabetes. A few proteins associated to these conformational diseases are part of a new category of proteins, called prionoids: proteins that share some, but not all, of the characteristics associated with prions. The p53 protein, a transcription factor that plays a major role in cancer, has recently been suggested to be a possible prionoid. The protein has been shown to accumulate in multiple cancer cell types, and its aggregation has also been reproduced in vitro by many independent groups. These observations suggest a role for p53 aggregates in cancer development. This study aims to test the «prion-like» features of p53. Our results show in vitro aggregation of the full length and N-terminally truncated protein (p53C, and penetration of these aggregates into cells. According to our findings, the aggregates enter cells using macropinocytosis, a non-specific pathway of entry. Lastly, we also show that once internalized by the cell, p53C aggregates can co-aggregate with endogenous p53 protein. Together, these findings suggest prion-like characteristics for p53 protein, based on the fact that p53 can spontaneously aggregate, these aggregates can penetrate cells and co-aggregate with cellular p53.

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples

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N. Mallouk

2018-07-01

Full Text Available A recently released kit (PerFix EXPOSE was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12. The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP of PGE1-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets. Keywords: Platelet signaling, VASP, Flow cytometry, Clopidogrel

Luminescent Organometallic Nanomaterials with Aggregation-Induced Emission.

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Shu, Tong; Wang, Jianxing; Su, Lei; Zhang, Xueji

2018-07-04

Recent researches in metal nanoclusters (NCs) have prompted their promising practical applications in biomedical fields as novel inorganic luminophores. More recently, to further improve the photoluminescence (PL) performance of NCs, the aggregation-induced emission (AIE) effect has been introduced to develop highly luminescent metal NCs and metal complex materials. In this review, we start our discussion from recent progresses on AIE materials developments. Then, we address our understandings on the PL properties of thiolated metal NCs. Subsequently, we link thiolated metal NCs with AIE effect. We also highlight some recent advances in synthesizing the AIE-type metal complex nanomaterials. We finally discuss visions and directions for future development of AIE-type metal complex nanomaterials.

Possible role of calcium dependent protein phosphorylation in the modulation of wound induced HRGP gene activation in potatoes after gamma irradiation

International Nuclear Information System (INIS)

Ussuf, K.K.; Laxmi, N.H.; Nair, P.M.

1996-01-01

Hydroxyproline rich glycoprotein (HRGP) gene is induced in both control and gamma irradiated potato tubers after wounding. The enhanced RNA synthesis in response to wounding correlated well with the accumulation of both HRGP gene transcripts and protein. Initially, the level of HRGP gene expression in gamma irradiated potatoes in response to wounding was 30% more than the corresponding controls. After post irradiation storage of 3-5 weeks, HRGP gene expression in response to wounding was significantly lower than the unirradiated samples. This low level of HRGP gene expression in irradiated potatoes was partially retrieved by 5 mM Ca 2+ treatment. Prior treatment with trifluoperazine, a calcium channel blocker resulted in 35% reduction in wound induced HRGP gene expression in control potatoes, further providing evidence for the involvement of Ca 2+ dependency for HRGP gene activation. A comparative study on in vivo protein phosphorylation induced by wounding in control and irradiated potatoes exhibited significant differences. A good correlation was observed in the modulation of phosphorylation and HRGP gene expression by Ca 2+ in irradiated potatoes. Wound induced signal transduction system and subsequent Ca 2+ dependent protein phosphorylation for the activation of HRGP gene is affected in potatoes after gamma irradiation, thus impairing the wound healing process adversely. (author). 25 refs., 5 figs

p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

International Nuclear Information System (INIS)

Ji, Guoping; Liu, Dongxu; Liu, Jing; Gao, Hui; Yuan, Xiao; Shen, Gang

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

Energy Technology Data Exchange (ETDEWEB)

Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

The roles of phosphorylation and SHAGGY-like protein kinases in geminivirus C4 protein induced hyperplasia.

Directory of Open Access Journals (Sweden)

Katherine Mills-Lujan

Full Text Available Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV. The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1 mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2 Ser49 is phosphorylated in planta; and 3 plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

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Deshet, Naamit; Lupu-Meiri, Monica; Espinoza, Ingrid; Fili, Oded; Shapira, Yuval; Lupu, Ruth; Gershengorn, Marvin C; Oron, Yoram

2008-09-01

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

Ebselen inhibits iron-induced tau phosphorylation by attenuating DMT1 up-regulation and cellular iron uptake.

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Xie, Ling; Zheng, Wei; Xin, Na; Xie, Jing-Wei; Wang, Tao; Wang, Zhan-You

2012-08-01

Dysregulation of iron homeostasis is involved in the pathological process of Alzheimer's disease (AD). We have recently reported that divalent metal transporter 1 (DMT1) is upregulated in an AD transgenic mouse brain, and that silencing of DMT1, which reduces cellular iron influx, results in inhibition of amyloidogenesis in vitro, suggesting a potential target of DMT1 for AD therapy. In the present study, we tested the hypothesis that inhibition of DMT1 with ebselen, a DMT1 transport inhibitor, could affect tau phosphorylation. Human neuroblastoma SH-SY5Y cells were pre-treated with ebselen and then treated with ferrous sulfate (dissolved in ascorbic acid), and the effects of ebselen on tau phosphorylation and the relative signaling pathways were examined. Our results showed that ebselen decreased iron influx, reduced iron-induced ROS production, inhibited the activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3β, and ultimately attenuated the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. The present study indicates that the neuroprotective effect of ebselen on AD is not only related to its antioxidant activity as reported previously, but is also associated with a reduction in tau phosphorylation by inhibition of DMT1. Copyright © 2012 Elsevier Ltd. All rights reserved.

Surface properties of heat-induced soluble soy protein aggregates of different molecular masses.

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Guo, Fengxian; Xiong, Youling L; Qin, Fang; Jian, Huajun; Huang, Xiaolin; Chen, Jie

2015-02-01

Suspensions (2% and 5%, w/v) of soy protein isolate (SPI) were heated at 80, 90, or 100 °C for different time periods to produce soluble aggregates of different molecular sizes to investigate the relationship between particle size and surface properties (emulsions and foams). Soluble aggregates generated in these model systems were characterized by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat treatment increased surface hydrophobicity, induced SPI aggregation via hydrophobic interaction and disulfide bonds, and formed soluble aggregates of different sizes. Heating of 5% SPI always promoted large-size aggregate (LA; >1000 kDa) formation irrespective of temperature, whereas the aggregate size distribution in 2% SPI was temperature dependent: the LA fraction progressively rose with temperature (80→90→100 °C), corresponding to the attenuation of medium-size aggregates (MA; 670 to 1000 kDa) initially abundant at 80 °C. Heated SPI with abundant LA (>50%) promoted foam stability. LA also exhibited excellent emulsifying activity and stabilized emulsions by promoting the formation of small oil droplets covered with a thick interfacial protein layer. However, despite a similar influence on emulsion stability, MA enhanced foaming capacity but were less capable of stabilizing emulsions than LA. The functionality variation between heated SPI samples is clearly related to the distribution of aggregates that differ in molecular size and surface activity. The findings may encourage further research to develop functional SPI aggregates for various commercial applications. © 2015 Institute of Food Technologists®

Effect of molecular aggregation on the photo-induced anisotropy in amorphous polymethacrylate bearing an aminonitroazobenzene moiety

CERN Document Server

Kim, B J; Choi, D H

2001-01-01

We investigated H-type molecular aggregation in a simply spin-coated amorphous homopolymer film of polymethacrylate containing push-pull azobenzene moieties. It was found that the aggregate formation was strongly influenced by thermal treatment and that the aggregate created in the polymer film could be easily disrupted by irradiation of a linearly polarized light. In the first writing cycle of aggregated polymer film, photo-induced birefringence showed a steep increase to the highest value followed by a gradual decrease to the certain asymptotic value under longer irradiation of linearly polarized light. This unique behavior could be attributed to the cooperative motion and the disruption of the aggregated molecules under continuous irradiation of light.

Effect of molecular aggregation on the photo-induced anisotropy in amorphous polymethacrylate bearing an aminonitroazobenzene moiety

International Nuclear Information System (INIS)

Kim, Beom Jun; Park, Soo Young; Choi, Dong Hoon

2001-01-01

We investigated H-type molecular aggregation in a simply spin-coated amorphous homopolymer film of polymethacrylate containing push-pull azobenzene moieties. It was found that the aggregate formation was strongly influenced by thermal treatment and that the aggregate created in the polymer film could be easily disrupted by irradiation of a linearly polarized light. In the first writing cycle of aggregated polymer film, photo-induced birefringence showed a steep increase to the highest value followed by a gradual decrease to the certain asymptotic value under longer irradiation of linearly polarized light. This unique behavior could be attributed to the cooperative motion and the disruption of the aggregated molecules under continuous irradiation of light

The indolinone MAZ51 induces cell rounding and G2/M cell cycle arrest in glioma cells without the inhibition of VEGFR-3 phosphorylation: involvement of the RhoA and Akt/GSK3β signaling pathways.

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Joo-Hee Park

Full Text Available MAZ51 is an indolinone-based molecule originally synthesized as a selective inhibitor of vascular endothelial growth factor receptor (VEGFR-3 tyrosine kinase. This study shows that exposure of two glioma cell lines, rat C6 and human U251MG, to MAZ51 caused dramatic shape changes, including the retraction of cellular protrusions and cell rounding. These changes were caused by the clustering and aggregation of actin filaments and microtubules. MAZ51 also induced G2/M phase cell cycle arrest. This led to an inhibition of cellular proliferation, without triggering significant cell death. These alterations induced by MAZ51 occurred with similar dose- and time-dependent patterns. Treatment of glioma cells with MAZ51 resulted in increased levels of phosphorylated GSK3β through the activation of Akt, as well as increased levels of active RhoA. Interestingly, MAZ51 did not affect the morphology and cell cycle patterns of rat primary cortical astrocytes, suggesting it selectively targeted transformed cells. Immunoprecipitation-western blot analyses indicated that MAZ51 did not decrease, but rather increased, tyrosine phosphorylation of VEGFR-3. To confirm this unanticipated result, several additional experiments were conducted. Enhancing VEGFR-3 phosphorylation by treatment of glioma cells with VEGF-C affected neither cytoskeleton arrangements nor cell cycle patterns. In addition, the knockdown of VEGFR-3 in glioma cells did not cause morphological or cytoskeletal alterations. Furthermore, treatment of VEGFR-3-silenced cells with MAZ51 caused the same alterations of cell shape and cytoskeletal arrangements as that observed in control cells. These data indicate that MAZ51 causes cytoskeletal alterations and G2/M cell cycle arrest in glioma cells. These effects are mediated through phosphorylation of Akt/GSK3β and activation of RhoA. The anti-proliferative activity of MAZ51 does not require the inhibition of VEGFR-3 phosphorylation, suggesting that it is

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

Science.gov (United States)

Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

2009-09-10

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

Salt-induced aggregation of stiff polyelectrolytes

International Nuclear Information System (INIS)

Fazli, Hossein; Mohammadinejad, Sarah; Golestanian, Ramin

2009-01-01

Molecular dynamics simulation techniques are used to study the process of aggregation of highly charged stiff polyelectrolytes due to the presence of multivalent salt. The dominant kinetic mode of aggregation is found to be the case of one end of one polyelectrolyte meeting others at right angles, and the kinetic pathway to bundle formation is found to be similar to that of flocculation dynamics of colloids as described by Smoluchowski. The aggregation process is found to favor the formation of finite bundles of 10-11 filaments at long times. Comparing the distribution of the cluster sizes with the Smoluchowski formula suggests that the energy barrier for the aggregation process is negligible. Also, the formation of long-lived metastable structures with similarities to the raft-like structures of actin filaments is observed within a range of salt concentration.

Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

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Yan, C; Han, R

1997-01-01

Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium

DEFF Research Database (Denmark)

Perez-Moreno, M; Avila, A; Islas, S

1998-01-01

and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased...

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

International Nuclear Information System (INIS)

Wang Hongmin; Monteiro, Mervyn J.

2007-01-01

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases

Aggregation-induced chemical reactions: acid dissociation in growing water clusters.

Science.gov (United States)

Forbert, Harald; Masia, Marco; Kaczmarek-Kedziera, Anna; Nair, Nisanth N; Marx, Dominik

2011-03-23

Understanding chemical reactivity at ultracold conditions, thus enabling molecular syntheses via interstellar and atmospheric processes, is a key issue in cryochemistry. In particular, acid dissociation and proton transfer reactions are ubiquitous in aqueous microsolvation environments. Here, the full dissociation of a HCl molecule upon stepwise solvation by a small number of water molecules at low temperatures, as relevant to helium nanodroplet isolation (HENDI) spectroscopy, is analyzed in mechanistic detail. It is found that upon successive aggregation of HCl with H(2)O molecules, a series of cyclic heteromolecular structures, up to and including HCl(H(2)O)(3), are initially obtained before a precursor state for dissociation, HCl(H(2)O)(3)···H(2)O, is observed upon addition of a fourth water molecule. The latter partially aggregated structure can be viewed as an "activated species", which readily leads to dissociation of HCl and to the formation of a solvent-shared ion pair, H(3)O(+)(H(2)O)(3)Cl(-). Overall, the process is mostly downhill in potential energy, and, in addition, small remaining barriers are overcome by using kinetic energy released as a result of forming hydrogen bonds due to aggregation. The associated barrier is not ruled by thermal equilibrium but is generated by athermal non-equilibrium dynamics. These "aggregation-induced chemical reactions" are expected to be of broad relevance to chemistry at ultralow temperature much beyond HENDI spectroscopy.

Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

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Thomas H Sanderson

Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

Science.gov (United States)

Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

2016-09-01

Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.

Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

Science.gov (United States)

Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

2004-07-01

A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

International Nuclear Information System (INIS)

Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

2014-01-01

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

Palytoxin and the sodium/potassium pump—phosphorylation and potassium interaction

International Nuclear Information System (INIS)

Rodrigues, Antônio M; De Almeida, Antônio-Carlos G; Infantosi, Antonio F C

2009-01-01

We proposed a reaction model for investigating interactions between K + and the palytoxin–sodium–potassium (PTX–Na + /K + ) pump complex under conditions where enzyme phosphorylation may occur. The model is composed of (i) the Albers–Post model for Na + /K + –ATPase, describing Na + and K + pumping; (ii) the reaction model proposed for Na + /K + –ATPase interactions with its ligands (Na + , K + , ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the pump ligands. The simulations allow interpretation of the simultaneous action of Na + /K + –ATPase phosphorylation and K + on the PTX-induced channels. The results suggest that (i) phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K + occlusion rate, and (ii) K + causes channel blockage, increases the toxin dissociation rate and impedes the induced channel phosphorylation, implying reduction of the PTX toxic effect

Model of fractal aggregates induced by shear

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Wan Zhanhong

2013-01-01

Full Text Available It is an undoubted fact that particle aggregates from marine, aerosol, and engineering systems have fractal structures. In this study, fractal geometry is used to describe the morphology of irregular aggregates. The mean-field theory is employed to solve coagulation kinetic equation of aggregates. The Taylor-expansion method of moments in conjunction with the self-similar fractal characteristics is used to represent the particulate field. The effect of the target fractal dimensions on zeroth-order moment, second-order moment, and geometric standard deviation of the aggregates is explored. Results show that the developed moment method is an efficient and powerful approach to solving such evolution equations.

Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

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Adi Ben Yehuda

Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

Investigation into process-induced de-aggregation of cohesive micronised API particles.

Science.gov (United States)

Hoffmann, Magnus; Wray, Patrick S; Gamble, John F; Tobyn, Mike

2015-09-30

The aim of this study was to assess the impact of unit processes on the de-aggregation of a cohesive micronised API within a pharmaceutical formulation using near-infrared chemical imaging. The impact on the primary API particles was also investigated using an image-based particle characterization system with integrated Raman analysis. The blended material was shown to contain large, API rich domains which were distributed in-homogeneously across the sample, suggesting that the blending process was not aggressive enough to disperse aggregates of micronised drug particles. Cone milling, routinely used to improve the homogeneity of such cohesive formulations, was observed to substantially reduce the number and size of API rich domains; however, several smaller API domains survived the milling process. Conveyance of the cone milled formulation through the Alexanderwerk WP120 powder feed system completely dispersed all remaining aggregates. Importantly, powder feed transmission of the un-milled formulation was observed to produce an equally homogeneous API distribution. The size of the micronised primary drug particles remained unchanged during powder feed transmission. These findings provide further evidence that this powder feed system does induce shear, and is in fact better able to disperse aggregates of a cohesive micronised API within a blend than the blend-mill-blend step. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

Science.gov (United States)

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

International Nuclear Information System (INIS)

Deaciuc, I.V.; Spitzer, J.A.

1989-01-01

The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

Photo-induced reorganization of molecular packing of amphi-PIC J-aggregates (single J-aggregate spectroscopy)

International Nuclear Information System (INIS)

Malyukin, Yu.V.; Sorokin, A.V.; Yefimova, S.L.; Lebedenko, A.N.

