Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

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De Santanu

2012-01-01

Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

14-3-3ε boosts bleomycin-induced DNA damage response by inhibiting the drug-resistant activity of MVP.

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Tang, Siwei; Bai, Chen; Yang, Pengyuan; Chen, Xian

2013-06-07

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC-MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein-protein interactions.

14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

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Michael P Grant

Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

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Schmitz, Matthias; Ebert, Elisabeth; Stoeck, Katharina; Karch, André; Collins, Steven; Calero, Miguel; Sklaviadis, Theodor; Laplanche, Jean-Louis; Golanska, Ewa; Baldeiras, Ines; Satoh, Katsuya; Sanchez-Valle, Raquel; Ladogana, Anna; Skinningsrud, Anders; Hammarin, Anna-Lena; Mitrova, Eva; Llorens, Franc; Kim, Yong Sun; Green, Alison; Zerr, Inga

2016-05-01

At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/μL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3.

P53 suppresses expression of the 14-3-3gamma oncogene

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Qi Wenqing

2011-08-01

Full Text Available Abstract Background 14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro. Methods qRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC. Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter. Results Quantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3gamma expression. Conclusion Increased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

Comparative interactomics: analysis of arabidopsis 14-3-3 complexes reveals highly conserved 14-3-3 interactions between humans and plants.

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Paul, Anna-Lisa; Liu, Li; McClung, Scott; Laughner, Beth; Chen, Sixue; Ferl, Robert J

2009-04-01

As a first step in the broad characterization of plant 14-3-3 multiprotein complexes in vivo, stringent and specific antibody affinity purification was used to capture 14-3-3s together with their interacting proteins from extracts of Arabidopsis cell suspension cultures. Approximately 120 proteins were identified as potential in vivo 14-3-3 interacting proteins by mass spectrometry of the recovered complexes. Comparison of the proteins in this data set with the 14-3-3 interacting proteins from a similar study in human embryonic kidney cell cultures revealed eight interacting proteins that likely represent reasonably abundant, fundamental 14-3-3 interaction complexes that are highly conserved across all eukaryotes. The Arabidopsis 14-3-3 interaction data set was also compared to a yeast in vivo 14-3-3 interaction data set. Four 14-3-3 interacting proteins are conserved in yeast, humans, and Arabidopsis. Comparisons of the data sets based on biochemical function revealed many additional similarities in the human and Arabidopsis data sets that represent conserved functional interactions, while also leaving many proteins uniquely identified in either Arabidopsis or human cells. In particular, the Arabidopsis interaction data set is enriched for proteins involved in metabolism.

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

DEFF Research Database (Denmark)

Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

2012-01-01

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...... and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate...

Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

Czech Academy of Sciences Publication Activity Database

Řežábková, L.; Kacířová, M.; Šulc, Miroslav; Herman, P.; Večeř, J.; Štěpánek, M.; Obšilová, Veronika; Obšil, T.

2012-01-01

Roč. 103, č. 9 (2012), s. 1960-1969 ISSN 0006-3495 Institutional support: RVO:61388971 ; RVO:67985823 Keywords : phosducin * 14-3-3 protein * fluorescence Subject RIV: CE - Biochemistry Impact factor: 3.668, year: 2012

Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response.

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Liu, Ziyan; Jia, Yuxin; Ding, Yanglin; Shi, Yiting; Li, Zhen; Guo, Yan; Gong, Zhizhong; Yang, Shuhua

2017-04-06

In plant cells, changes in fluidity of the plasma membrane may serve as the primary sensor of cold stress; however, the precise mechanism and how the cell transduces and fine-tunes cold signals remain elusive. Here we show that the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1) phosphorylates 14-3-3 proteins. The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus, where they interact with and destabilize the key cold-responsive C-repeat-binding factor (CBF) proteins. Consistent with this, the crpk1 and 14-3-3κλ mutants show enhanced freezing tolerance, and transgenic plants overexpressing 14-3-3λ show reduced freezing tolerance. Further study shows that CRPK1 is essential for the nuclear translocation of 14-3-3 proteins and for 14-3-3 function in freezing tolerance. Thus, our study reveals that the CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus to modulate CBF stability, which ensures a faithfully adjusted response to cold stress of plants. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

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Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

2009-06-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

Czech Academy of Sciences Publication Activity Database

Alblová, Miroslava; Šmídová, Aneta; Dočekal, V.; Veselý, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

2017-01-01

Roč. 114, č. 46 (2017), E9811-E9820 ISSN 0027-8424 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA17-00726S Institutional support: RVO:67985823 Keywords : 14-3-3 protein * trehalase * crystal structure * enzyme * allostery Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemical research methods Impact factor: 9.661, year: 2016

Do 14-3-3 proteins and plasma membrane H+-ATPases interact in the barley epidermis in response to the barley powdery mildew fungus?