2005-01-01

Confocal luminescence microscopy has been used to excite and collect luminescence from single amphi-PIC J-aggregate. Two types of J-aggregates have been revealed in the luminescence image: bead-like J-aggregates, which diameter is less than 1 μm and rod-like ones, which length is about 3 μm and diameter is less than 1 μm. It has been found that single rod-like and bead-like J-aggregates exhibit different luminescence bands with different decay parameters. At the off-resonance blue tail excitation, the J-aggregate exciton luminescence disappeared within a certain time period and a new band appeared, which cannot be attributed to the monomer emission. The luminescence image shows that the J-aggregate is not destroyed. However, J-aggregate storage in darkness does not recover its exciton luminescence

Antiplatelet Activity of Morus alba Leaves Extract, Mediated via Inhibiting Granule Secretion and Blocking the Phosphorylation of Extracellular-Signal-Regulated Kinase and Akt

Science.gov (United States)

Rhee, Man Hee; Sung, Yoon-Young; Yang, Won-Kyung; Kim, Seung Hyung; Kim, Ho-Kyoung

2014-01-01

Ethnopharmacological Relevance. Morus alba L. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema, diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis. Materials and Methods. The anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrin α IIb β 3 and phosphorylation of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The thrombus formation in vivo was also evaluated in arteriovenous shunt model of rats. Results. HPLC chromatographic analysis revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE also attenuated serotonin secretion and thromboxane A2 formation. In addition, the extract in vivo activity showed that MAE at 100, 200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3% (P < 0.001), 28.3% (P < 0.01), and 19.1% (P < 0.05), respectively. Conclusions. MAE inhibit platelet activation, TXB2 formation, serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and antithrombotic agent. PMID:24701244

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

Science.gov (United States)

Li, Chang-Ling; Yang, Jin-Gang; Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

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Chang-Ling Li

Full Text Available Megakaryocytes (MKs are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1 at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in

Propofol directly increases tau phosphorylation.

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Robert A Whittington

2011-01-01

Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

International Nuclear Information System (INIS)

Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

2009-01-01

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

Detection of pH-induced aggregation of "smart" gold nanoparticles with photothermal optical coherence tomography.

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Xiao, Peng; Li, Qingyun; Joo, Yongjoon; Nam, Jutaek; Hwang, Sekyu; Song, Jaejung; Kim, Sungjee; Joo, Chulmin; Kim, Ki Hean

2013-11-01

We report the feasibility of a novel contrast agent, namely "smart" gold nanoparticles (AuNPs), in the detection of cancer cells with photothermal optical coherence tomography (PT-OCT). "Smart" AuNPs form aggregation in low pH condition, which is typical for cancer cells, and this aggregation results in a shift of their absorption spectrum. A PT-OCT system was developed to detect this pH-induced aggregation by combining an OCT light source and a laser with 660 nm in wavelength for photothermal excitation. Optical detection of pH-induced aggregation was tested with solution samples at two different pH conditions. An increase in optical path length (OPL) variation was measured at mild acidic condition, while there was not much change at neutral condition. Detection of cancer cells was tested with cultured cell samples. HeLa and fibroblast cells, as cancer and normal cells respectively, were incubated with "smart" gold nanoparticles and measured with PT-OCT. An elevated OPL variation signal was detected with the HeLa cells while not much of a signal was detected with the fibroblast cells. With the novel optical property of "smart" AuNPs and high sensitivity of PT-OCT, this technique is promising for cancer cell detection.

Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues

International Nuclear Information System (INIS)

Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

1990-01-01

The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [ 32 P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32 P incorporation and the electrophoretic patterns were dependent on 32 P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K m values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins

Potent homocysteine-induced ERK phosphorylation in cultured neurons depends on self-sensitization via system Xc-

International Nuclear Information System (INIS)

Gu Li; Hu Xiaoling; Xue Zhanxia; Yang Jun; Wan Lishu; Ren Yan; Hertz, Leif; Peng Liang

2010-01-01

Homocysteine is increased during pathological conditions, endangering vascular and cognitive functions, and elevated homocysteine during pregnancy may be correlated with an increased incidence of schizophrenia in the offspring. This study showed that millimolar homocysteine concentrations in saline medium cause phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK 1/2 ) in cerebellar granule neurons, inhibitable by metabotropic but not ionotropic glutamate receptor antagonists. These findings are analogous to observations by , that similar concentrations cause neuronal death. However, these concentrations are much higher than those occurring clinically during hyperhomocysteinemia. It is therefore important that a ∼ 10-fold increase in potency occurred in the presence of the glutamate precursor glutamine, when ERK 1/2 phosphorylation became inhibitable by NMDA or non-NMDA antagonists and dependent upon epidermal growth factor (EGF) receptor transactivation. However, glutamate release to the medium was reduced, suggesting that reversal of the cystine/glutamate antiporter, system X c - could be involved in potentiation of the response by causing a localized release of initially accumulated homocysteine. In agreement with this hypothesis further enhancement of ERK 1/2 phosphorylation occurred in the additional presence of cystine. Pharmacological inhibition of system X c - prevented the effect of micromolar homocysteine concentrations, and U0126-mediated inhibition of ERK 1/2 phosphorylation enhanced homocysteine-induced death. In conclusion, homocysteine interacts with system X c - like quisqualate (Venkatraman et al. 1994), by 'self-sensitization' with initial accumulation and subsequent release in exchange with cystine and/or glutamate, establishing high local homocysteine concentrations, which activate adjacent ionotropic glutamate receptors and cause neurotoxicity.

[Interleukin-37 induces apoptosis and autophagy of SMMC-7721 cells by inhibiting phosphorylation of mTOR].

Science.gov (United States)

Li, Tingting; Zhu, Di; Mou, Tong; Guo, Zhen; Pu, Junliang; Wu, Zhongjun

2017-04-01

Objective To investigate the underlying mechanism by which interleukin-37 (IL-37) induces the apoptosis and autophagy in SMMC-7721 cells. Methods SMMC-7721 cells were incubated in vitro and divided into two groups, IL-37 treated group and control group. The cells were treated with (50, 100, 200) ng/mL of recombinant human interleukin-37 (rhIL-37). CCK-8 assay was used to detect the cell proliferation of SMMC-7721 cells. Cell apoptosis was measured by flow cytometry. Western blot analysis was performed to examine the expressions of apoptosis-related proteins, Bax, Bcl-2, and autophagy related proteins, microtubule-associated proteins 1 light chain 3 (LC3), beclin 1 and mammalian target of rapamycin (mTOR). Transmission electron microscopy (TEM) was used to observe the ultrastructures of autophagosomes. Results The rhIL-37 inhibited the proliferation of hepatocellular carcinoma SMMC-7721 cells. It induced the apoptosis and autophagy in SMMC-7721 cells. In the IL-37 treated group, the levels of Bax, LC3 and beclin 1 increased but Bcl-2 decreased. The phosphorylation of mTOR was inhibited in the IL-37 treated group. Autophagosome was obvious in the IL-37 treated group. Conclusion IL-37 induces the apoptosis and autophagy in SMMC-7721 cells, which may be related to the phosphorylation of mTOR.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

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Cho, Seong-Jun; Kang, Hana [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Kim, Min Young [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Pyo, Suhkneung [College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do (Korea, Republic of); Yang, Kwang Hee, E-mail: kwangheey@khnp.co.kr [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of)

2016-04-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a {sup 137}Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

Science.gov (United States)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-04-01

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast. Copyright © 2016 Elsevier Inc. All rights reserved.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

International Nuclear Information System (INIS)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-01-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a "1"3"7Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Mineral trioxide aggregate induces osteoblastogenesis via Atf6

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Toyonobu Maeda

2015-06-01

Full Text Available Mineral trioxide aggregate (MTA has been recommended for various uses in endodontics. To understand the effects of MTA on alveolar bone, we examined whether MTA induces osteoblastic differentiation using MC3T3-E1 cells. MTA enhanced mineralization concomitant with alkaline phosphatase activity in a dose- and time-dependent manner. MTA increased production of collagens (Type I and Type III and matrix metalloproteinases (MMP-9 and MMP-13, suggesting that MTA affects bone matrix remodeling. MTA also induced Bglap (osteocalcin but not Bmp2 (bone morphogenetic protein-2 mRNA expression. We observed induction of Atf6 (activating transcription factor 6, an endoplasmic reticulum (ER stress response transcription factor mRNA expression and activation of Atf6 by MTA treatment. Forced expression of p50Atf6 (active form of Atf6 markedly enhanced Bglap mRNA expression. Chromatin immunoprecipitation assay was performed to investigate the increase in p50Atf6 binding to the Bglap promoter region by MTA treatment. Furthermore, knockdown of Atf6 gene expression by introduction of Tet-on Atf6 shRNA expression vector abrogated MTA-induced mineralization. These results suggest that MTA induces in vitro osteoblastogenesis through the Atf6–osteocalcin axis as ER stress signaling. Therefore, MTA in endodontic treatment may affect alveolar bone healing in the resorbed region caused by pulpal infection.

Aggregation induced enhancement of linear and nonlinear optical emission from a hexaphenylene derivative

NARCIS (Netherlands)

Duan, Y.; Ju, C.G.; Yang, G.; Fron, E.; Coutino-Gonzalez, E.; Semin, S.V.; Fan, C.C.; Balok, R.S.; Cremers, J.; Tinnemans, P.T.; Feng, Y.Q.; Li, Y.L.; Hofkens, J.; Rowan, A.E.; Rasing, T.H.M.; Xu, J.L.

2016-01-01

The discovery of the phenomenon known as aggregation-induced emission (AIE) has opened the door to a variety of brilliant organic solid-state light-emitting materials. While AIE is well established in linear optics, the development of AIE luminogens (AIEgens) with highly efficient nonlinear optical

Lycium barbarum Polysaccharides Protect Rat Corneal Epithelial Cells against Ultraviolet B-Induced Apoptosis by Attenuating the Mitochondrial Pathway and Inhibiting JNK Phosphorylation

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Shaobo Du

2017-01-01

Full Text Available Lycium barbarum polysaccharides (LBPs have been shown to play a key role in protecting the eyes by reducing the apoptosis induced by certain types of damage. However, it is not known whether LBPs can protect damaged corneal cells from apoptosis. Moreover, no reports have focused on the role of LBPs in guarding against ultraviolet B- (UVB- induced apoptosis. The present study aimed to investigate the protective effect and underlying mechanism of LBPs against UVB-induced apoptosis in rat corneal epithelial (RCE cells. The results showed that LBPs significantly prevented the loss of cell viability and inhibited cell apoptosis induced by UVB in RCE cells. LBPs also inhibited UVB-induced loss of mitochondrial membrane potential, downregulation of Bcl-2, and upregulation of Bax and caspase-3. Finally, LBPs attenuated the phosphorylation of c-Jun NH2-terminal kinase (JNK triggered by UVB. In summary, LBPs protect RCE cells against UVB-induced damage and apoptosis, and the underlying mechanism involves the attenuation of the mitochondrial apoptosis pathway and the inhibition of JNK phosphorylation.

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

Science.gov (United States)

Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

2016-10-01

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

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Alexey Navdaev

Full Text Available von Willebrand factor/ristocetin (vWF/R induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

Science.gov (United States)

Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

2014-01-01

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

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Sebastian König

Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

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Fukuda Shin

2009-11-01

Full Text Available Abstract Background Theca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK. Methods Ovarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells. Results Akt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

International Nuclear Information System (INIS)

Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

2014-01-01

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation

Gingival tissue-produced inhibition of platelet aggregation and the loss of inhibition in streptozotocin-induced diabetic rats

Energy Technology Data Exchange (ETDEWEB)

Kawamura, Keiichiroh; Tamai, Kazuharu; Shirakawa, Masaharu; Okamoto, Hiroshi; Dohi, Toshihiro; Tsujimoto, Akira

1988-01-01

Addition of medium incubated with normal rat gingival tissue to platelet-rich plasma inhibited ADP-induced platelet aggregation. The ability of rat gingiva to produce activity inhibiting platelet aggregation was enhanced by the addition of arachidonic acid. Diabetic rat gingiva failed to inhibit platelet aggregation but did produce the anti-platelet aggregating activity in the presence of arachidonic acid. Indomethacin blocked the production of anti-platelet aggregating activity. There was no difference in conversion of (1-/sup 14/C)arachidonic acid to prostaglandins by normal and diabetic rat gingiva. These results suggest that an arachidonic acid metabolite released from gingiva during incubation inhibits platelet aggregation, and the synthesis of the metabolite is impaired in diabetic rat gingiva. A decrease in availability of arachidonic acid may be a causal factor of the defect in diabetic rat gingiva.

Gingival tissue-produced inhibition of platelet aggregation and the loss of inhibition in streptozotocin-induced diabetic rats

International Nuclear Information System (INIS)

Kawamura, Keiichiroh; Tamai, Kazuharu; Shirakawa, Masaharu; Okamoto, Hiroshi; Dohi, Toshihiro; Tsujimoto, Akira

1988-01-01

Addition of medium incubated with normal rat gingival tissue to platelet-rich plasma inhibited ADP-induced platelet aggregation. The ability of rat gingiva to produce activity inhibiting platelet aggregation was enhanced by the addition of arachidonic acid. Diabetic rat gingiva failed to inhibit platelet aggregation but did produce the anti-platelet aggregating activity in the presence of arachidonic acid. Indomethacin blocked the production of anti-platelet aggregating activity. There was no difference in conversion of [1- 14 C]arachidonic acid to prostaglandins by normal and diabetic rat gingiva. These results suggest that an arachidonic acid metabolite released from gingiva during incubation inhibits platelet aggregation, and the synthesis of the metabolite is impaired in diabetic rat gingiva. A decrease in availability of arachidonic acid may be a causal factor of the defect in diabetic rat gingiva. (author)

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

OpenAIRE

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2012-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substra...

Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

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Yu-Ting Huang

2007-12-01

Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

Glycogen synthase kinase 3-{beta} phosphorylates novel S/T-P-S/T domains in Notch1 intracellular domain and induces its nuclear localization

Energy Technology Data Exchange (ETDEWEB)

Han, Xiangzi [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Preventive Medicine, Yanbian University College of Medicine, Yanji (China); Ju, Ji-hyun [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of)

2012-06-29

Highlights: Black-Right-Pointing-Pointer Novel S/T-P-S/T domains were identified in NICD. Black-Right-Pointing-Pointer Phosphorylation of NICD on the S/T-P-S/T domains induced nuclear localization. Black-Right-Pointing-Pointer GSK-3{beta} phosphorylated S and T residues in NICD S/T-P-S/T domains. -- Abstract: We identified two S/T-P-S/T domains (2122-2124, 2126-2128) inducing Notch intracellular domain (NICD) nuclear localization. The GFP-NICD (1963-2145) fusion protein deletion mutant without classical NLS was localized in the nucleus like the full length GFP-NICD. However, quadruple substitution mutant (T2122A T2124A S2126A T2128A) showed increased cytoplasmic localization. GSK-3{beta} enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3{beta} phosphorylated S and T residues in NICD S/T-P-S/T domains. These results suggest that the novel S/T-P-S/T domain, phosphorylated by GSK-3{beta} is also involved in the nuclear localization of NICD as well as classical NLS.

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

Science.gov (United States)

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

Science.gov (United States)

Bakthisaran, Raman; Akula, Kranthi Kiran; Tangirala, Ramakrishna; Rao, Ch Mohan

2016-01-01

αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin

Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

Science.gov (United States)

Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

2014-06-01

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

Science.gov (United States)

Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

2015-02-01

Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data

Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

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Sebastian Gehlert

Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

Fabrication of fluorescent silica nanoparticles with aggregation-induced emission luminogens for cell imaging.

Science.gov (United States)

Chen, Sijie; Lam, Jacky W Y; Tang, Ben Zhong

2013-01-01

Fluorescence-based techniques have found wide applications in life science. Among various luminogenic materials, fluorescent nanoparticles have attracted much attention due to their fabulous emission properties and potential applications as sensors. Here, we describe the fabrication of fluorescent silica nanoparticles (FSNPs) containing aggregation-induced emission (AIE) luminogens. By employing surfactant-free sol-gel reaction, FSNPs with uniform size and high surface charge and colloidal stability are generated. The FSNPs emit strong light upon photoexcitation, due to the AIE characteristic of the silole -aggregates in the hybrid nanoparticles. The FSNPs are cytocompatible and can be utilized as fluorescent visualizer for intracellular imaging for HeLa cells.

Phototropism and Protein Phosphorylation in Higher Plants: Unilateral Blue Light Irradiation Generates a Directional Gradient of Protein Phosphorylation Across the Oat Coleoptile

International Nuclear Information System (INIS)

Salomon, M.; Zacherl, M.; Rüdiger, W.

1997-01-01

Blue light induces the phosphorylation of a 116 kDa oat protein found in plasma membrane preparations from coleoptile tips. We developed a very sensitive in vitro method that allowed us to determine the tissue distribution of protein phosphorylation after applying unilateral and bilateral blue light pulses in vivo. We found that following unilateral in vivo irradiation the degree in phosphorylation of the 116 kDa protein is significantly higher at the irradiated than at the shaded side of the coleoptile tip. This asymmetry can be considered as previously missing criterion that protein phosphorylation represents an early event within the transduction chain for phototropism. (author)

SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.

Science.gov (United States)

Thomas, J Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R; Moos, Malcolm

2016-01-01

In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Energy Technology Data Exchange (ETDEWEB)

Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

2012-11-01

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation. �

Novel hybrids of oxoisoaporphine-tryptamine as acetylcholinesterase-induced β-amyloid aggregation inhibitors with improved antioxidant properties

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Zhao Hai-Tao

2015-01-01

Full Text Available A series of dual binding site acetylcholinesterase (AChE inhibitors have been designed, synthesized, and tested for their antioxidant ability and inhibitory potency on AChE and AChE-induced b-amyloid (Ab aggregation. The new hybrids consist of a unit of 1-azabenzanthrone and a tryptamine or its derivative, connected through a a,w - alkyldiamide bridge. These hybrids exhibit moderate AChE inhibitory activity with IC50 values in the micromolar range and significant in vitro inhibitory activity toward the AChE-induced Ab aggregation. Moreover, six out of the nine hybrids of this series exhibit a higher oxygen radical absorbance capacity than trolox, which makes them promising anti-Alzheimer drug candidates.

Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Chung, Myung-Hoon; Kim, Do-Hee [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Na, Hye-Kyung [Department of Food and Nutrition, Sungshin Women' s University, Seoul (Korea, Republic of); Kim, Jung-Hwan; Kim, Ha-Na [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Haegeman, Guy [LEGEST, University of Gent (Belgium); Surh, Young-Joon, E-mail: surh@snu.ac.kr [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

2014-10-15

Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

Core Cross-Linked Multiarm Star Polymers with Aggregation-Induced Emission and Temperature Responsive Fluorescence Characteristics

KAUST Repository

Zhang, Zhen; Bilalis, Panagiotis; Zhang, Hefeng; Gnanou, Yves; Hadjichristidis, Nikolaos

2017-01-01

Aggregation-induced emission (AIE) active core cross-linked multiarm star polymers, carrying polystyrene (PS), polyethylene (PE), or polyethylene-b-polycaprolactone (PE-b-PCL) arms, have been synthesized through an “arm-first” strategy, by atom

Neurexin-1β Binding to Neuroligin-1 Triggers the Preferential Recruitment of PSD-95 versus Gephyrin through Tyrosine Phosphorylation of Neuroligin-1

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Grégory Giannone

2013-06-01

Full Text Available Adhesion between neurexin-1β (Nrx1β and neuroligin-1 (Nlg1 induces early recruitment of the postsynaptic density protein 95 (PSD-95 scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1β or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1β binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1 can be phosphorylated at a unique tyrosine (Y782, preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.

Activation of H2O2-induced VSOR Cl- currents in HTC cells require phospholipase Cgamma1 phosphorylation and Ca2+ mobilisation

DEFF Research Database (Denmark)

Varela, Diego; Simon, Felipe; Olivero, Pablo

2007-01-01

)R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCgamma1 activation and/or [Ca(2+)](i) rise, abolished H(2)O(2)-induced VSOR Cl(-) currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCgamma1, H(2)O(2) did not induce activation......Volume-sensitive outwardly rectifying (VSOR) Cl(-) channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H(2)O(2)) in VSOR Cl(-) channel...... activation. The aim of this study was to determine the signalling pathways responsible for H(2)O(2)-induced VSOR Cl(-) channel activation. In rat hepatoma (HTC) cells, H(2)O(2) elicited a transient increase in tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) that was blocked by PP2, a Src...

Synthesis of new N-heteroaromatic attached tetraphenylethene based luminogens having aggregation induced emission and their applications in organic light emitting diodes

Energy Technology Data Exchange (ETDEWEB)

Odabas, Serhat [Department of Chemistry, Middle East Technical University, 06800 Ankara (Turkey); The Scientific and Technological Research Council of Turkey (TUBITAK), Marmara Research Center, Institute of Chemical Technology, P.K. 21, 41470 Gebze, Kocaeli (Turkey); Tekin, Emine [The Scientific and Technological Research Council of Turkey (TUBITAK), Marmara Research Center, Material Institute, P.K. 21, 41470 Gebze, Kocaeli (Turkey); Turksoy, Figen, E-mail: figen.turksoy@tubitak.gov.tr [The Scientific and Technological Research Council of Turkey (TUBITAK), Marmara Research Center, Institute of Chemical Technology, P.K. 21, 41470 Gebze, Kocaeli (Turkey); Tanyeli, Cihangir, E-mail: tanyeli@metu.edu.tr [Department of Chemistry, Middle East Technical University, 06800 Ankara (Turkey)

2016-08-15

Aggregation induced emission is an auxiliary property that can eliminate the aggregation caused quenching effect. It is a common undesired emission characteristic of most bulky aromatic molecules. Tetraphenylethene, with its unique features, is the most commonly studied molecule having aggregation induced emission property. In this work four N heteroaromatic attached tetraphenylethene derivatives were synthesized via inexpensive C–N bond formation reactions. The synthesized luminogens showed good thermal properties, having high Td values between 366 °C and 505 °C. All four tetraphenylethene derivatives exhibited excellent aggregation induced emission properties and there was an up to 200-fold increase in their fluorescence intensities. According to the time-resolved emission decay experimental analyses, all four luminogens showed two relaxation pathways in a THF – water mixture (10–90%). Non-doped turquoise and light green OLED devices using the synthesized luminogens as a light emitting layer exhibited a maximum brightness up to 2600 cd/m{sup 2}, a maximum current efficiency up to 3.6 cd/A, turn-on voltages between 7.0 and 10.0 V, and external quantum efficiencies up to 1.5%. - Highlights: • A series of N-heterocyclic attached tetraphenylethene cored luminogens are synthesized and characterized. • Excellent thermal and aggregation induced emission properties are observed. • OLEDs are fabricated using synthesized luminogens as non-doped emitting layers.

Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

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Deng Xiaolu

2011-04-01

Full Text Available Abstract Background Tumor necrosis factor-α (TNF-α, a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC barrier dysfunction. Methods Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL. RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER. p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability. Results We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER. Conclusions Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF

Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells

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Magdalena Riedl

2017-01-01

Discussion: Therefore, our findings of (i neutrophils adhering to complement-activated endothelial cells, (ii the formation of neutrophil-platelet aggregates on endothelial cells, and (iii the ability of aHUS serum to induce similar effects identify a possible role for neutrophils in aHUS manifestation.

UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein

International Nuclear Information System (INIS)

Carty, M.P.; Zernik-Kobak, M.; McGrath, S.; Dixon, K.

1994-01-01

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus. (Author)

SB203580 Modulates p38 MAPK Signaling and Dengue Virus-Induced Liver Injury by Reducing MAPKAPK2, HSP27, and ATF2 Phosphorylation.

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Gopinathan Pillai Sreekanth

Full Text Available Dengue virus (DENV infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs, including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury.

Brain glucose transport and phosphorylation under acute insulin-induced hypoglycemia in mice: an 18F-FDG PET study.

Science.gov (United States)

Alf, Malte F; Duarte, João M N; Schibli, Roger; Gruetter, Rolf; Krämer, Stefanie D

2013-12-01

We addressed the questions of how cerebral glucose transport and phosphorylation change under acute hypoglycemia and what the underlying mechanisms of adaptation are. Quantitative (18)F-FDG PET combined with the acquisition of real-time arterial input function was performed on mice. Hypoglycemia was induced and maintained by insulin infusion. PET data were analyzed with the 2-tissue-compartment model for (18)F-FDG, and the results were evaluated with Michaelis-Menten saturation kinetics. Glucose clearance from plasma to brain (K1,glc) and the phosphorylation rate constant increased with decreasing plasma glucose (Gp), in particular at a Gp of less than 2.5 mmol/L. Estimated cerebral glucose extraction ratios taking into account an increased cerebral blood flow (CBF) at a Gp of less than 2 mmol/L were between 0.14 and 0.79. CBF-normalized K1,glc values were in agreement with saturation kinetics. Phosphorylation rate constants indicated intracellular glucose depletion at a Gp of less than 2-3 mmol/L. When brain regions were compared, glucose transport under hypoglycemia was lowest in the hypothalamus. Alterations in glucose transport and phosphorylation, as well as intracellular glucose depletion, under acute hypoglycemia can be modeled by saturation kinetics taking into account an increase in CBF. Distinct transport kinetics in the hypothalamus may be involved in its glucose-sensing function.

Multivalent scaffolds induce galectin-3 aggregation into nanoparticles

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Candace K. Goodman

2014-07-01

Full Text Available Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer–galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.

Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

Science.gov (United States)

Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

2015-07-01

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Involvement of nuclear factor κB in platelet CD40 signaling

International Nuclear Information System (INIS)

Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

2012-01-01

Highlights: ► sCD40L induces TRAF2 association to CD40 and NF-κB activation in platelets. ► IκBα phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. ► IκBα is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.

Lack of arginine vasopressin-induced phosphorylation of aquaporin-2 mutant AQP2-R254L explains dominant nephrogenic diabetes insipidus.

NARCIS (Netherlands)

Mattia, F.P. de; Savelkoul, P.J.M.; Kamsteeg, E.J.; Konings, I.B.M.; Sluijs, P. van der; Mallmann, R.; Oksche, A.; Deen, P.M.T.

2005-01-01

Water homeostasis in humans is regulated by vasopressin, which induces the translocation of homotetrameric aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells. For this process, phosphorylation of AQP2 at S256 by cAMP-dependent protein kinase

Strategy of ring-shaped aggregates in excitation energy transfer for removing disorder-induced shielding

International Nuclear Information System (INIS)

Tei, Go; Nakatani, Masatoshi; Ishihara, Hajime

2013-01-01

Peripheral light harvesting complex (LH2), which is found in photosynthetic antenna systems of purple photosynthetic bacteria, has important functions in the photosynthetic process, such as harvesting sunlight and transferring its energy to the photosynthetic reaction center. The key component in excitation energy transfer (EET) between LH2s is B850, which is a characteristic ring-shaped aggregate of pigments usually formed by 18 or 16 bacteriochlorophylls in LH2. We theoretically study the strategy of the ring-shaped aggregate structure, which maximizes EET efficiency, by using the standard Frenkel exciton model and the self-consistent calculation method for the Markovian quantum master equation and Maxwell equation. As a result, we have revealed a simple but ingenious strategy of the ring-shaped aggregate structure. The combination of three key properties of the ring unit system maximizes the EET efficiency, namely the large dipole moment of aggregates causes the basic improvement of EET efficiency, and the isotropic nature and the large occupying area are critically effective to remove the disorder-induced shielding that inhibits EET in the presence of the randomness of orientation and alignment of carriers of excitation energy. (paper)

Strategy of ring-shaped aggregates in excitation energy transfer for removing disorder-induced shielding

Science.gov (United States)

Tei, Go; Nakatani, Masatoshi; Ishihara, Hajime

2013-06-01

Peripheral light harvesting complex (LH2), which is found in photosynthetic antenna systems of purple photosynthetic bacteria, has important functions in the photosynthetic process, such as harvesting sunlight and transferring its energy to the photosynthetic reaction center. The key component in excitation energy transfer (EET) between LH2s is B850, which is a characteristic ring-shaped aggregate of pigments usually formed by 18 or 16 bacteriochlorophylls in LH2. We theoretically study the strategy of the ring-shaped aggregate structure, which maximizes EET efficiency, by using the standard Frenkel exciton model and the self-consistent calculation method for the Markovian quantum master equation and Maxwell equation. As a result, we have revealed a simple but ingenious strategy of the ring-shaped aggregate structure. The combination of three key properties of the ring unit system maximizes the EET efficiency, namely the large dipole moment of aggregates causes the basic improvement of EET efficiency, and the isotropic nature and the large occupying area are critically effective to remove the disorder-induced shielding that inhibits EET in the presence of the randomness of orientation and alignment of carriers of excitation energy.

Rapid and selective detection of cysteine based on its induced aggregates of cetyltrimethylammonium bromide capped gold nanoparticles

International Nuclear Information System (INIS)

Wang Jian; Li Yuanfang; Huang Chengzhi; Wu Tong

2008-01-01

A detection method of cysteine is reported in this contribution with water-soluble positively charged gold nanoparticles (Au-NPs) that were prepared by seed-mediated method and capped with cetyltrimethylammonium bromide (CTAB). In aqueous medium of pH 4.2, the CTAB-capped Au-NPs display greatly different features from those of generally prepared citrate-coated Au-NPs. It was found that in a medium of high salt concentration, the presence of cysteine could induce aggregation of CTAB-capped Au-NPs, while citrate-coated Au-NPs could get aggregation soon even if without the presence of cysteine. The cysteine-induced aggregates of CTAB-capped Au-NPs display strong plasmon resonance light scattering (PRLS) signals characterized at 566.0 nm when excited by a light beam, and the PRLS intensities of the aggregates are in proportion to the concentration of cysteine in the range of 0.01-0.40 μg mL -1 with the limit of detection (3σ) being 2.9 ng mL -1 . No amino acids in the samples interfere with the detection, and cysteine in artificial samples could be detected with the recovery between 95.3% and 105.9%, and R.S.D. is less than 3.6%

Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53

International Nuclear Information System (INIS)

O'Hagan, Heather M.; Ljungman, Mats

2004-01-01

It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation

Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: A study of DNAJB6 chaperone

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Rasha Mohamed Hussein

2015-07-01

Full Text Available Alzheimer’s disease is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly Q peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with α-synuclein at the single molecule level

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Nübling Georg

2012-07-01

Full Text Available Abstract Background Fibrillar amyloid-like deposits and co-deposits of tau and α-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood. Results We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3β exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers. Conclusions Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

Science.gov (United States)

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism.

Science.gov (United States)

Volovelsky, Oded; Cohen, Gili; Kenig, Ariel; Wasserman, Gilad; Dreazen, Avigail; Meyuhas, Oded; Silver, Justin; Naveh-Many, Tally

2016-04-01

Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6. Copyright © 2016 by the American Society of Nephrology.

ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

International Nuclear Information System (INIS)

Colonna, Cecilia; Podesta, Ernesto J.

2005-01-01

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics

N-acetyl-L-cysteine prevents stress-induced desmin aggregation in cellular models of desminopathy.

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Bertrand-David Segard

Full Text Available Mutations within the human desmin gene are responsible for a subcategory of myofibrillar myopathies called desminopathies. However, a single inherited mutation can produce different phenotypes within a family, suggesting that environmental factors influence disease states. Although several mouse models have been used to investigate organ-specific desminopathies, a more general mechanistic perspective is required to advance our knowledge toward patient treatment. To improve our understanding of disease pathology, we have developed cellular models to observe desmin behaviour in early stages of disease pathology, e.g., upon formation of cytoplasmic desmin aggregates, within an isogenic background. We cloned the wildtype and three mutant desmin cDNAs using a Tet-On Advanced® expression system in C2C12 cells. Mutations were selected based on positioning within desmin and capacity to form aggregates in transient experiments, as follows: DesS46Y (head domain; low aggregation, DesD399Y (central rod domain; high aggregation, and DesS460I (tail domain; moderate aggregation. Introduction of these proteins into a C2C12 background permitted us to compare between desmin variants as well as to determine the role of external stress on aggregation. Three different types of stress, likely encountered during muscle activity, were introduced to the cell models-thermal (heat shock, redox-associated (H2O2 and cadmium chloride, and mechanical (stretching stresses-after which aggregation was measured. Cells containing variant DesD399Y were more sensitive to stress, leading to marked cytoplasmic perinuclear aggregations. We then evaluated the capacity of biochemical compounds to prevent this aggregation, applying dexamethasone (an inducer of heat shock proteins, fisetin or N-acetyl-L-cysteine (antioxidants before stress induction. Interestingly, N-acetyl-L-cysteine pre-treatment prevented DesD399Y aggregation during most stress. N-acetyl-L-cysteine has recently been

Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

International Nuclear Information System (INIS)

Hill, Jennifer J.; Callaghan, Deborah A.; Ding Wen; Kelly, John F.; Chakravarthy, Balu R.

2006-01-01

Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in Brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3β, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is First report of a phosphorylation site on complexin 2

Aggregation of soy protein-isoflavone complexes and gel formation induced by glucono-δ-lactone in soymilk

Science.gov (United States)

Hsia, Sheng-Yang; Hsiao, Yu-Hsuan; Li, Wen-Tai; Hsieh, Jung-Feng

2016-10-01

This study investigated the glucono-δ-lactone (GDL)-induced aggregation of isoflavones and soy proteins in soymilk. High-performance liquid chromatography (HPLC) analysis indicated that isoflavones mixed with β-conglycinin (7S) and glycinin (11S) proteins formed 7S-isoflavone and 11S-isoflavone complexes in soymilk supernatant fraction (SSF). Most of the soy protein-isoflavone complexes then precipitated into the soymilk pellet fraction (SPF) following the addition of 4 mM GDL, whereupon the pH value of the soymilk dropped from 6.6 to 5.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis suggest that the addition of 4 mM GDL induced the aggregation of most 7S (α’, α and β subunits), 11S acidic and 11S basic proteins as well as isoflavones, including most aglycones, including daidzein, glycitein, genistein and a portion of glucosides, including daidzin, glycitin, genistin, malonyldaidzin and malonylgenistin. These results provide an important reference pertaining to the effects of GDL on the aggregation of soy protein-isoflavone complexes and could benefit future research regarding the production of tofu from soymilk.

Synthetic food additive dye "Tartrazine" triggers amorphous aggregation in cationic myoglobin.

Science.gov (United States)

Al-Shabib, Nasser Abdulatif; Khan, Javed Masood; Khan, Mohd Shahnawaz; Ali, Mohd Sajid; Al-Senaidy, Abdulrahman M; Alsenaidy, Mohammad A; Husain, Fohad Mabood; Al-Lohedan, Hamad A

2017-05-01

Protein aggregation, a characteristic of several neurodegenerative diseases, displays vast conformational diversity from amorphous to amyloid-like aggregates. In this study, we have explored the interaction of tartrazine with myoglobin protein at two different pHs (7.4 and 2.0). We have utilized various spectroscopic techniques (turbidity, Rayleigh light scattering (RLS), intrinsic fluorescence, Congo Red and far-UV CD) along with microscopy techniques i.e. atomic force microscopy (AFM) and transmission electron microscopy (TEM) to characterize the tartrazine-induced aggregation in myoglobin. The results showed that higher concentrations of tartrazine (2.0-10.0mM) induced amorphous aggregation in myoglobin at pH 2.0 via electrostatic interactions. However, tartrazine was not able to induce aggregation in myoglobin at pH 7.4; because of strong electrostatic repulsion between myoglobin and tartrazine at this pH. The tartrazine-induced amorphous aggregation process is kinetically very fast, and aggregation occurred without the formation of a nucleus. These results proposed that the electrostatic interaction is responsible for tartrazine-induced amorphous aggregation. This study may help in the understanding of mechanistic insight of aggregation by tartrazine. Copyright © 2017 Elsevier B.V. All rights reserved.