DEFF Research Database (Denmark)

Finnie, C.; Andersen, C.H.; Borch, J.

2002-01-01

14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon...... inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHA1), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus......, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H...

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro.

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Meller, R; Schindler, C K; Chu, X P; Xiong, Z G; Cameron, J A; Simon, R P; Henshall, D C

2003-05-01

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

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.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program.

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Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

2015-01-01

Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD.

CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

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Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

2016-09-01

Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases.

Structural and biophysical characterization of the PI4KB:14-3-3 protein complex

Czech Academy of Sciences Publication Activity Database

Chalupská, Dominika; Eisenreichová, Andrea; Rozycki, B.; Řežábková, L.; Humpolíčková, Jana; Klíma, Martin; Bouřa, Evžen

2017-01-01

Roč. 284, Suppl 1 (2017), s. 191 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : PI4KB * 14-3-3 proteins Subject RIV: CE - Biochemistry

Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response.

Science.gov (United States)

Wang, Chen; Skinner, Craig; Easlon, Erin; Lin, Su-Ju

2009-12-01

Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from age-dependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1Delta-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1Delta mutant also displays a decreased reactive oxygen species level and increased heat-shock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR- and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

Structure of a 14-3-3σ–YAP phosphopeptide complex at 1.15 Å resolution

International Nuclear Information System (INIS)

Schumacher, Benjamin; Skwarczynska, Malgorzata; Rose, Rolf; Ottmann, Christian

2010-01-01

The first structure of a 14-3-3 protein–phosphopeptide complex is reported at 1.15 Å resolution. The YAP 14-3-3-binding motif is revealed for the first time using crystallographic tools. The 14-3-3 proteins are a class of eukaryotic acidic adapter proteins, with seven isoforms in humans. 14-3-3 proteins mediate their biological function by binding to target proteins and influencing their activity. They are involved in pivotal pathways in the cell such as signal transduction, gene expression, enzyme activation, cell division and apoptosis. The Yes-associated protein (YAP) is a WW-domain protein that exists in two transcript variants of 48 and 54 kDa in humans. By transducing signals from the cytoplasm to the nucleus, YAP is important for transcriptional regulation. In both variants, interaction with 14-3-3 proteins after phosphorylation of Ser127 is important for nucleocytoplasmic trafficking, via which the localization of YAP is controlled. In this study, 14-3-3σ has been cloned, purified and crystallized in complex with a phosphopeptide from the YAP 14-3-3-binding domain, which led to a crystal that diffracted to 1.15 Å resolution. The crystals belonged to space group C222 1 , with unit-cell parameters a = 82.3, b = 112.1, c = 62.9 Å

Changes in Brain 14-3-3 Proteins in Response to Insulin Resistance Induced by a High Palatable Diet.

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Bock, Hugo; Zimmer, Aline Rigon; Zimmer, Eduardo Rigon; de Souza, Diogo Onofre Gomes; Portela, Luis Valmor Cruz; Saraiva-Pereira, Maria Luiza

2015-08-01

The 14-3-3 protein family takes part in a wide range of cellular processes and is expressed in all eukaryotic organisms. In mammals, seven isoforms (β, ε, η, γ, τ, ζ, and σ) have been identified. 14-3-3 proteins are suggested to modulate the insulin-signaling cascade in the brain. The aim of this study was to investigate whether insulin resistance state induced by high palatable diet modulates expression of the 14-3-3 proteins in brain. Wistar male rats (n = 8) were divided into two experimental groups: insulin resistant (IR), induced by high palatable diet, and control (CO) group. Biochemical parameters (glucose tolerance test and plasma lipid profile) were evaluated after 130 days. Brain structures (cortex and hippocampus) were dissected for evaluation of messenger RNA (mRNA) and protein levels of different 14-3-3 proteins. Statistical analyses included Student t test and Pearson correlation. Significant decrease was observed in Ywhah and in Ywahq mRNA levels in the cortex of IR group, while no changes were observed in the hippocampus. Significant increase of θ isoform was observed in hippocampus IR group by immunodetection, while no differences were detected in the remaining isoforms. Inverse correlation was observed between blood glucose levels in cortex IR group and both Ywhah and Ywhaq mRNA levels. Protein levels of Creb and phosphatidylinositide 3-kinases (PI3K) showed to be increased in the hippocampus. These alterations may be due to a compensatory effect of impaired insulin signaling. We demonstrated differential expression of 14-3-3 isoforms throughout brain regions of rats with IR. As a whole, our results indicate that brain 14-3-3 levels are influenced by different diets.