Reconstruction and analysis of nutrient-induced phosphorylation networks in Arabidopsis thaliana.

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Guangyou eDuan

2013-12-01

Full Text Available Elucidating the dynamics of molecular processes in living organisms in response to external perturbations is a central goal in modern systems biology. We investigated the dynamics of protein phosphorylation events in Arabidopsis thaliana exposed to changing nutrient conditions. Phosphopeptide expression levels were detected at five consecutive time points over a time interval of 30 minutes after nutrient resupply following prior starvation. The three tested inorganic, ionic nutrients NH4+, NO3-, PO43- elicited similar phosphosignaling responses that were distinguishable from those invoked by the sugars mannitol, sucrose. When embedded in the protein-protein interaction network of Arabidopsis thaliana, phosphoproteins were found to exhibit a higher degree compared to average proteins. Based on the time-series data, we reconstructed a network of regulatory interactions mediated by phosphorylation. The performance of different network inference methods was evaluated by the observed likelihood of physical interactions within and across different subcellular compartments and based on gene ontology semantic similarity. The dynamic phosphorylation network was then reconstructed using a Pearson correlation method with added directionality based on partial variance differences. The topology of the inferred integrated network corresponds to an information dissemination architecture, in which the phosphorylation signal is passed on to an increasing number of phosphoproteins stratified into an initiation, processing, and effector layer. Specific phosphorylation peptide motifs associated with the distinct layers were identified indicating the action of layer-specific kinases. Despite the limited temporal resolution, combined with information on subcellular location, the available time-series data proved useful for reconstructing the dynamics of the molecular signaling cascade in response to nutrient stress conditions in the plant Arabidopsis thaliana.

Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-1791

Science.gov (United States)

Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

2015-01-01

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non–small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1–induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1–mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1–mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1–induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1–mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1–induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1–induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

Phosphorylation-induced conformational changes in short peptides probed by fluorescence resonance energy transfer in the 10A domain.

Science.gov (United States)

Sahoo, Harekrushna; Nau, Werner M

2007-03-26

Phosphorylation-induced conformational changes in short polypeptides were probed by a fluorescence resonance energy transfer (FRET) method by employing a short-distance FRET pair (R(0) approximately 10 A) based on tryptophan as natural donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as synthetic acceptor. Two substrates for kinases, LeuArgArgTrpSerLeuGly-Dbo (peptide I) and TrpLysArgThrLeuArgArg-Dbo (peptide II), were investigated, with serine and threonine, respectively, as phosphorylation sites. Steady-state and time-resolved fluorescence experiments in H(2)O revealed a decrease in FRET efficiency for peptide I and an increase for peptide II; this suggested that the effective distances between donor and acceptor increased and decreased, respectively. The same trends and similar absolute variations in effective donor-acceptor distances were observed in propylene glycol, a less polar and highly viscous solvent; this suggested that the variations are due to intrinsic structural preferences. Fitting of the time-resolved decay traces according to a distribution function model (Gaussian distribution) provided the mean donor-acceptor distances, which showed an increase upon phosphorylation for peptide I (from 9.7 to 10.5 A) and a decrease for peptide II (from 10.9 to 9.3 A) in H(2)O. The broadness (half-width) of the distributions, which provides a measure of the rigidity of the peptides, remained similar upon phosphorylation of peptide I (3.0 versus 3.1 A), but decreased for peptide II (from 3.1 to 0.73 A in H(2)O); this suggests a more compact, structured conformation upon phosphorylation of the latter peptide. The elongation of the peptide backbone (by ca. 0.7 A) for peptide I is attributed to an increase in steric demand upon phosphorylation, which favors an extended conformation. The contraction (by ca. 1.4 A) and structural rigidification of peptide II is attributed to attractive Coulombic interactions and hydrogen bonding between the

Silicification-induced cell aggregation for the sustainable production of H2 under aerobic conditions.

Science.gov (United States)

Xiong, Wei; Zhao, Xiaohong; Zhu, Genxing; Shao, Changyu; Li, Yaling; Ma, Weimin; Xu, Xurong; Tang, Ruikang

2015-10-05

Photobiological hydrogen production is of great importance because of its promise for generating clean renewable energy. In nature, green algae cannot produce hydrogen as a result of the extreme sensitivity of hydrogenase to oxygen. However, we find that silicification-induced green algae aggregates can achieve sustainable photobiological hydrogen production even under natural aerobic conditions. The core-shell structure of the green algae aggregates creates a balance between photosynthetic electron generation and hydrogenase activity, thus allowing the production of hydrogen. This finding provides a viable pathway for the solar-driven splitting of water into hydrogen and oxygen to develop green energy alternatives by using rationally designed cell-material complexes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SCO2 induces p53-mediated apoptosis by Thr845 phosphorylation of ASK-1 and dissociation of the ASK-1-Trx complex.

Science.gov (United States)

Madan, Esha; Gogna, Rajan; Kuppusamy, Periannan; Bhatt, Madan; Mahdi, Abbas Ali; Pati, Uttam

2013-04-01

p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

Cholesterol impairment contributes to neuroserpin aggregation

Science.gov (United States)

Giampietro, Costanza; Lionetti, Maria Chiara; Costantini, Giulio; Mutti, Federico; Zapperi, Stefano; La Porta, Caterina A. M.

2017-03-01

Intraneural accumulation of misfolded proteins is a common feature of several neurodegenerative pathologies including Alzheimer’s and Parkinson’s diseases, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). FENIB is a rare disease due to a point mutation in neuroserpin which accelerates protein aggregation in the endoplasmic reticulum (ER). Here we show that cholesterol depletion induced either by prolonged exposure to statins or by inhibiting the sterol reg-ulatory binding-element protein (SREBP) pathway also enhances aggregation of neuroserpin proteins. These findings can be explained considering a computational model of protein aggregation under non-equilibrium conditions, where a decrease in the rate of protein clearance improves aggregation. Decreasing cholesterol in cell membranes affects their biophysical properties, including their ability to form the vesicles needed for protein clearance, as we illustrate by a simple mathematical model. Taken together, these results suggest that cholesterol reduction induces neuroserpin aggregation, even in absence of specific neuroserpin mutations. The new mechanism we uncover could be relevant also for other neurodegenerative diseases associated with protein aggregation.

Lactobacillus rhamnosus GG (ATCC 53103) and platelet aggregation in vitro.

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Korpela, R; Moilanen, E; Saxelin, M; Vapaatalo, H

1997-06-17

Lactobacillus rhamnosus GG is an experimentally and clinically well documented probiotic used in different dairy products. The present study aimed to investigate the safety aspects of Lactobacillus rhamnosus GG, particularly with respect to platelet aggregation, the initiating event in thrombosis. Platelet rich plasma was separated from the blood of healthy volunteers, and the effects of Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus rhamnosus (ATCC 7469) and Enterococcus faecium T2L6 in different dilutions on spontaneous, ADP- and adrenaline-induced aggregation were tested. The bacteria did not influence spontaneous aggregation. Only Enterococcus faecium T2L6 enhanced the adrenaline-induced aggregation, with a less clear effect on ADP-induced aggregation.

Synthesis and Properties of Gelators Derived from Tetraphenylethylene and Gallic Acid with Aggregation-Induced Emission

Science.gov (United States)

Luo, Miao; Zhou, Xie; Chi, Zhenguo; Ma, Chunping; Zhang, Yi; Liu, Siwei; Xu, Jiarui

2013-09-01

Two novel organogelators (TEG and TAG) based on tetraphenylethylene and 3,4,5-tris(dodecyloxy) benzoic acid were synthesized through ester bond and amido bond linkages, respectively. Compounds TEG and TAG were able to induce gelation in ethanol. Aggregation-induced enhanced emission was observed in these organogelator molecules, with increased fluorescence intensity from the solutions to the gels. The completely thermoreversible gelation occurred due to the aggregation of the organogelators. In the process, a fibrous network was formed by a combination of intermolecular hydrogen bonding, π-π stacking and van der Waals interactions. These phenomena were observed in the xerogels by field-emission scanning electron microscopy and Fourier-transform infrared spectroscopy. The results of differential scanning calorimetry and polarized optical microscopy indicated that compound TAG exhibited stable liquid crystalline phases over a wide temperature range. The linking groups have severe influence on the properties of the organogelators, which was mainly attributed to the hydrogen bonding interaction in compound TAG.

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

International Nuclear Information System (INIS)

Martin, S.A.; Russell, J.B.

1987-01-01

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments, indicated that separate phosphotransferases systems existed for glucose, maltose, and sucrose. [ 14 C]maltose transport was inhibited by excess glucose and to a lesser extent by sucrose. [ 14 C]glucose and [ 14 C]sucrose transports were not inhibited by an excess of maltose. Since [ 14 C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of P/sub i/ was increased from 0 to 100 mM, a maltose phosphorylase was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for α-glucose 1-phosphate. Only sucrose-grown cells possessed sucrose hydrolase activity, and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities

Axin-mediated CKI phosphorylation of beta-catenin at Ser 45

DEFF Research Database (Denmark)

Amit, Sharon; Hatzubai, Ada; Birman, Yaara

2002-01-01

The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin...... and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta......-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta...

Phosphorylation-induced changes in the energetic frustration in human Tank binding kinase 1.

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Husain, Shahrukh; Kumar, Vijay; Hassan, Md Imtaiyaz

2018-07-14

Tank binding kinase 1 (TBK-1) plays an important role in immunity, inflammation, autophagy, cell growth and proliferation. Nevertheless, a key molecular and structural detail of TBK-1 phosphorylation and activation has been largely unknown. Here we investigated the energy landscape of phosphorylated (active) and unphosphorylated (inactive) forms of human TBK-1 to characterize the interplay between phosphorylation and local frustration. By employing the algorithm equipped with energy function and implemented in Frustratometer web-server (http://www.frustratometer.tk), we quantify the role of frustration in the activation of TBK-1. Accordingly, the conformational changes were observed in phosphoregulated active and inactive TBK-1. Substantial changes in frustration, flexibility and interatomic motions were observed among different forms of TBK-1. Structurally rigid kinase domain constitutes a minimally frustrated hub in the core of the catalytic domain, and highly frustrated clusters mainly at the C-lobe might enable the conformational transitions during activation. Also, a large network of highly frustrated interactions is found in the SDD domain of TBK-1 involved in protein-protein interactions and dimerization. The contact maps of the activation loop and α-C helix of kinase domain showed significant changes upon phosphorylation. Cross correlation analysis indicate that both intra and inter subunit correlated motions increases with phosphorylation of TBK-1. Phosphorylation thus introduces subtle changes in long-range contacts that might lead to significant conformational change of TBK-1. Copyright © 2018 Elsevier Ltd. All rights reserved.

Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

International Nuclear Information System (INIS)

Gupta, Preeti; Deep, Shashank

2014-01-01

Highlights: • HCAII forms amyloid-like aggregates at moderate concentration of trifluoroethanol. • Protein adopts a state between β-sheet and α-helix at moderate % of TFE. • Hydrophobic surface(s) of partially structured conformation forms amyloid. • High % of TFE induces stable α-helical state preventing aggregation. - Abstract: In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII

Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study.

Science.gov (United States)

Hubbard, Gary P; Wolffram, Siegfried; de Vos, Ric; Bovy, Arnaud; Gibbins, Jonathan M; Lovegrove, Julie A

2006-09-01

Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertaken. Subjects ingested a soup containing either a high or a low amount of quercetin. Plasma quercetin concentrations and platelet aggregation and signalling were assessed after soup ingestion. The high-quercetin soup contained 69 mg total quercetin compared with the low-quercetin soup containing 5 mg total quercetin. Plasma quercetin concentrations were significantly higher after high-quercetin soup ingestion than after low-quercetin soup ingestion and peaked at 2.59 (sem 0.42) mumol/l. Collagen-stimulated (0.5 mug/ml) platelet aggregation was inhibited after ingestion of the high-quercetin soup in a time-dependent manner. Collagen-stimulated tyrosine phosphorylation of a key component of the collagen-signalling pathway via glycoprotein VI, Syk, was significantly inhibited by ingestion of the high-quercetin soup. The inhibition of Syk tyrosine phosphorylation was correlated with the area under the curve for the high-quercetin plasma profile. In conclusion, the ingestion of quercetin from a dietary source of onion soup could inhibit some aspects of collagen-stimulated platelet aggregation and signalling ex vivo. This further substantiates the epidemiological data suggesting that those who preferentially consume high amounts of quercetin-containing foods have a reduced risk of thrombosis and potential CVD risk.

Phosphorylation of ETS-1 is a critical event in DNA polymerase iota-induced invasion and metastasis of esophageal squamous cell carcinoma.

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He, Chao; Wu, Shuhua; Gao, Aidi; Su, Ye; Min, Han; Shang, Zeng-Fu; Wu, Jinchang; Yang, Li; Ding, Wei-Qun; Zhou, Jundong

2017-12-01

An aberrantly elevated expression of DNA polymerase ι (Pol ι) is significantly associated with poor prognosis of patients with esophageal squamous cell carcinoma (ESCC), yet the mechanisms behind this phenomenon remain obscure. Based on the RNA-Seq transcriptome and real-time PCR analysis, we identified ETS-1 as a candidate gene involved in Pol ι-mediated progression of ESCC. Wound-healing and transwell assay indicated that downregulation of ETS-1 attenuates Pol ι-mediated invasiveness of ESCC. Signaling pathway analysis showed that Pol ι enhances ETS-1 phosphorylation at threonine-38 through the Erk signaling pathway in ESCC cells. Kaplan-Meier analysis, based on 93 clinical tissue samples, revealed that ETS-1 phosphorylation at threonine-38 is associated with poor prognosis of ESCC patients. The present study thus demonstrates that phosphorylation of ETS-1 is a critical event in the Pol ι-induced invasion and metastasis of ESCC. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Pyrene-Phosphonate Conjugate: Aggregation-Induced Enhanced Emission, and Selective Fe3+ Ions Sensing Properties

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Sachin D. Padghan

2017-08-01

Full Text Available A new pyrene-phosphonate colorimetric receptor 1 has been designed and synthesized in a one-step process via amide bond formation between pyrene butyric acid chloride and phosphonate-appended aniline. The pyrene-phosphonate receptor 1 showed aggregation-induced enhanced emission (AIEE properties in water/acetonitrile (ACN solutions. Dynamic light scattering (DLS characterization revealed that the aggregates of receptor 1 at 80% water fraction have an average size of ≈142 nm. Field emission scanning electron microscopy (FE-SEM analysis confirmed the formation of spherical aggregates upon solvent evaporation. The sensing properties of receptor 1 were investigated by UV-vis, fluorescence emission spectroscopy, and other optical methods. Among the tested metal ions, receptor 1 is capable of recognizing the Fe3+ ion selectively. The changes in spectral measurements were explained on the basis of complex formation. The composition of receptor 1 and Fe3+ ions was determined by using Job’s plot and found to be 1:1. The receptor 1–Fe3+ complex showed a reversible UV-vis response in the presence of EDTA.

γ-aminobutyric acidA (GABAA) receptor regulates ERK1/2 phosphorylation in rat hippocampus in high doses of Methyl Tert-Butyl Ether (MTBE)-induced impairment of spatial memory

International Nuclear Information System (INIS)

Zheng Gang; Zhang Wenbin; Zhang Yun; Chen Yaoming; Liu Mingchao; Yao Ting; Yang Yanxia; Zhao Fang; Li Jingxia; Huang Chuanshu; Luo Wenjing; Chen Jingyuan

2009-01-01

Experimental and occupational exposure to Methyl Tert-Butyl Ether (MTBE) has been reported to induce neurotoxicological and neurobehavioral effects, such as headache, nausea, dizziness, and disorientation, etc. However, the molecular mechanisms involved in MTBE-induced neurotoxicity are still not well understood. In the present study, we investigated the effects of MTBE on spatial memory and the expression and function of GABA A receptor in the hippocampus. Our results demonstrated that intraventricular injection of MTBE impaired the performance of the rats in a Morris water maze task, and significantly increased the expression of GABA A receptor α1 subunit in the hippocampus. The phosphorylation of ERK1/2 decreased after the MTBE injection. Furthermore, the decreased ability of learning and the reduction of phosphorylated ERK1/2 level of the MTBE-treated rats was partly reversed by bicuculline injected 30 min before the training. These results suggested that MTBE exposure could result in impaired spatial memory. GABA A receptor may play an important role in the MTBE-induced impairment of learning and memory by regulating the phosphorylation of ERK in the hippocampus.

Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling.

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Chen, Xiaoyu; Bai, Bo; Tian, Yanjun; Du, Hui; Chen, Jing

2014-11-07

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

International Nuclear Information System (INIS)

Jung, Th.; Streffer, C.

1992-01-01

To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G 2 block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G 2 block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author)

Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

Energy Technology Data Exchange (ETDEWEB)

Jung, Th. (AFRC Institute of Animal Physiology and Genetics Research, Babraham (United Kingdom)); Streffer, C. (Essen Univ (Germany). Inst. fuer Medizinische Strahlenbiolgie)

1992-08-01

To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G[sub 2] block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G[sub 2] block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author).

Spontaneous and Induced Platelet Aggregation during Pregnancy and Labor

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T. P. Bondar

2016-01-01

Full Text Available Objective: to evaluate changes in characteristics of spontaneous platelet (Pt aggregation in patients with obstetric complications associated with hereditary thrombophilia.Materials and methods. Blood samples were taken from 52 recently confined women on the first day after labor; at that, ethic regulations for the preanalytical phase were followed. Determination of PlA1/ PlA2 polymorphism enotype was performed by means of amplificationrestriction analysis. Geometrical characteristics of patients' peripheral blood Pt aggregation were studied by means of AFM Integra Prima. The degree of confidence of the parameters under test was determined using the ttest, and the significance level was considered valid at P<0.05.Results. A statistical analysis of the findings demonstrated that the length of Pt aggregates in healthy pregnant women was significantly higher than that in healthy nonpregnant women at all study phases. Patients with the P1A1/P1A2 polymorphism in the GP IIb/IIIa Pt receptor gene demonstrated increased widthm height, and density of Pt aggregates. The changes were most significant during the incubation phase lasting for 15 and 30 minutes. The study of geometric parameters of different exposures demonstrated the following: the longer the incubation period, the greater the difference between geometric parameters of the aggregates (e.g. height, length, and width. Conclusion. The analysis of obtained data demonstrated that the presence of P1A1/P1A2 polymorphism in GP IIb/IIIa Pt gene receptor contributes to the decrease in the platelet response threshold and enhances the spontaneous Pt aggregation. The imaging of aggregates provides strong evidence for the accelerated growth of the aggregates in thrombotic complications of pregnancy.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

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Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Size- and time-dependent growth properties of human induced pluripotent stem cells in the culture of single aggregate.

Science.gov (United States)

Nath, Suman C; Horie, Masanobu; Nagamori, Eiji; Kino-Oka, Masahiro

2017-10-01

Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 10 1 -(2.8 ± 0.5) × 10 1 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 10 1 -(6.8 ± 1.1) × 10 2 cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 10 3 -(3.5 ± 1.1) × 10 3 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

International Nuclear Information System (INIS)

Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar; Lang, Florian

2012-01-01

Highlights: ► Akt/SGK dependent phosphorylation of GSK3α,β regulates T lymphocytes. ► T cells from mice expressing Akt/SGK insensitive GSK3α,β (gsk3 KI ) release less IL-2. ► CD4 + cells from gsk3 KI mice express less CD62L. ► CD8 + cells from gsk3 KI mice are relatively resistant to activation induced cell death. ► Perforin expression is enhanced in gsk3 KI T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3α,β. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3α,β inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3 KI ). T cells from gsk3 KI mice were compared to T cells from corresponding wild type mice (gsk3 WT ). As a result, in gsk3 KI CD4 + cells surface CD62L (L-selectin) was significantly less abundant than in gsk3 WT CD4 + cells. Upon activation in vitro T cells from gsk3 KI mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3 KI T cells, suggesting that GSK3 induces effector function in CD8 + T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3α,β is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

Involvement of nuclear factor {kappa}B in platelet CD40 signaling

Energy Technology Data Exchange (ETDEWEB)

Hachem, Ahmed [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Yacoub, Daniel [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Zaid, Younes [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Mourad, Walid [Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Merhi, Yahye, E-mail: yahye.merhi@icm-mhi.org [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada)

2012-08-17

Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Given that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo

Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

Science.gov (United States)

Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

2009-06-01

Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

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Zhou Bihong

2011-02-01

Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2.

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Ambarish Nag

Full Text Available The linker for activation of T cells (LAT, the linker for activation of B cells (LAB, and the linker for activation of X cells (LAX form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate. We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

Therapeutic effect of methyl salicylate 2-O-β-d-lactoside on LPS-induced acute lung injury by inhibiting TAK1/NF-kappaB phosphorylation and NLRP3 expression.

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Yang, Shengqian; Yu, Ziru; Yuan, Tianyi; Wang, Lin; Wang, Xue; Yang, Haiguang; Sun, Lan; Wang, Yuehua; Du, Guanhua

2016-11-01

Acute lung injury (ALI), characterized by pulmonary edema and inflammatory cell infiltration, is a common syndrome of acute hypoxemic respiratory failure. Methyl salicylate 2-O-β-d-lactoside (MSL), a natural derivative of salicylate extracted from Gaultheria yunnanensis (Franch.) Rehder, was reported to have potent anti-inflammatory effects on the progression of collagen or adjuvant-induced arthritis in vivo and in vitro. The aim of this study is to investigate the therapeutic effect of MSL on lipopolysaccharide (LPS)-induced acute lung injury and reveal underlying molecular mechanisms. Our results showed that MSL significantly ameliorated pulmonary edema and histological severities, and inhibited IL-6 and IL-1β production in LPS-induced ALI mice. MSL also reduced MPO activity in lung tissues and the number of inflammatory cells in BALF. Moreover, we found that MSL significantly inhibited LPS-induced TAK1 and NF-κB p65 phosphorylation, as well as the expression of NLRP3 protein in lung tissues. Furthermore, MSL significantly inhibited LPS-induced TAK1 and NF-κB p65 phosphorylation in Raw264.7 cells. In addition, MSL significantly inhibited nuclear translocation of NF-κB p65 in cells treated with LPS in vitro. Taken together, our results suggested that MSL exhibited a therapeutic effect on LPS-induced ALI by inhibiting TAK1/NF-κB phosphorylation and NLRP3 expression. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

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Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

2009-11-30

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

The influence of N-terminal acetylation on micelle-induced conformational changes and aggregation of α-Synuclein.

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David Ruzafa

Full Text Available The biological function of α-Synuclein has been related to binding to lipids and membranes but these interactions can also mediate α-Synuclein aggregation, which is associated to Parkinson's disease and other neuropathologies. In brain tissue α-Synuclein is constitutively N-acetylated, a modification that plays an important role in its conformational propensity, lipid and membrane binding, and aggregation propensity. We studied the interactions of the lipid-mimetic SDS with N-acetylated and non-acetylated α-Synuclein, as well as their early-onset Parkinson's disease variants A30P, E46K and A53T. At low SDS/protein ratios α-Synuclein forms oligomeric complexes with SDS micelles with relatively low α-helical structure. These micellar oligomers can efficiently nucleate aggregation of monomeric α-Synuclein, with successive formation of oligomers, protofibrils, curly fibrils and mature amyloid fibrils. N-acetylation reduces considerably the rate of aggregation of WT α-Synuclein. However, in presence of any of the early-onset Parkinson's disease mutations the protective effect of N-acetylation against micelle-induced aggregation becomes impaired. At higher SDS/protein ratios, N-acetylation favors another conformational transition, in which a second type of α-helix-rich, non-aggregating oligomers become stabilized. Once again, the Parkinson's disease mutations disconnect the influence of N-acetylation in promoting this transition. These results suggest a cooperative link between the N-terminus and the region of the mutations that may be important for α-Synuclein function.

Suppression of Kasha's rule as a mechanism for fluorescent molecular rotors and aggregation-induced emission

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Qian, Hai; Cousins, Morgan E.; Horak, Erik H.; Wakefield, Audrey; Liptak, Matthew D.; Aprahamian, Ivan

2017-01-01

Although there are some proposed explanations for aggregation-induced emission, a phenomenon with applications that range from biosensors to organic light-emitting diodes, current understanding of the quantum-mechanical origin of this photophysical behaviour is limited. To address this issue, we assessed the emission properties of a series of BF2-hydrazone-based dyes as a function of solvent viscosity. These molecules turned out to be highly efficient fluorescent molecular rotors. This property, in addition to them being aggregation-induced emission luminogens, enabled us to probe deeper into their emission mechanism. Time-dependent density functional theory calculations and experimental results showed that the emission is not from the S1 state, as predicted from Kasha's rule, but from a higher energy (>S1) state. Furthermore, we found that suppression of internal conversion to the dark S1 state by restricting the rotor rotation enhances fluorescence, which leads to the proposal that suppression of Kasha's rule is the photophysical mechanism responsible for emission in both viscous solution and the solid state.

Regulation of the autophagy protein LC3 by phosphorylation

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Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

2010-01-01

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

Novel Role of Src in Priming Pyk2 Phosphorylation.

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Ming Zhao

Full Text Available Proline-rich tyrosine kinase 2 (Pyk2 is a member of the focal adhesion kinase (FAK family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

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Vendrell, Iolanda; Carrascal, Montserrat; Campos, Francisco; Abian, Joaquin; Sunol, Cristina

2010-01-01

Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 ± 199, 345 ± 47, and 243 ± 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 ± 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation

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Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung; Rhee, Man Hee; Kim, Yun-Bae

2013-01-01

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-depende...

Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

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Kumari Ratna

2010-07-01

Full Text Available Abstract Background p53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5 is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment. Results To determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well. Conclusion Present study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

Transcutaneous electrical nerve stimulation on Yongquan acupoint reduces CFA-induced thermal hyperalgesia of rats via down-regulation of ERK2 phosphorylation and c-Fos expression.

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Yang, Lin; Yang, Lianxue; Gao, Xiulai

2010-07-01

Activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and its involvement in regulating gene expression in spinal dorsal horn, cortical and subcortical neurons by peripheral noxious stimulation contribute to pain hypersensitivity. Transcutaneous electrical nerve stimulation (TENS) is a treatment used in physiotherapy practice to promote analgesia in acute and chronic inflammatory conditions. In this study, a total number of 114 rats were used for three experiments. Effects of complete Freund's adjuvant (CFA)-induced inflammatory pain hypersensitivity and TENS analgesia on ERK1/2 phosphorylation and c-Fos protein expression were examined by using behavioral test, Western blot, and immunostaining methods. We found that CFA injection caused an area of localized swelling, erythema, hypersensitivity to thermal stimuli, the decreased response time of hind paw licking (HPL), as well as upregulation of c-Fos protein expression and ERK2 phosphorylation in the ipsilateral spinal dorsal horn and the contralateral primary somatosensory area of cortex and the amygdala of rats. TENS on Yongquan acupoint for 20 min produced obvious analgesic effects as demonstrated with increased HPL to thermal stimuli of CFA-treated rats. In addition, TENS application suppressed the CFA-induced ERK2 activation and c-Fos protein expression. These results suggest that down-regulation of ERK2 phosphorylation and c-Fos expression were involved in TENS inhibition on CFA-induced thermal hyperalgesia of rats.

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells.

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Kim, Jong-Sik; Baek, Seung Joon; Bottone, Frank G; Sali, Tina; Eling, Thomas E

2005-09-01

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.

Intrathecal administration of rapamycin inhibits the phosphorylation of DRG Nav1.8 and attenuates STZ-induced painful diabetic neuropathy in rats.

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He, Wan-You; Zhang, Bin; Xiong, Qing-Ming; Yang, Cheng-Xiang; Zhao, Wei-Cheng; He, Jian; Zhou, Jun; Wang, Han-Bing

2016-04-21

The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation and protein synthesis, and it is specifically inhibited by rapamycin. In chronic pain conditions, mTOR-mediated local protein synthesis is crucial for neuronal hyperexcitability and synaptic plasticity. The tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8 plays a major role in action potential initiation and propagation and cellular excitability in DRG (dorsal root ganglion) neurons. In this study, we investigated if mTOR modulates the phosphorylation of Nav1.8 that is associated with neuronal hyperexcitability and behavioral hypersensitivity in STZ-induced diabetic rats. Painful diabetic neuropathy (PDN) was induced in Sprague-Dawley rats by intraperitoneal injection with streptozotocin (STZ) at 60mg/kg. After the onset of PDN, the rats received daily intrathecal administrations of rapamycin (1μg, 3μg, or 10μg/day) for 7 days; other diabetic rats received the same volumes of dimethyl sulfoxide (DMSO). Herein, we demonstrate a marked increase in protein expression of total mTOR and phospho-mTOR (p-mTOR) together with the up-regulation of phosphor-Nav1.8 (p-Nav1.8) prior to the mechanical withdrawal threshold reaching a significant reduction in dorsal root ganglions (DRGs). Furthermore, the intrathecal administration of rapamycin, inhibiting the activity of mTOR, suppressed the phosphorylation of DRG Nav1.8, reduced the TTX-R current density, heightened the voltage threshold for activation and lowered the voltage threshold for inactivation and relieved mechanical hypersensitivity in diabetic rats. An intrathecal injection (i.t.) of rapamycin inhibited the phosphorylation and enhanced the functional availability of DRG Nav1.8 attenuated STZ-induced hyperalgesia. These results suggest that rapamycin is a potential therapeutic intervention for clinical PDN. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Macrophage triggering by aggregated immunoglobulins. II. Comparison of IgE and IgG aggregates or immune complexes.

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Pestel, J; Dessaint, J P; Joseph, M; Bazin, H; Capron, A

1984-01-01

Macrophages incubated with complexed or aggregated IgE released beta-glucuronidase (beta-G) within 30 min. In contrast in the presence of aggregated or complexed IgG, macrophages liberated equivalent amount of beta-G only after 6 h incubation. In addition the rapid macrophage stimulation induced by aggregated IgE was also followed by a faster 3H-glucosamine incorporation when compared to the delayed activation caused by aggregated IgG. However, macrophages stimulated either by IgG or by IgE oligomers produced the same percentage of plasminogen activator at 24 h. In contrast, while the interaction between macrophages and aggregated IgE was only followed by a peak of cyclic GMP and a beta-G release during the first 30 min of incubation, the interaction between macrophages and IgG oligomers was accompanied by a simultaneous increase of cyclic GMP and AMP nucleotides and by an absence of beta-G exocytosis. Moreover, the beta-G release induced by aggregated IgE was increased when macrophages were preincubated with aggregated IgG. This additive effect was not observed in the reverse situation. Finally macrophages activated by IgG oligomers were demonstrated to exert a cytotoxic effect on tumour cells and to kill schistosomula in the presence of a low level of complement. Taken together these results underline the peculiar ability of aggregated or complexed IgE to trigger rapidly the macrophage activation compared to aggregated IgG and can explain the important role of complexed IgE in some macrophage dependent cytotoxicity mechanisms (i.e. in parasitic diseases). PMID:6088135

NMR of α-synuclein–polyamine complexes elucidates the mechanism and kinetics of induced aggregation

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Fernández, Claudio O; Hoyer, Wolfgang; Zweckstetter, Markus; Jares-Erijman, Elizabeth A; Subramaniam, Vinod; Griesinger, Christian; Jovin, Thomas M

2004-01-01

The aggregation of α-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of α-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with α-synuclein by NMR and assigned the binding site to C-terminal residues 109–140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 → +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by ∼104 and the rate of monomer addition ∼40-fold. Significant secondary structure is not induced in monomeric α-synuclein by polyamines at 15°C. Instead, NMR reveals changes in a region (aa 22–93) far removed from the polyamine binding site and presumed to adopt the β-sheet conformation characteristic of fibrillar α-synuclein. We conclude that the C-terminal domain acts as a regulator of α-synuclein aggregation. PMID:15103328

Stat5 phosphorylation is responsible for the excessive potency of HB-EGF.

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Heo, Jeongyeon; Kim, Jae Geun; Kim, Sunghwan; Kang, Hara

2017-12-23

Heparin-binding EGF-like growth factor (HB-EGF) is a potent growth factor involved in wound healing and tumorigenesis. Despite the sequence similarity between HB-EGF and EGF, HB-EGF induces cellular proliferation and migration more potently than EGF. However, the differential regulation by HB-EGF and EGF has not been thoroughly elucidated. In this study, we compared signaling pathways activated by HB-EGF and EGF to understand the details of the molecular mechanism of the high potency induced by HB-EGF. HB-EGF specifically induced the phosphorylation of EGFR-Y1045 and activated Stat5, which is responsible for promoting cell proliferation, and migration. The competition of phosphorylated EGFR-Y1045 inhibited Stat5 activation and consequently lowered the effect of HB-EGF on cell proliferation, suggesting that the phosphorylation of EGFR-Y1045 is essential for the activation of Stat5. The phosphorylation of EGFR-Y1045 and Stat5 induced by HB-EGF was prevented by sequestering the heparin-binding domain, suggesting that the heparin-binding domain is critical for HB-EGF-mediated signaling and cellular responses. In conclusion, the heparin-binding domain of HB-EGF was responsible for EGFR-mediated Stat5 activation, resulting in a more potent cellular proliferation, and migration than that mediated by EGF. This molecular mechanism is useful for understanding ligand-specific EGFR signaling and developing biomedicines for wound healing or cancer therapy. © 2017 Wiley Periodicals, Inc.

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

International Nuclear Information System (INIS)

Jeong, Jin Boo; Jeong, Hyung Jin

2010-01-01

Research highlights: → 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. → 2M4VP inhibited hyper-phosphorylation of Rb protein. → 2M4VP induced cell cycle arrest from G1 to S. → 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. → 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

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Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

2010-10-01

Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress

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Claudia Rodrigues

2016-12-01

Full Text Available Aquaporin-5 (AQP5 is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis in different organs and systems. Despite its wide distribution pattern and physiological importance, AQP5 short-term regulation was not reported and mechanisms underlying its involvement in cancer are not well defined. In this work, we expressed rat AQP5 in yeast and investigated mechanisms of gating, as well as AQP5’s ability to facilitate H2O2 plasma membrane diffusion. We found that AQP5 can be gated by extracellular pH in a phosphorylation-dependent manner, with higher activity at physiological pH 7.4. Moreover, similar to other mammalian AQPs, AQP5 is able to increase extracellular H2O2 influx and to affect oxidative cell response with dual effects: whereas in acute oxidative stress conditions AQP5 induces an initial higher sensitivity, in chronic stress AQP5 expressing cells show improved cell survival and resistance. Our findings support the involvement of AQP5 in oxidative stress and suggest AQP5 modulation by phosphorylation as a novel tool for therapeutics.