Detection of the circulating antigen 14-3-3 protein of Schistosoma japonicum by time-resolved fluoroimmunoassay in rabbits

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Wang Jie

2011-05-01

Full Text Available Abstract Background Schistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum. Results The detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection. Conclusions A sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

Czech Academy of Sciences Publication Activity Database

Petrvalská, Olivia; Košek, Dalibor; Kukačka, Zdeněk; Tošner, Z.; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

2016-01-01

Roč. 291, č. 39 (2016), s. 20753-20765 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-10061S Institutional support: RVO:67985823 ; RVO:61388971 Keywords : 14-3-3 protein * apoptosis signal-regulating kinase 1 (ASK1) * fluorescence * nuclear magnetic resonance (NMR) * protein cross-linking * small-angle x-ray scattering (SAXS) Subject RIV: CE - Biochemistry Impact factor: 4.125, year: 2016

14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

2010-02-19

14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

14-3-3σ controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

International Nuclear Information System (INIS)

Xin, Ying; Lu, Qingxian; Li, Qiutang

2010-01-01

14-3-3σ (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3σ mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3σ activity in corneal epithelial cells by overexpressing dominative negative 14-3-3σ led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3σ mutant-expressing corneal epithelial cells. We conclude that 14-3-3σ is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

Czech Academy of Sciences Publication Activity Database

Kacířová, Miroslava; Košek, Dalibor; Kádek, Alan; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

2015-01-01

Roč. 290, č. 26 (2015), s. 16246-16260 ISSN 0021-9258 Grant - others:GA ČR(CZ) GAP305/11/0708 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : Phosducin * 14-3-3 protein * fluorescence spectroscopy * SAXS * hydrogen-deuterium exchange Subject RIV: CE - Biochemistry Impact factor: 4.258, year: 2015

Role of the EF-hand-like Motif in the 14-3-3 Protein- mediated Activation of Yeast Neutral Trehalase Nth1

Czech Academy of Sciences Publication Activity Database

Kopecká, Miroslava; Košek, Dalibor; Kukačka, Zdeněk; Řežábková, Lenka; Man, Petr; Novák, Petr; Obšil, T.; Obšilová, Veronika

2014-01-01

Roč. 289, č. 20 (2014), s. 13948-13961 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GAP207/11/0455 Grant - others:Univerzita Karlova(CZ) 644313; Univerzita Karlova(CZ) 800413 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : calcium * enzyme mechanisms * mass spectrometry (MS) * protein cross-linking * protein structure * 14-3-3 * Bmh * H/D exchange * neutral trehalase * SAXS Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

Science.gov (United States)

Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

2016-08-20

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

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Gi Won Seo

2016-08-01

Full Text Available The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

Altered expression of 14-3-3ζ protein in spinal cords of rat fetuses with spina bifida aperta.

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Li-na Wu

Full Text Available BACKGROUND: A large number of studies have confirmed that excessive apoptosis is one of the reasons for deficient neuronal function in neural tube defects (NTDs. A previous study from our laboratory used 2-D gel electrophoresis to demonstrate that 14-3-3ζ expression was low in the spinal cords of rat fetuses with spina bifida aperta at embryonic day (E 17. As a member of the 14-3-3 protein family, 14-3-3ζ plays a crucial role in the determination of cell fate and anti-apoptotic activity. However, neither the expression of 14-3-3ζ in defective spinal cords, nor the correlation between 14-3-3ζ and excessive apoptosis in NTDs has been fully confirmed. METHODOLOGY/PRINCIPAL FINDINGS: We used immunoblotting and quantitative real-time PCR (qRT-PCR to quantify the expression of 14-3-3ζ and double immunofluorescence to visualize 14-3-3ζ and apoptosis. We found that, compared with controls, 14-3-3ζ was down-regulated in spina bifida between E12 and E15. Excessive apoptotic cells and low expression of 14-3-3ζ were observed in the dorsal region of spinal cords with spina bifida during the same time period. To initially explore the molecular mechanisms of apoptosis in NTDs, we investigated the expression of microRNA-7 (miR-7, microRNA-375 (miR-375 and microRNA-451 (miR-451, which are known to down-regulate 14-3-3ζ in several different cell types. We also investigated the expression of p53, a molecule that is downstream of 14-3-3ζ and can be down-regulated by it. We discovered that, in contrast to the reduction of 14-3-3ζ expression, the expression of miR-451, miR-375 and p53 increased in spina bifida rat fetuses. CONCLUSIONS/SIGNIFICANCE: These data suggest that the reduced expression of 14-3-3ζ plays a role in the excessive apoptosis that occurs in spina bifida and may be partly regulated by the over-expression of miR-451 and miR-375, and the consequent up-regulation of p53 might further promote apoptosis in spina bifida.

Use of 14-3-3 and other brain-specific proteins in CSF in the diagnosis of variant Creutzfeldt-Jakob disease

Science.gov (United States)

Green, A; Thompson, E; Stewart, G; Zeidler, M; McKenzie, J; MacLeod, M; Ironside, J; Will, R; Knight, R

2001-01-01

OBJECTIVES—The detection of the protein 14-3-3 in the CSF has been shown to be a reliable and sensitive marker for sporadic Creutzfeldt-Jakob disease (CJD). Other brain-specific proteins such as neuron specific enolase (NSE), S-100b, and tau protein have also been reported to be increased in the CSF of patients with sporadic CJD. In 1996a variant of CJD (vCJD) was described which is likely to be causally linked to the bovine spongiform encephalopathy agent. This study reports and compares the findings of CSF brain specific protein analysis in 45 patients with vCJD and in 34 control patients.
METHODS—The CSF from 45 patients with vCJD and 34 controls were investigated for the presence of 14-3-3 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with chemiluminescent detection. Tau protein, S-100b, and NSE concentrations in CSF were measured using enzyme immunoassays.
RESULTS—Protein 14-3-3 was detected in the CSF of 22/45 patients with vCJD and in 3/34 controls. The mean concentrations of NSE, S-100b, and tau protein in CSF were significantly raised in patients with vCJD compared with controls. The positive predictive value of CSF 14-3-3 was 86% and the negative predictive value was 63%. These values are lower than those reported for sporadic CJD. An increased CSF tau had a positive predictive value of 93% and a negative predictive value of 81%. The combination of CSF 14-3-3 and/or increased CSF tau had a positive predictive value of 91% and a negative predictive value of 84%.
CONCLUSIONS—CSF protein 14-3-3 is not as useful a marker for vCJD as it is for sporadic CJD. Increased concentration of CSF tau was found to be a sensitive marker of vCJD but as concentrations may be increased in many forms of non-CJD dementia, this may limit its usefulness as a diagnostic test.

 PMID:11385008

Structural Basis for the 14-3-3 Protein-Dependent Inhibition of Phosducin Function

Czech Academy of Sciences Publication Activity Database

Kacířová, Miroslava; Nováček, J.; Man, Petr; Obšilová, Veronika; Obšil, Tomáš

2017-01-01

Roč. 112, č. 7 (2017), s. 1339-1349 ISSN 0006-3495 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : phosducin * 14-3-3 protein * NMR spectroscopy * limited proteolysis Subject RIV: EB - Genetics ; Molecular Biology; CE - Biochemistry (MBU-M) OBOR OECD: Biochemical research methods; Biochemistry and molecular biology (MBU-M) Impact factor: 3.656, year: 2016

The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

KAUST Repository

Fiorillo, Annarita

2014-03-21

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

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Annarita Fiorillo

Full Text Available The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3, unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

KAUST Repository

Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

2014-01-01

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

Human 14-3-3 paralogs differences uncovered by cross-talk of phosphorylation and lysine acetylation.

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Marina Uhart

Full Text Available The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta's network, the number of acetylated partners (and the number of modify lysines is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli.

Interaction between 14-3-3β and PrP influences the dimerization of 14-3-3 and fibrillization of PrP106-126.