Effect of acacia nilotica leaves extract on hyperglycaemia, lipid profile and platelet aggregation in streptozotocin induced diabetic rats

International Nuclear Information System (INIS)

Asad, M.; Munir, T.A.; Nadeem, A.

2011-01-01

To consider new hypoglycaemic, anti-hyperlipidaemic and anti-platelet aggregation sources, aqueous methanol extract of Acacia Nilotica (AN) leaves was investigated in streptozotocin induced diabetic rats. Methods: Diabetes mellitus was induced in 90 out of 120 male albino rats by administering 50 mg/Kg body weight (bw) streptozotocin intraperitoneal y, and was confirmed by measuring fasting blood glucose level >200 mg/dL on fourth post-induction day. The rats were equally divided into 4 groups, A (normal control), B (diabetic control), C (diabetics rats treated with plant extract) and group D (diabetics rats treated with glyburide). The rats of group C and D were given single dose of 300 mg/Kg bw, An extract, and 900 micro g/Kg bw glyburide respectively for 3 weeks. Blood glucose levels were measured by gluco meter, platelet aggregation by Dia Med method, beta-thrombo globulin and insulin by ELISA technique, and lipid components were measured by enzymatic calorimetric method. Results: Significant differences (p<0.05) were noticed in blood glucose, serum insulin, platelet aggregation and triglyceride levels in diabetic rats treated with AN extract and glyburide as compared to diabetic controlled rats. A significant difference (p<0.05) in beta-thrombo globulin and LDL levels was also noticed in rats treated with glyburide than the diabetic controlled rats. The levels of fasting blood glucose, beta-thrombo globulin and platelet aggregation were significantly reduced (p<0.05) in diabetic rats treated with glyburide than AN extract treated rats. Conclusions: Administration of AN leaves extract showed hypoglycaemic and anti-platelet aggregation activity in diabetic rats as that of glyburide. (author)

Aggregation-induced emission active tetraphenylethene-based sensor for uranyl ion detection.

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Wen, Jun; Huang, Zeng; Hu, Sheng; Li, Shuo; Li, Weiyi; Wang, Xiaolin

2016-11-15

A novel tetraphenylethene-based fluorescent sensor, TPE-T, was developed for the detection of uranyl ions. The selective binding of TPE-T to uranyl ions resulted in a detectable signal owing to the quenching of its aggregation-induced emission. The developed sensor could be used to visually distinguish UO2(2+) from lanthanides, transition metals, and alkali metals under UV light; the presence of other metal ions did not interfere with the detection of uranyl ions. In addition, TPE-T was successfully used for the detection of uranyl ions in river water, illustrating its potential applications in environmental systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Aggregation and fusion of modified low density lipoprotein.

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Pentikäinen, M O; Lehtonen, E M; Kovanen, P T

1996-12-01

In atherogenesis, low density lipoprotein (LDL, diameter 22 nm) accumulates in the extracellular space of the arterial intima in the form of aggregates of lipid droplets (droplet diameter up to 400 nm). Here we studied the effects of various established in vitro LDL modifications on LDL aggregation and fusion. LDL was subjected to vortexing, oxidation by copper ions, proteolysis by alpha-chymotrypsin, lipolysis by sphingomyelinase, and nonenzymatic glycosylation, and was induced to form adducts with malondialdehyde or complexes with anti-apoB-100 antibodies. To assess the amount of enlarged LDL-derived structures formed (due to aggregation or fusion), we measured the turbidity of solutions containing modified LDL, and quantified the proportion of modified LDL that 1) sedimented at low-speed centrifugation (14,000 g), 2) floated at an increased rate at high-speed centrifugation (rate zonal flotation at 285,000 gmax), 3) were excluded in size-exclusion column chromatography (exclusion limit 40 MDa), or 4) failed to enter into 0.5%. Fast Lane agarose gel during electrophoresis. To detect whether particle fusion had contributed to the formation of the enlarged LDL-derived structures, particle morphology was examined using negative staining and thin-section transmission electron microscopy. We found that 1) aggregation was induced by the formation of LDL-antibody complexes, malondialdehyde treatment, and glycosylation of LDL; 2) fusion of LDL was induced by proteolysis of LDL by alpha-chymotrypsin; and 3) aggregation and fusion of LDL were induced by vortexing, oxidation by copper ions, and lipolysis by sphingomyclinase of LDL. The various modifications of LDL differed in their ability to induce aggregation and fusion.

Enhancement of Aggregation-Induced Emission by Introducing Multiple o-Carborane Substitutions into Triphenylamine

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Kenta Nishino

2017-11-01

Full Text Available The enhancement of aggregation-induced emission (AIE is presented on the basis of the strategy for improving solid-state luminescence by employing multiple o-carborane substituents. We synthesized the modified triphenylamines with various numbers of o-carborane units and compared their optical properties. From the optical measurements, the emission bands from the twisted intramolecular charge transfer (TICT state were obtained from the modified triphenylamines. It was notable that emission efficiencies of the multi-substituted triphenylamines including two or three o-carborane units were enhanced 6- to 8-fold compared to those of the mono-substituted triphenylamine. According to mechanistic studies, it was proposed that the single o-carborane substitution can load the AIE property via the TICT mechanism. It was revealed that the additional o-carborane units contribute to improving solid-state emission by suppressing aggregation-caused quenching (ACQ. Subsequently, intense AIEs were obtained. This paper presents a new role of the o-carborane substituent in the enhancement of AIEs.

Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

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Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

2015-11-01

Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. Copyright © 2015 Elsevier B.V. All rights reserved.

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

Science.gov (United States)

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Observation of aggregation triggered by Resonance Energy Transfer (RET) induced intermolecular pairing force.

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Pan, Xiaoyong; Wang, Weizhi; Ke, Lin; Zhang, Nan

2017-07-20

In this report, we showed the existence of RET induced intermolecular pairing force by comparing their fluorescence behaviors under room illumination vs standing in dark area for either PFluAnt solution or PFluAnt&PFOBT mixture. Their prominent emission attenuation under room illumination brought out the critical role of photo, i.e. RET induced intermolecular pairing force in induction of polymer aggregation. Constant UV-Vis absorption and fluorescence spectra in terms of both peak shapes and maximum wavelengths implied no chemical decomposition was involved. Recoverable fluorescence intensity, fluorescence lifetime as well as NMR spectra further exclude photo induced decomposition. The controllable on/off state of RET induced intermolecular pairing force was verified by the masking effect of outside PFluAnt solution which function as filter to block the excitation of inside PFluAnt and thus off the RET induced intermolecular pairing force. Theoretical calculation suggest that magnitude of RET induced intermolecular pairing force is on the same scale as that of van der Waals interaction. Although the absolute magnitude of RET induced intermolecular pairing force was not tunable, its effect can be magnified by intentionally turn it "on", which was achieved by irradiance with 5 W desk lamp in this report.

Small-angle X-ray scattering studies of metastable intermediates of beta-lactoglobulin isolated after heat-induced aggregation

DEFF Research Database (Denmark)

Carrotta, R.; Arleth, L.; Pedersen, J.S.

2003-01-01

Small-angle x-ray scattering was used for studying intermediate species, isolated after heat-induced aggregation of the A variant of bovine P-lactoglobulin. The intermediates were separated in two fractions, the heated metastable dimer and heated metastable oligomers larger than the dimer. The pa...

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

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Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

Buddleja globosa (matico) prevents collagen-induced platelet activation by decreasing phospholipase C-gamma 2 and protein kinase C phosphorylation signaling.

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Fuentes, Manuel; Sepúlveda, Cesar; Alarcón, Marcelo; Palomo, Iván; Fuentes, Eduardo

2018-01-01

Platelets play a key role in thrombosis and cardiovascular diseases. Medicinal plants could be one of the most important factors that influence risks for platelet activation. Buddleja globosa (known as "matico") is a medicinal plant with many biological activities. The high content of polyphenols suggest that matico could have antiplatelet activity. The present study was aimed at evaluating mechanisms of antiplatelet action of an extract of matico. We demonstrated that matico extract at low concentrations and in a concentration dependent manner (0.05-1 mg/mL) was a potent inhibitor of platelet aggregation in response to collagen, convulsion and ADP (IC 50 values was 61 μg/mL, 72 μg/mL and 290 μg/mL, respectively). In this sense matico extract exerted the greatest antiaggregant activity induced by collagen. Similarly, matico showed a decrease in % of positive platelet for P-selectina (vehicle, 0.01, 0.05, 0.1, 0.5 and 1 mg/mL were 32 ± 2%, 29 ± 2 (p < 0.05), 19 ± 1 (p < 0.01), 15 ± 2 (p < 0.01), 10 ± 1% (p < 0.01) and 7 ± 2% (p < 0.01), respectively) and PAC-1 binding (vehicle, 0.01, 0.05, 0.1, 0.5 and 1 mg/mL were 59 ± 1, 58 ± 3 (n.s), 55 ± 2 (p < 0.05), 50 ± 2 (p < 0.01), 38 ± 1 (p < 0.01), 36 ± 2 (p < 0.01). The cellular mechanism for the antiplatelet activity of matico might be mediated by the inhibition of phospholipase C-gamma 2 and protein kinase C phosphorylation. This beneficial property of matico may be of importance in thrombosis, in which platelet activation and aggregation are important determinants of thrombus initiation and development, and may contribute to the beneficial effects of matico intake in the prevention of cardiovascular diseases.

Buddleja globosa (matico prevents collagen-induced platelet activation by decreasing phospholipase C-gamma 2 and protein kinase C phosphorylation signaling

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Manuel Fuentes

2018-01-01

Full Text Available Platelets play a key role in thrombosis and cardiovascular diseases. Medicinal plants could be one of the most important factors that influence risks for platelet activation. Buddleja globosa (known as “matico” is a medicinal plant with many biological activities. The high content of polyphenols suggest that matico could have antiplatelet activity. The present study was aimed at evaluating mechanisms of antiplatelet action of an extract of matico. We demonstrated that matico extract at low concentrations and in a concentration dependent manner (0.05–1 mg/mL was a potent inhibitor of platelet aggregation in response to collagen, convulsion and ADP (IC50 values was 61 μg/mL, 72 μg/mL and 290 μg/mL, respectively. In this sense matico extract exerted the greatest antiaggregant activity induced by collagen. Similarly, matico showed a decrease in % of positive platelet for P-selectina (vehicle, 0.01, 0.05, 0.1, 0.5 and 1 mg/mL were 32 ± 2%, 29 ± 2 (p < 0.05, 19 ± 1 (p < 0.01, 15 ± 2 (p < 0.01, 10 ± 1% (p < 0.01 and 7 ± 2% (p < 0.01, respectively and PAC-1 binding (vehicle, 0.01, 0.05, 0.1, 0.5 and 1 mg/mL were 59 ± 1, 58 ± 3 (n.s, 55 ± 2 (p < 0.05, 50 ± 2 (p < 0.01, 38 ± 1 (p < 0.01, 36 ± 2 (p < 0.01. The cellular mechanism for the antiplatelet activity of matico might be mediated by the inhibition of phospholipase C-gamma 2 and protein kinase C phosphorylation. This beneficial property of matico may be of importance in thrombosis, in which platelet activation and aggregation are important determinants of thrombus initiation and development, and may contribute to the beneficial effects of matico intake in the prevention of cardiovascular diseases.

Anoxic microniches in marine sediments induced by aggregate settlement

DEFF Research Database (Denmark)

Lehto, Niklas; Glud, Ronnie N.; Nordi, Gunnvør á

2014-01-01

an exponential increase in the mineralisation rate constant and a gradual exhaustion of reactive organic material. The peak value of the time-dependent reaction rate constant of organic matter mineralisation (kOMM) was seven to sixty times greater than analogous values measured previously in water column...... experiments. The validated model was used to investigate how the size and reactivity of parcels of organic matter influence the formation of anoxic microniches at the sediment–water interface of typical deep-sea environments. As well as kOMM, the concentration of reactive organic matter in the aggregate, its...... size and porosity were also critical in determining the likelihood of anoxic microniche formation. For the optimum fitted parameters describing kOMM and the concentration of reactive organic atter, the minimum diameter of the parcel to induce anoxia was 1.8 mm, hereas it was 2.8 mm to make...

Tunneling electron induced molecular electroluminescence from individual porphyrin J-aggregates

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Meng, Qiushi; Zhang, Chao; Zhang, Yang, E-mail: zhyangnano@ustc.edu.cn, E-mail: zcdong@ustc.edu.cn; Zhang, Yao; Liao, Yuan; Dong, Zhenchao, E-mail: zhyangnano@ustc.edu.cn, E-mail: zcdong@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at the Microscale and Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China)

2015-07-27

We investigate molecular electroluminescence from individual tubular porphyrin J-aggregates on Au(111) by tunneling electron excitations in an ultrahigh-vacuum scanning tunneling microscope (STM). High-resolution STM images suggest a spiral tubular structure for the porphyrin J-aggregate with highly ordered “brickwork”-like arrangements. Such aggregated nanotube is found to behave like a self-decoupled molecular architecture and shows red-shifted electroluminescence characteristics of J-aggregates originated from the delocalized excitons. The positions of the emission peaks are found to shift slightly depending on the excitation sites, which, together with the changes in the observed spectral profiles with vibronic progressions, suggest a limited exciton coherence number within several molecules. The J-aggregate electroluminescence is also found unipolar, occurring only at negative sample voltages, which is presumably related to the junction asymmetry in the context of molecular excitations via the carrier injection mechanism.

Phosphorylation of p53 at serine 15 in A549 pulmonary epithelial cells exposed to vanadate: Involvement of ATM pathway

International Nuclear Information System (INIS)

Suzuki, Katsura; Inageda, Kiyoshi; Nishitai, Gen; Matsuoka, Masato

2007-01-01

When A549 cells were exposed to sodium metavanadate (NaVO 3 ), the pentavalent species of vanadium (vanadate), phosphorylation of p53 protein at Ser15 was found in a time (8-48 h)- and dose (10-200 μM)-dependent manner. After the incubation with 50 or 100 μM NaVO 3 for 48 h, accumulation of p53 protein was accompanied with Ser15 phosphorylation. Among serines in p53 protein immunoprecipitated from A549 cells treated with 100 μM NaVO 3 for 48 h, only Ser15 was markedly phosphorylated. Treatment with other vanadate compounds, sodium orthovanadate (Na 3 VO 4 ) and ammonium metavanadate (NH 4 VO 3 ), also induced Ser15 phosphorylation and accumulation of p53 protein. While phosphorylation of extracellular signal-regulated protein kinase (ERK) was found in cells treated with NaVO 3 , treatment with U0126 did not suppress Ser15 phosphorylation. On the other hand, treatment with wortmannin or caffeine, the inhibitors to phosphatidylinositol 3-kinase related kinases (PIKKs), suppressed both NaVO 3 -induced Ser15 phosphorylation and accumulation of p53 protein. The silencing of ataxia telangiectasia mutated (ATM) expression using short-interference RNA resulted in the marked suppression of Ser15 phosphorylation in A549 cells exposed to NaVO 3 . However, treatment with antioxidants such as catalase and N-acetylcysteine did not suppress NaVO 3 -induced Ser15 phosphorylation. Transcriptional activation of p53 and DNA fragmentation in A549 cells treated with NaVO 3 were suppressed only slightly by S15A mutation, suggesting that Ser15 phosphorylation is not essential for these responses. The present results showed that vanadate induces the phosphorylation of p53 at Ser15 depending on ATM, one of the members of PIKK family, in this human pulmonary epithelial cell line

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

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Li, Xinhong; Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Yang, Qiangzhen; Li, Sisi; Zhang, Yukun

2016-05-16

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment

DEFF Research Database (Denmark)

Kelstrup, Christian D; Hekmat, Omid; Francavilla, Chiara

2011-01-01

Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site......-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions...... contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra...

Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 in human skeletal muscle

DEFF Research Database (Denmark)

Treebak, Jonas Thue; Pehmøller, Christian; Kristensen, Jonas Møller

2014-01-01

We investigated the phosphorylation signatures of two Rab GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers e...

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

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Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

2012-08-17

Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

Photochemical induced growth and aggregation of metal nanoparticles in diode-array spectrophotometer via excited dimethyl-sulfoxide.

Science.gov (United States)

Zidki, Tomer; Cohen, Haim; Meyerstein, Dan

2010-10-21

Ag(0) and Au(0) nanoparticles suspended in dilute aqueous solutions containing (CH(3))(2)SO are photochemically unstable. The light source of a diode-array spectrophotometer induces, within less than a minute, particle growth and aggregation. The results indicate that this process is triggered by UV light absorption by the (CH(3))(2)SO.

p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats.

Science.gov (United States)

Kiyomoto, Masaaki; Shinoda, Masamichi; Honda, Kuniya; Nakaya, Yuka; Dezawa, Ko; Katagiri, Ayano; Kamakura, Satoshi; Inoue, Tomio; Iwata, Koichi

2015-08-12

Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain. Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1β over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration. These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

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Hector N Aguilar

Full Text Available The 'phosphate-binding tag' (phos-tag reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT and calpeptin (Calp induce RLC kinase (MLCK- and rho-kinase (ROK-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.

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Yongbin Chen

2011-06-01

Full Text Available Hedgehog (Hh signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo, but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.

Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

International Nuclear Information System (INIS)

Koch, J.A.; Waxman, D.J.

1989-01-01

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [ 32 P]orthophosphate in the presence of N 6 , O 2' -dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-induced P-450 form PB-4 (P-450 gene IIB1). Two-dimensional gel electrophoresis revealed that these 32 P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 μM. Phosphoamino acid analysis of the 32 P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2. In vitro analyses revealed that phosphorylation of P-450 PB-4 leads to a loss of monooxygenase activity, suggesting that the posttranslational modification of this P-450 enzyme by cAMP-dependent protein kinase may play a role in the modulation of P-450-dependent monooxygenase activity in vivo

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation.

Science.gov (United States)

Hrynets, Yuliya; Ndagijimana, Maurice; Betti, Mirko

2015-01-01

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.

Increased histone H3 phosphorylation in neurons in specific brain structures after induction of status epilepticus in mice.

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Tetsuji Mori

Full Text Available Status epilepticus (SE induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3(+ (PH3(+ neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3(+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3(+ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation.

What favors convective aggregation and why?

Science.gov (United States)

Muller, Caroline; Bony, Sandrine

2015-07-01

The organization of convection is ubiquitous, but its physical understanding remains limited. One particular type of organization is the spatial self-aggregation of convection, taking the form of cloud clusters, or tropical cyclones in the presence of rotation. We show that several physical processes can give rise to self-aggregation and highlight the key features responsible for it, using idealized simulations. Longwave radiative feedbacks yield a "radiative aggregation." In that case, sufficient spatial variability of radiative cooling rates yields a low-level circulation, which induces the upgradient energy transport and radiative-convective instability. Not only do vertically integrated radiative budgets matter but the vertical profile of cooling is also crucial. Convective aggregation is facilitated when downdrafts below clouds are weak ("moisture-memory aggregation"), and this is sufficient to trigger aggregation in the absence of longwave radiative feedbacks. These results shed some light on the sensitivity of self-aggregation to various parameters, including resolution or domain size.

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

Science.gov (United States)

Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

2011-11-01

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways.

Science.gov (United States)

Wang, Y; Li, J; Song, W; Yu, J

2014-06-01

The aim of this study was to investigate effects of mineral trioxide aggregate (MTA) on odonto/osteogenic differentiation of bone marrow stromal cells (BMSCs) from craniofacial bones. Craniofacial BMSCs were isolated from rat mandible and effects of MTA on their proliferation, differentiation and MAPK pathway involvement were subsequently investigated, in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazoliumbromide) assay was performed to evaluate proliferation of the MTA-treated cells. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction and western blot assays were used to assess differentiation capacity as well as MAPK pathway involvement. 0.02 mg/ml MTA-treated BMSCs had significantly higher ALP activity and formed more mineralized nodules than the untreated group. Odonto/osteoblastic marker genes/proteins (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN and Dspp/DSP respectively) in MTA-treated cells were remarkably upregulated compared to untreated ones. Mechanistically, phosphorylated Jun N-terminal kinase (P-JNK) and phosphorylated extracellular regulated protein kinases (P-ERK) in MTA-treated BMSCs increased significantly in a time-dependent manner, while inhibition of JNK and ERK MAPK pathways dramatically blocked MTA-induced odonto/osteoblastic differentiation, as indicated by reduced ALP levels, weakened mineralization capacity and downregulated levels of odonto/osteoblastic marker genes (Alp, Runx2, Osx, Ocn and Dspp). Mineral trioxide aggregate promoted odonto/osteogenic capacity of craniofacial BMSCs via JNK and ERK MAPK signalling pathways. © 2014 John Wiley & Sons Ltd.

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

Energy Technology Data Exchange (ETDEWEB)

Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

2011-10-14

Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation

CSIR Research Space (South Africa)

Owen, GR

2014-10-01

Full Text Available –deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide...

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

Science.gov (United States)

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

Science.gov (United States)

She, Hua; Mao, Zixu

2017-01-01

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

Science.gov (United States)

Ku, H; Meier, K E

2000-04-14

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

Science.gov (United States)

Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

2017-06-01

Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

DEFF Research Database (Denmark)

Pedersen, Nis Borbye; Hofmeister, Marlene Vind; Rosenbaek, Lena L

2010-01-01

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites...

Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

International Nuclear Information System (INIS)

Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.; Kuo, M.-L.; Ho, Y.-S.; Lee, W.-S.

2008-01-01

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

Science.gov (United States)

Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

Science.gov (United States)

Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

2010-01-01

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

International Nuclear Information System (INIS)

Brush, G.S.; Morrow, D.M.; Hieter, P.; Kelly, T.J.

1996-01-01

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T

Aggregation induced emission enhancement of pyrene-appended Schiff base luminophore and its photovoltaic effect

Energy Technology Data Exchange (ETDEWEB)

Panda, Uttam; Roy, Suman; Mallick, Debashis [Department of Chemistry, Jadavpur University, Kolkata 700032 (India); Dalapati, Pradip [Condensed Matter Physics Research Center, Department of Physics, Jadavpur University, Kolkata 700032 (India); Biswas, Subir [Immunology Lab, Department of Zoology, University of Calcutta, Kolkata 700019 (India); Manik, Nabin Baran [Condensed Matter Physics Research Center, Department of Physics, Jadavpur University, Kolkata 700032 (India); Bhattacharyya, Arindam [Immunology Lab, Department of Zoology, University of Calcutta, Kolkata 700019 (India); Sinha, Chittaranjan, E-mail: c_r_sinha@yahoo.com [Department of Chemistry, Jadavpur University, Kolkata 700032 (India)

2016-07-15

Pyrene appended Schiff base, 1,3-bis-((E)-pyrene-1-ylmethyleneamino)propan-2-ol (HL), a fluorophore, shows aggregation induced emission enhancement upon addition of water in DMF (4:1, v/v) solution; the quantum yield (Φ) is increased by 0.034–0.450. Under illumination of solar radiation HL (10{sup −3} M) impregnated photovoltaic cell generates 4.4 mA/6.29 V electricity. DFT and TD-DFT computation of optimized geometry of HL has explained the photophysical properties.

Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin.

Science.gov (United States)

Rodrigues, Antônio M; Almeida, Antônio-Carlos G; Infantosi, Antonio F C; Teixeira, Hewerson Z; Duarte, Mario A

2008-02-01

The ATP hydrolysis reactions responsible for the Na(+)/K(+)-ATPase phosphorylation, according to recent experimental evidences, also occur for the PTX-Na(+)/K(+) pump complex. Moreover, it has been demonstrated that PTX interferes with the enzymes phosphorylation status. However, the reactions involved in the PTX-Na(+)/K(+) pump complex phosphorylation are not very well established yet. This work aims at proposing a reaction model for PTX-Na(+)/K(+) pump complex, with similar structure to the Albers-Post model, to contribute to elucidate the PTX effect over Na(+)/K(+)-ATPase phosphorylation and dephosphorylation. Computational simulations with the proposed model support several hypotheses and also suggest: (i) phosphorylation promotes an increase of the open probability of induced channels; (ii) PTX reduces the Na(+)/K(+) pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na(+)/K(+)-ATPase may not be phosphorylated; (iv) PTX can bind to substates of the two principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity sites. The proposed model also allows previewing the behavior of the PTX-pump complex substates for different levels of intracellular ATP concentrations.

Detergent-induced aggregation of an amyloidogenic intrinsically ...

Indian Academy of Sciences (India)

Shruti Arya

2017-11-02

Nov 2, 2017 ... the effect of a well-known anionic lipid mimetic, sodium dodecyl sulfate (SDS), on the aggregation ... for the fabrication of nano-structures for a wide variety ...... M 2014 Classification of intrinsically disordered regions.

Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

Energy Technology Data Exchange (ETDEWEB)

Ilowski, Maren; Putz, Christine [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Weiss, Thomas S. [Department of Surgery, University of Regensburg Hospital, Regensburg (Germany); Brand, Stephan [Department of Internal Medicine II, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, TU Dortmund University, Dortmund (Germany); Thasler, Wolfgang Erwin, E-mail: wolfgang.thasler@med.uni-muenchen.de [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany)

2010-04-16

Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

The role of Golgi reassembly and stacking protein 65 phosphorylation in H2O2-induced cell death and Golgi morphological changes.

Science.gov (United States)

Ji, Guang; Zhang, Weiwei; Quan, Moyuan; Chen, Yang; Qu, Hui; Hu, Zhiping

2016-12-01

This study aimed to investigate the effects of H 2 O 2 -induced oxidative stress on cell viability and survival, as well as changes in the distribution of Golgi apparatus and in the level of Golgi reassembly and stacking protein 65 (GRASP65). Cell viability of cultured N2a cells treated with H 2 O 2 was measured by the MTT assay. Apoptosis was measured by flow cytometry analyses. Cells labeled by indirect immunofluorescence were observed under confocal microscope to detect any Golgi morphological alterations; electron microscopy of Golgi apparatus was also done. Expression of GRASP65 and phospho-GRASP65 was examined by immunoblotting. H 2 O 2 treatment reduced the cell viability and raised the cell mortality of N2a cells in a time-dependent manner. Notable changes were only observed in the distribution and morphology of Golgi apparatus at 6 h after H 2 O 2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H 2 O 2 treatment, peaked at 3 h, and finally dropped at 6 h. Taken together, GRASP65 phosphorylation may have a critical role in inducing cell death at the early stage after H 2 O 2 treatment, while its role in H 2 O 2 -induced Golgi morphological changes may be complex.

Collagen induced aggregation of platelets and release of 14C serotonin from platelets depending on temperature and pH during in vitro storage of platelets

International Nuclear Information System (INIS)

Krause, J.

1978-01-01

The paper investigates collagen-induced platelet aggregation and 14 C serotonin release in dependence of age, temperature, and pH value during the storage of the conserved platelets. The optimum pH (with adjusted CO 2 /air mixture) for platelet storage is found to be pH 6.9. The optimum temperature for platelet storage is 4-8 0 C. After 12, 24, or 48 hours of storage at pH 6.9 and 4-8 0 C and subsequent heating of the platelet-rich plasma to 37 0 C for 30 minutes, the values determined for collagen-induced platelet aggregation and 14 C serotonin release rarely differed from the initial values before storage. Cold-induced spontaneous platelet aggregation and serotonin release of the platelets stored at 4-8 0 C can be avoided by 30-60 minutes pre-incubation of the platelets at 37 0 C before transfusions. The in vitro findings for collagen-induced platelet aggregation and 14 C serotonin release indicate that platelet storage for 24-48 hours at pH 6.9 and 4-8 0 C may be permissible also for clinical purposes. The problem remains open whether the clinical effect of these platelets is still sufficient after 48 hours of storage, but literature findings suggest that this may well be the case. (orig.) [de

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

Science.gov (United States)

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-01-01

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells. PMID:26943035

Aggregation in thin-film silver: Induced by chlorine and inhibited by alloying with two dopants

International Nuclear Information System (INIS)

Koike, Katsuhiko; Shimada, Koichi; Fukuda, Shin

2009-01-01

The Ag aggregation mechanism triggered by chlorine (Cl) is discussed. The frontier orbital theory by K. Fukui is applied in order to determine the growing point in the silver (Ag) cluster. Ag in the thin-film silver would grow to Ag n Cl and stack, triggered by Cl from the outside according to the mechanism described. This would lead to an aggregate with a high Ag density. It is suggested that this would be the generating mechanism of the silver-gray aggregate consisting mostly of Ag, which is generated by exposing it to Cl. Two tactics in order to prevent restrain aggregation induced by Cl according to the mechanism are proposed. Tactic 1 is a restraining of structure change to a plane in the process of Ag 6 Cl + Ag → Ag 7 Cl. Tactic 2 is the trapping of Cl before it generates a bond to Ag. The ability of the two combined dopants with the abilities of tactics 1 and 2, such as in an Ag alloy including palladium and copper (APC), and including neodymium and gold (ANA) is expected to be very high. The aggregation resistance of an Ag alloy including two dopants is evaluated by a salt water immersion test. The APC and ANA demonstrated a very high resistance to Cl, because of the combination of the dopants working with tactic 1 (Pd, Au) and tactic 2 (Cu, Nd). The multilayer sputter coating with an ANA layer demonstrated a very interesting profile where the light transmittance and the electrical sheet resistance are almost the same as the multilayer sputter coating with a pure Ag. The multilayer sputter coating with AIS also demonstrates a very interesting profile, where the light transmittance is higher than the multilayer sputter coating with a pure Ag.

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.

Science.gov (United States)

Tõugu, Vello; Karafin, Ann; Zovo, Kairit; Chung, Roger S; Howells, Claire; West, Adrian K; Palumaa, Peep

2009-09-01

Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling

Science.gov (United States)

Chen, Qing; Su, Yi; Wesslowski, Janine; Hagemann, Anja I; Ramialison, Mirana; Wittbrodt, Joachim; Scholpp, Steffen; Davidson, Gary

2014-01-01

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6. Subject Categories Membrane & Intracellular Transport; Post-translational Modifications, Proteolysis & Proteomics PMID:25391905

Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS).

Science.gov (United States)

Cooper, John C; Boustead, Jared N; Yu, Chao-Lan

2006-06-01

Cytokine-inducible SH2-containing protein (CIS) is the first identified member of genes encoding for the suppressor of cytokine signaling (SOCS). CIS is also a well-known target gene of signal transducer and activator of transcription 5 (STAT5) pathways, providing normal negative feedback control of signaling by cytokines and growth factors. Three other SOCS genes, SOCS1, SOCS2, and SOCS3, can be silenced by DNA hypermethylation in human cancers, suggesting a potential mechanism for constitutive STAT activation. However, it is not known whether CIS expression is similarly perturbed in tumor cells. We report here the absence of CIS expression in T lymphoma LSTRA that overexpresses the Lck protein tyrosine kinase and exhibits elevated STAT5 activity. Pervanadate-induced CIS expression and STAT5 binding to the CIS promoter in vivo over a short time course implies that mechanisms other than DNA hypermethylation may contribute to defective CIS expression in LSTRA cells. Comparison with cytokine-dependent BaF3 cells stimulated with interleukin-3 (IL-3) further reveals that CIS induction correlates with specific STAT5b post-translational modifications. It exhibits as the slowest migrating form through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This distinctly modified STAT5b is the predominant form that binds to the consensus STAT5 sites in the CIS promoter and accumulates in the nucleus. In vitro phosphatase assays and phosphoamino acid analysis suggest the involvement of phosphorylation on residues other than the highly conserved tyrosine and serine sites in this distinct STAT5b mobility shift. All together, our results provide a novel link between incomplete STAT5b phosphorylation and defective SOCS gene expression in cancer cells.

Phosphorylated nano-diamond/ Polyimide Nanocomposites

International Nuclear Information System (INIS)

Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

2014-01-01

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

DEFF Research Database (Denmark)

Confalonieri, S; Salcini, A E; Puri, C

2000-01-01

for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function....... Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization...... of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular...

HSP20 phosphorylation and airway smooth muscle relaxation

Directory of Open Access Journals (Sweden)

Mariam Ba

2009-06-01

Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP.

Science.gov (United States)

Ottaiano, Tatiana F; Andrade, Sheila S; de Oliveira, Cleide; Silva, Mariana C C; Buri, Marcus V; Juliano, Maria A; Girão, Manoel J B C; Sampaio, Misako U; Schmaier, Alvin H; Wlodawer, Alexander; Maffei, Francisco H A; Oliva, Maria Luiza V

2017-04-01

Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin α IIb β 3 through interactions with the KGD/KGE sequence motif in huPK. Integrin α IIb β 3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS 473 , ERK1/2, and p38 MAPK, and to Ca 2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and α IIb β 3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis.

Science.gov (United States)

Tanaka, Saori; Hosogi, Shigekuni; Sawabe, Yukinori; Shimamoto, Chikao; Matsumura, Hitoshi; Inui, Toshio; Marunaka, Yoshinori; Nakahari, Takashi

2016-01-01

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

Comparison of the effects of isobutylmethylxanthine and milrinone on ischaemia-induced arrhythmias and platelet aggregation in anaesthetized rabbits.

Science.gov (United States)

Holbrook, M.; Coker, S. J.

1989-01-01

1. The aim of this study was to compare the effects of the non-selective phosphodiesterase (PDE) inhibitor, isobutylmethylxanthine (IBMX) and the selective PDE III inhibitor, milrinone, in a rabbit model of acute myocardial ischaemia. 2. Coronary artery occlusion caused changes in the ST-segment of the ECG and ectopic activity in all control rabbits. Ventricular fibrillation occurred in 10 out of 14 (71%) of these animals. Pretreatment with IBMX 100 micrograms kg-1 plus 10 micrograms kg-1 min-1, starting 10 min before coronary artery occlusion, reduced ischaemia-induced ST-segment changes and ventricular fibrillation occurred in only 10% of this group (n = 10). A similar dose of milrinone had no antiarrhythmic activity, whereas with a lower dose of milrinone, 30 micrograms kg-1 plus 3 micrograms kg-1 min-1 (n = 10), only 30% of rabbits fibrillated and ST-segment changes were attenuated. 3. Acute administration of both IBMX and milrinone reduced arterial blood pressure. With the higher dose of milrinone a significant effect was still present after 10 min of drug infusion. A greater hypotensive response to the higher dose of milrinone was observed in the rabbits which subsequently fibrillated during ischaemia. A marked tachycardia was also observed after administration of the higher dose of milrinone. 4. At the end of the experiment platelet aggregation was studied ex vivo. ADP-induced aggregation was reduced by pretreatment of the rabbits with milrinone but not IBMX. Both PDE inhibitors enhanced the ability of isoprenaline to inhibit ADP-induced platelet aggregation but milrinone was more effective, particularly at the higher dose.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2478245

Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

International Nuclear Information System (INIS)

Singer, H.A.