Science.gov (United States)

Han, Jun; Song, Qin-Qin; Sun, Peng; Zhang, Jin; Wang, Xu; Song, Juan; Li, Gong-Qi; Liu, Ying-Hui; Mei, Guo-Yong; Shi, Qi; Tian, Chan; Chen, Cao; Gao, Chen; Zhao, Bo; Dong, Xiao-Ping

2014-02-01

Proteins of the 14-3-3 family are universal participate in multiple cellular processes. However, their exact role in the pathogenesis of prion diseases remains unclear. In this study, we proposed that human PrP was able to form molecular complex with 14-3-3β. The domains responsible for the interactions between PrP and 14-3-3β were mapped at the segments of amino acid (aa) residues 106-126 within PrP and aa 1-38 within 14-3-3β. Homology modeling revealed that the key aa residues for molecular interaction were D22 and D23 in 14-3-3β as well as K110 in PrP. Mutations in these aa residues inhibited the interaction between the two proteins in vitro. Our results also showed that recombinant PrP encouraged 14-3-3β dimer formation, whereas PrP106-126 peptide inhibited it. Recombinant 14-3-3β disaggregated the mature PrP106-126 fibrils in vitro. Moreover, the PrP-14-3-3 protein complexes were observed in the brain tissues of normal and scrapie agent 263K infected hamsters. Colocalization of PrP and 14-3-3 was seen in the cytoplasm of human neuroblastoma cell line SH-SY5Y, as well as human cervical cancer cell line HeLa transiently expressing full-length human PrP. Our current data suggest the neuroprotection of PrPC and neuron damage caused by PrPSc may be associated with their functions of 14-3-3 dimerization regulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

14-3-3 Proteins and a 13-lipoxygenase form associations in a phosphorylation-dependent manner

NARCIS (Netherlands)

Holtman, W.L.; Roberts, M.R.; Wang, M.

2000-01-01

Recently, we have demonstrated by two different methods that lipoxgenases (LOXs) and 14-3-3 proteins form interactions in barley embryos [Holtman, Roberts, Oppedijk, Testerink, van Zeijl and Wang (2000) FEBS Lett. 474, 48-52]. It was shown by both co-immunoprecipitations and surface-plasmon

Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

Science.gov (United States)

Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

2015-04-03

The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

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Laura Civiero

2017-12-01

Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

Molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in bovine mammary epithelial cells.

Science.gov (United States)

Yu, Cuiping; Luo, Chaochao; Qu, Bo; Khudhair, Nagam; Gu, Xinyu; Zang, Yanli; Wang, Chunmei; Zhang, Na; Li, Qingzhang; Gao, Xuejun

2014-12-15

14-3-3γ, an isoform of the 14-3-3 protein family, was proved to be a positive regulator of mTOR pathway. Here, we analyzed the function of 14-3-3γ in protein synthesis using bovine mammary epithelial cells (BMECs). We found that 14-3-3γ interacted with eIF1AX and RPS7 by 14-3-3γ coimmunoprecipitation (CoIP) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) peptide mass fingerprinting analysis. These interactions of 14-3-3γ with eIF1AX and RPS7 were further confirmed by colocalization and fluorescence resonance energy transfer (FRET) analysis. We also found that methionine could promote protein synthesis and trigger the protein expression levels of 14-3-3γ, eIF1AX and RPS7. Analysis of overexpression and inhibition of 14-3-3γ confirmed that it positively affected the protein expression levels of eIF1AX, RPS7, Stat5 and mTOR pathway to promote protein synthesis and cell proliferation in BMECs. We further showed that overexpression of eIF1AX and RPS7 also triggered protein translation and cell proliferation. From these results, we conclude that molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in BMECs. Copyright © 2014 Elsevier Inc. All rights reserved.

Transgenic overexpression of 14-3-3 zeta protects hippocampus against endoplasmic reticulum stress and status epilepticus in vivo.

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Gary P Brennan

Full Text Available 14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ isoform has been linked to endoplasmic reticulum (ER function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress.

Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

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Karunesh Kumar

Full Text Available 14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3, sorghum (Sb14-3-3 and maize (Zm14-3-3, respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize

Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

Science.gov (United States)

Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

2015-01-01

14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization.

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Sandur, Santosh K; Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

2010-01-01

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

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Sakai Keiko

2008-04-01

Full Text Available Abstract Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.