1990-01-01

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms

Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

Directory of Open Access Journals (Sweden)

Javier Modrego

Full Text Available OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA may be associated with a different ability of platelets to generate nitric oxide (NO. PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26 and ASA-resistant (n = 24 using a platelet functionality test (PFA-100. RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3 was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2 isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786 → C in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser(1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018 of NOS3 phosphorylation at Ser(1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177.

The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

Directory of Open Access Journals (Sweden)

Krishna Madhuri Manda

Full Text Available Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD. Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synuclein (112-syn in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.

Effect of homogenisation in formation of thermally induced aggregates in a non- and low- fat milk model system with microparticulated whey proteins

DEFF Research Database (Denmark)

Celigueta Torres, Isabel; Nieto, Gema; Nylander, Tommy

2017-01-01

in the formation of protein aggregates were studied by polyacrylamide gel electrophoresis and the final complexes visualised by darkfield microscopy. Homogenisation of non-fat milk systems led to partial adsorption of caseins onto microparticles, independently of the type of microparticle. On the contrary...... are responsible for the formation of heat-induced aggregates that influence the texture and sensory characteristics of the final product. The formation of heat-induced complexes was studied in non- and low-fat milk model systems, where microparticulated whey protein (MWP) was used as fat replacer. Five MWP types......, homogenisation of low-fat milk resulted in preferential adsorption of caseins onto fat globules, rather than onto microparticles. Further heating of the milk, led to the formation of heat induced complexes with different sizes and characteristics depending on the type of MWP and the presence or not of fat...

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

Science.gov (United States)

Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

2011-01-01

Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

KAUST Repository

Shah, Dhawal

2011-09-21

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP\\'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine\\'s preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine\\'s interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

KAUST Repository

Shah, Dhawal; Li, Jianguo; Shaikh, Abdul Rajjak; Rajagopalan, Raj

2011-01-01

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP's indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

Ras-Induced and Extracellular Signal-Regulated Kinase 1 and 2 Phosphorylation-Dependent Isomerization of Protein Tyrosine Phosphatase (PTP)-PEST by PIN1 Promotes FAK Dephosphorylation by PTP-PEST ▿

Science.gov (United States)

Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X.; Lu, Zhimin

2011-01-01

Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression. PMID:21876001

ABA-Induced Stomatal Closure Involves ALMT4, a Phosphorylation-Dependent Vacuolar Anion Channel of Arabidopsis[OPEN

Science.gov (United States)

Baetz, Ulrike; Huck, Nicola V.; Zhang, Jingbo

2017-01-01

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2−. Efflux of Mal2− from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2− release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2− efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2− efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2− efflux during ABA-induced stomatal closure and its activity depends on phosphorylation. PMID:28874508

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation.

Science.gov (United States)

Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung; Rhee, Man Hee; Kim, Yun-Bae

2013-12-01

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

OpenAIRE

En-Ju Chou; Liang-Yi Hung; Chieh-Ju C. Tang; Wen-Bin Hsu; Hsin-Yi Wu; Pao-Chi Liao; Tang K. Tang

2016-01-01

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. In...

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

Science.gov (United States)

Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

2013-03-01

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Damage-induced BRCA1 phosphorylation by Chk2 contributes to the timing of end resection.

Science.gov (United States)

Parameswaran, Balaji; Chiang, Huai-Chin; Lu, Yunzhe; Coates, Julia; Deng, Chu-Xia; Baer, Richard; Lin, Hui-Kuan; Li, Rong; Paull, Tanya T; Hu, Yanfen

2015-01-01

The BRCA1 tumor suppressor plays an important role in homologous recombination (HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN) and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCF(Skp2) and that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs. BRCA1 is known to inhibit Mre11 nuclease activity. SCF(Skp2) activity appears at late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal of BRCA1 from DSBs by SCF(Skp2)-mediated degradation terminates BRCA1-mediated inhibition of Mre11 nuclease activity, allowing for end resection and restricting the initiation of HR to the S/G2 phases of the cell cycle.

Selective inhibition of phosphodiesterases 4, 5 and 9 induces HSP20 phosphorylation and attenuates amyloid beta 1-42 mediated cytotoxicity

OpenAIRE

Cameron, Ryan T.; Whiteley, Ellanor; Day, Jon P.; Parachikova, Anna I.; Baillie, George S.

2017-01-01

Phosphodiesterase (PDE) inhibitors are currently under evaluation as agents that may facilitate the improvement of cognitive impairment associated with Alzheimer's disease. Our aim was to determine whether inhibitors of PDEs 4, 5 and 9 could alleviate the cytotoxic effects of amyloid beta 1?42 (A?1?42) via a mechanism involving the small heatshock protein HSP20. We show that inhibition of PDEs 4, 5 and 9 but not 3 induces the phosphorylation of HSP20 which, in turn, increases the colocalisati...

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

Science.gov (United States)

Villalobo, A; Lehninger, A L

1980-03-25

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

Regulation of Glioma Cell Migration by Seri ne-Phosphorylated P3111

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Wendy S. McDonough

2005-09-01

Full Text Available P311, an 8-kDa polypeptide, was previously shown to be highly expressed in invasive glioma cells. Here, we report the functional characteristics of P311 with regard to influencing glioma cell migration. P311 is constitutively serine-phosphorylated; decreased phosphorylation is observed in migration-activated glioma cells. The primary amino acid sequence of P311 indicates a putative serine phosphorylation site (S59 near the PEST domain. Site-directed mutagenesis of S59A retarded P311 degradation, induced glioma cell motility. In contrast, S59D mutation resulted in the rapid degradation of P311, reduced glioma cell migration. Coimmunoprecipitation coupled with matrixassisted laser desorption/ionization time-of-flight mass spectrometry analysis identified Filamin A as a binding partner of P311, immunofluorescence studies showed that both proteins colocalized at the cell periphery. Moreover, P311-induced cell migration was abrogated by inhibition of β1 integrin function using TACβ1A, a dominant-negative inhibitor of β1 integrin signaling, suggesting that P311 acts downstream of β1 signaling. Finally, overexpression of P311 or P311 S59A mutant protein activates Raci GTPase; small interfering RNA-mediated depletion of Raci suppresses P311-induced motility. Collectively, these results suggest a role for levels of P311 in regulating glioma motility, invasion through the reorganization of actin cytoskeleton at the cell periphery.

The Role of Aggregates of Therapeutic Protein Products in Immunogenicity: An Evaluation by Mathematical Modeling

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Liusong Yin

2015-01-01

Full Text Available Therapeutic protein products (TPP have been widely used to treat a variety of human diseases, including cancer, hemophilia, and autoimmune diseases. However, TPP can induce unwanted immune responses that can impact both drug efficacy and patient safety. The presence of aggregates is of particular concern as they have been implicated in inducing both T cell-independent and T cell-dependent immune responses. We used mathematical modeling to evaluate several mechanisms through which aggregates of TPP could contribute to the development of immunogenicity. Modeling interactions between aggregates and B cell receptors demonstrated that aggregates are unlikely to induce T cell-independent immune responses by cross-linking B cell receptors because the amount of signal transducing complex that can form under physiologically relevant conditions is limited. We systematically evaluate the role of aggregates in inducing T cell-dependent immune responses using a recently developed multiscale mechanistic mathematical model. Our analysis indicates that aggregates could contribute to T cell-dependent immune response by inducing high affinity epitopes which may not be present in the nonaggregated TPP and/or by enhancing danger signals to break tolerance. In summary, our computational analysis is suggestive of novel insights into the mechanisms underlying aggregate-induced immunogenicity, which could be used to develop mitigation strategies.

Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

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Abskharon, Romany; Dang, Johnny; Elfarash, Ameer; Wang, Zerui; Shen, Pingping; Zou, Lewis S; Hassan, Sedky; Wang, Fei; Fujioka, Hisashi; Steyaert, Jan; Mulaj, Mentor; Surewicz, Witold K; Castilla, Joaquín; Wohlkonig, Alexandre; Zou, Wen-Quan

2017-10-04

The infectious prion protein (PrP Sc or prion) is derived from its cellular form (PrP C ) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrP C to PrP Sc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrP C (BVPrP) is highly susceptible to PrP Sc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts

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Jong Yuh Cherng

2012-08-01

Full Text Available UVC irradiation induces oxidative stress and leads to cell death through an apoptotic pathway. This apoptosis is caused by activation of caspase-3 and formation of poly(ADP-ribose polymerase-1 (PARP-1. In this study, the underlying mechanisms of Chlorella derived peptide (CDP activity against UVC-induced cytotoxicity were investigated. Human skin fibroblasts were treated with CDP, vitamin C, or vitamin E after UVC irradiation for a total energy of 15 J/cm². After the UVC exposure, cell proliferation and caspase-3 activity were measured at 12, 24, 48, and 72 h later. Expression of phosphorylated FADD and cleaved PARP-1 were measured 16 h later. DNA damage (expressed as pyrimidine (6-4 pyrimidone photoproducts DNA concentration and fragmentation assay were performed 24 h after the UVC exposure. Results showed that UVC irradiation induced cytotoxicity in all groups except those treated with CDP. The caspase-3 activity in CDP-treated cells was inhibited from 12 h onward. Expression of phosphorylated FADD and cleaved PARP-1 were also reduced in CDP-treated cells. Moreover, UVC-induced DNA damage and fragmentation were also prevented by the CDP treatment. This study shows that treatment of CDP provides protective effects against UVC-induced cytotoxicity through the inhibition of caspase-3 activity and the reduction of phosphorylated FADD and cleaved PARP-1 expression.

Stabilization and activation of p53 are regulated independently by different phosphorylation events

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Chernov, Mikhail V.; Ramana, Chilakamarti V.; Adler, Victor V.; Stark, George R.

1998-01-01

Treatment of mouse or human cells with the protein kinase C (PKC) inhibitors H7 or bisindolylmaleimide I induced an increase in the lifetime of p53, leading to its accumulation. In inhibitor-treated cells, p53 translocated to the nuclei and bound to DNA but was not competent to induce transcription. However, transactivation could be induced by subsequent DNA damage. Phorbol ester, a potent activator of PKC, significantly inhibited the accumulation of p53 after DNA damage. Therefore, constitutive PKC-dependent phosphorylation of p53 itself, or of a protein that interacts with p53, is required for the rapid degradation of p53 in untreated cells. Furthermore, an increase in the lifetime of p53 is not accompanied necessarily by its activation. Treatment with the PKC inhibitors decreased the overall level of p53 phosphorylation but led to the appearance of a phosphopeptide not seen in tryptic digests of p53 from untreated cells. Therefore, the lifetime and activities of p53 are likely to be regulated by distinct alterations of the phosphorylation pattern of p53, probably caused by the actions of different kinases. PMID:9482877

Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle.

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Treebak, Jonas T; Pehmøller, Christian; Kristensen, Jonas M; Kjøbsted, Rasmus; Birk, Jesper B; Schjerling, Peter; Richter, Erik A; Goodyear, Laurie J; Wojtaszewski, Jørgen F P

2014-01-15

We investigated the phosphorylation signatures of two Rab-GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 phospho-sites that (1) did not respond to exercise or postprandial increase in insulin (TBC1D4: S666), (2) responded to insulin only (TBC1D4: S318), (3) responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and (4) responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin-stimulated leg, Akt phosphorylation of both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2/β2/γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK regulated phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species is problematic.

Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

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Alexei Vazquez

Full Text Available The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

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Chihara, Kazuyasu [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan); Kimura, Yukihiro [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Division of Otorhinolaryngology Head and Neck Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Honjoh, Chisato [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Third Department of Internal Medicine, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Yamauchi, Shota; Takeuchi, Kenji [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan); Sada, Kiyonao, E-mail: ksada@u-fukui.ac.jp [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan)

2014-03-10

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 446} in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr{sup 183} and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 426} of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr{sup 426} is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr{sup 426} was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr{sup 426} following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation.

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

International Nuclear Information System (INIS)

Chihara, Kazuyasu; Kimura, Yukihiro; Honjoh, Chisato; Yamauchi, Shota; Takeuchi, Kenji; Sada, Kiyonao

2014-01-01

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr 174 , Tyr 183 and Tyr 446 in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr 183 and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr 174 , Tyr 183 and Tyr 426 of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr 426 is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr 426 was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr 426 following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation

Trihydrophobin 1 Phosphorylation by c-Src Regulates MAPK/ERK Signaling and Cell Migration

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Wu, Weibin; Sun, Zhichao; Wu, Jingwen; Peng, Xiaomin; Gan, Huacheng; Zhang, Chunyi; Ji, Lingling; Xie, Jianhui; Zhu, Haiyan; Ren, Shifang

2012-01-01

c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src. PMID:22238675

Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β-Lactoglobulin.

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Chihi, Mohamed-Lazhar; Mession, Jean-luc; Sok, Nicolas; Saurel, Rémi

2016-04-06

The present work investigates the formation of protein aggregates (85 °C, 60 min incubation) upon heat treatment of β-lactoglobulin (βlg)-pea globulins (Glob) mixtures at pH 7.2 and 5 mM NaCl from laboratory-prepared protein isolates. Various βlg/Glob weight ratios were applied, for a total protein concentration of 2 wt % in admixture. Different analytical methods were used to determine the aggregation behavior of "mixed" aggregates, that is, surface hydrophobicity and also sulfhydryl content, protein interactions by means of SDS-PAGE electrophoresis, and molecule size distribution by DLS and gel filtration. The production of "mixed" thermal aggregates would involve both the formation of new disulfide bonds and noncovalent interactions between the denatured βlg and Glob subunits. The majority of "mixed" soluble aggregates displayed higher molecular weight and smaller diameter than those for Glob heated in isolation. The development of pea-whey protein "mixed" aggregates may help to design new ingredients for the control of innovative food textures.

Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

International Nuclear Information System (INIS)

Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin

2006-01-01

Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions

Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

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Ni Zhu

2018-03-01

Full Text Available Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI, a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1, a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a

The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

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Ann De Vos

2015-04-01

Full Text Available Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

Spinal cord homogenates from SOD1 familial amyotrophic lateral sclerosis induce SOD1 aggregation in living cells.

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Edward Pokrishevsky

Full Text Available Mutant Cu/Zn superoxide dismutase (SOD1 can confer its misfolding on wild-type SOD1 in living cells; the propagation of misfolding can also be transmitted between cells in vitro. Recent studies identified fluorescently-tagged SOD1G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, in vitro and in vivo. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1. We observed that human spinal cord homogenates prepared from SOD1 familial ALS (FALS can induce significantly more intracellular reporter protein aggregation than spinal cord homogenates from sporadic ALS, Alzheimer's disease, multiple system atrophy or healthy control individuals. We also determined that the induction of reporter protein aggregation by SOD1-FALS tissue homogenates can be attenuated by incubating the cells with the SOD1 misfolding-specific antibody 3H1, or the small molecule 5-fluorouridine. Our study further implicates SOD1 as the seeding particle responsible for the spread of SOD1-FALS neurodegeneration from its initial onset site(s, and demonstrates two potential therapeutic strategies for SOD1-mediated disease. This work also comprises a medium-throughput cell-based platform of screening potential therapeutics to attenuate propagated aggregation of SOD1.

A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment

DEFF Research Database (Denmark)

Korshunova, Irina; Gjørlund, Michelle D; Jacobsen, Sylwia Owczarek

2015-01-01

neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit...... in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site...... receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated...

Oxidative phosphorylation revisited

DEFF Research Database (Denmark)

Nath, Sunil; Villadsen, John

2015-01-01

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

Science.gov (United States)

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation.

Science.gov (United States)

Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V; Liang, Jie; Arkowitz, Robert A; Stone, David E

2016-04-12

Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion. Copyright © 2016, American Association for the Advancement of Science.

Suppression of AKT phosphorylation restores rapamycin-based synthetic lethality in SMAD4-defective pancreatic cancer cells.

Science.gov (United States)

Le Gendre, Onica; Sookdeo, Ayisha; Duliepre, Stephie-Anne; Utter, Matthew; Frias, Maria; Foster, David A

2013-05-01

mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling. ©2013 AACR.

Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

International Nuclear Information System (INIS)

Tomita, Masanori

2010-01-01

Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

Learning about individuals' health from aggregate data.

Science.gov (United States)

Colbaugh, Rich; Glass, Kristin

2017-07-01

There is growing awareness that user-generated social media content contains valuable health-related information and is more convenient to collect than typical health data. For example, Twitter has been employed to predict aggregate-level outcomes, such as regional rates of diabetes and child poverty, and to identify individual cases of depression and food poisoning. Models which make aggregate-level inferences can be induced from aggregate data, and consequently are straightforward to build. In contrast, learning models that produce individual-level (IL) predictions, which are more informative, usually requires a large number of difficult-to-acquire labeled IL examples. This paper presents a new machine learning method which achieves the best of both worlds, enabling IL models to be learned from aggregate labels. The algorithm makes predictions by combining unsupervised feature extraction, aggregate-based modeling, and optimal integration of aggregate-level and IL information. Two case studies illustrate how to learn health-relevant IL prediction models using only aggregate labels, and show that these models perform as well as state-of-the-art models trained on hundreds or thousands of labeled individuals.

Effect of carbon sources on the aggregation of photo fermentative bacteria induced by L-cysteine for enhancing hydrogen production.