Structural analysis of phosphatidylinositol 4-kinase III beta (PI4KB) - 14-3-3 protein complex reveals internal flexibility and explains 14-3-3 mediated protection from degradation in vitro

Czech Academy of Sciences Publication Activity Database

Chalupská, Dominika; Eisenreichová, Andrea; Rozycki, B.; Řežábková, L.; Humpolíčková, Jana; Klíma, Martin; Bouřa, Evžen

2017-01-01

Roč. 200, č. 1 (2017), s. 36-44 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA17-05200S Institutional support: RVO:61388963 Keywords : lipid * kinase * PI4KB * 14-3-3 protein * phosphatidylinositol Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.767, year: 2016

Over-expression of 14-3-3zeta is an early event in oral cancer

International Nuclear Information System (INIS)

Matta, Ajay; Bahadur, Sudhir; Duggal, Ritu; Gupta, Siddhartha D; Ralhan, Ranju

2007-01-01

The functional and clinical significance of 14-3-3 proteins in human cancers remain largely undetermined. Earlier, we have reported differential expression of 14-3-3ζ mRNA in oral squamous cell carcinoma (OSCC) by differential display. The clinical relevance of 14-3-3ζ protein in oral tumorigenesis was determined by immunohistochemistry in paraffin embedded sections of oral pre-malignant lesions (OPLs), OSCCs and histologically normal oral tissues and corroborated by Western Blotting. Co-immunoprecipitation assays were carried out to determine its association with NFκB, β-catenin and Bcl-2. Intense immunostaining of 14-3-3ζ protein was observed in 61/89 (69%) OPLs and 95/120 (79%) OSCCs. Immunohistochemistry showed significant increase in expression of 14-3-3ζ protein from normal mucosa to OPLs to OSCCs (p trend < 0.001). Significant increase in expression of 14-3-3ζ protein was observed as early as in hyperplasia (p = 0.009), with further elevation in moderate and severe dysplasia, that was sustained in OSCCs. These findings were validated by Western blotting. Using Co-immunoprecipitation, we demonstrated that 14-3-3ζ protein binds to NFκB, β-catenin and Bcl-2, suggesting its involvement in cellular signaling, leading to proliferation of oral cancer cells. Our findings suggest that over-expression of 14-3-3ζ is an early event in oral tumorigenesis and may have an important role in its development and progression. Thus, 14-3-3ζ may serve as an important molecular target for designing novel therapy for oral cancer

Serum levels of 14-3-3η protein supplement C-reactive protein and rheumatoid arthritis-associated antibodies to predict clinical and radiographic outcomes in a prospective cohort of patients with recent-onset inflammatory polyarthritis.

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Carrier, Nathalie; Marotta, Anthony; de Brum-Fernandes, Artur J; Liang, Patrick; Masetto, Ariel; Ménard, Henri A; Maksymowych, Walter P; Boire, Gilles

2016-02-01

Age, C-Reactive Protein (CRP) and autoantibodies (Abs) are associated with worse prognosis in patients with recent-onset inflammatory polyarthritis (EPA). Serum 14-3-3η protein is a joint-derived biomarker that up-regulates cytokines and enzymes that perpetuate local and systemic inflammation and may contribute to joint damage. Our objective was to evaluate, over a 5-year prospective period of observation, the additional prognostic potential of serum 14-3-3η protein in EPA patients. Clinical variables, serum and radiographs (scored according to the Sharp/van der Heijde (SvH) method) were collected serially. Relationships between serum 14-3-3η protein and other biomarkers were computed with Spearman correlations. Outcomes were Simple Disease Activity Index (SDAI) scores and joint damage progression: ΔSvH for SvH score and ΔErosion for its Erosive component. The additional predictive contribution of 14-3-3η was defined using generalized estimating equations (GEE) and generalized linear mixed models (GLMM). Among 331 patients, baseline 14-3-3η was ≥0.19 and ≥0.50 ng/ml in 153 (46.2 %) and 119 (36.0 %), respectively; CRP was >8.0 mg/L in 207 (62.5 %), and at least one Ab (Rheumatoid Factor, anti-CCP2 or anti-Sa/citrullinated vimentin) was positive in 170 (51.5 %). Elevated 14-3-3η levels moderately correlated with positive Abs, but not with elevated CRP. Baseline 14-3-3η ≥0.19 ng/ml was associated with more radiographic progression over 5 years. The optimal levels of baseline 14-3-3η to predict radiographic progression was defined by ROC curves at 0.50 ng/ml. Levels of 14-3-3η ≥0.50 ng/ml at baseline were associated with lower likelihoods of ever reaching SDAI remission (RR 0.79 (95 % CI 0.64-0.98), p = 0.03) and higher subsequent progression of Total and Erosion SvH scores. Elevated levels of 14-3-3η during follow-up also predicted higher subsequent progression, even in patients in SDAI remission. Decreases of 14-3-3η levels by at least 0

Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease

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Sarada Subramanian

2016-01-01

Full Text Available Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD requires demonstration of infective prion protein (PrPSc in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy.This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001 in the CSF levels of 14-3-3 protein between the CJD cases (N= 50 and disease controls (N= 70. The receiver operating characteristic (ROC analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

Differential Effects of Ethanol on c-Jun N-Terminal Kinase, 14-3-3 Proteins, and Bax in Postnatal Day 4 and Postnatal Day 7 Rat Cerebellum

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Heaton, Marieta Barrow; Paiva, Michael; Kubovic, Stacey; Kotler, Alexandra; Rogozinski, Jonathan; Swanson, Eric; Madorsky, Vladimir; Posados, Michelle

2011-01-01

These studies investigated ethanol effects on upstream cellular elements and interactions which contribute to Bax-related apoptosis in neonatal rat cerebellum at ages of peak ethanol sensitivity (postnatal day 4 [P4]), compared to later ages of relative resistance (P7). Analyses were made of basal levels of the pro-apoptotic c-jun N-termimal kinase (JNK), Bax, and the 14-3-3 anchoring proteins, as well as the responsiveness of these substances to ethanol at P4 versus P7. Dimerization of Bax with 14-3-3 was also investigated at the two ages following ethanol treatment, a process which sequesters Bax in the cytosol, thus inhibiting its mitochondrial translocation and disruption of the mitochondrial membrane potential. Cultured cerebellar granule cells were used to examine the protective potential of JNK inhibition on ethanol-mediated cell death. Basal levels of JNK were significantly higher at P4 than P7, but no differences in the other proteins were found. Activated JNK, and cytosolic and mitochondrially-translocated Bax were increased in P4 but not P7 animals following ethanol exposure, while protective 14-3-3 proteins were increased only at P7. Ethanol treatment resulted in decreases in Bax:14-3-3 heterodimers at P4, but not at P7. Inhibition of JNK activity in vitro provided partial protection against ethanol neurotoxicity. Thus, differential temporal vulnerability to ethanol in this CNS region correlates with differences in both levels of apoptosis-related substances (e.g., JNK), and differential cellular responsiveness, favoring apoptosis at the most sensitive age and survival at the resistant age. The upstream elements contributing to this vulnerability can be targets for future therapeutic strategies. PMID:22169498

Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

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Branham-O'Connor, Melissa; Robichaux, William G; Zhang, Xian-Kui; Cho, Hyeseon; Kehrl, John H; Lanier, Stephen M; Blumer, Joe B

2014-04-11

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors.

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Reis, Henning; Pütter, Carolin; Megger, Dominik A; Bracht, Thilo; Weber, Frank; Hoffmann, Andreas-C; Bertram, Stefanie; Wohlschläger, Jeremias; Hagemann, Sascha; Eisenacher, Martin; Scherag, André; Schlaak, Jörg F; Canbay, Ali; Meyer, Helmut E; Sitek, Barbara; Baba, Hideo A

2015-06-01

Hepatocellular carcinoma (HCC) is a major lethal cancer worldwide. Despite sophisticated diagnostic algorithms, the differential diagnosis of small liver nodules still is difficult. While imaging techniques have advanced, adjuvant protein-biomarkers as glypican3 (GPC3), glutamine-synthetase (GS) and heat-shock protein 70 (HSP70) have enhanced diagnostic accuracy. The aim was to further detect useful protein-biomarkers of HCC with a structured systematic approach using differential proteome techniques, bring the results to practical application and compare the diagnostic accuracy of the candidates with the established biomarkers. After label-free and gel-based proteomics (n=18 HCC/corresponding non-tumorous liver tissue (NTLT)) biomarker candidates were tested for diagnostic accuracy in immunohistochemical analyses (n=14 HCC/NTLT). Suitable candidates were further tested for consistency in comparison to known protein-biomarkers in HCC (n=78), hepatocellular adenoma (n=25; HCA), focal nodular hyperplasia (n=28; FNH) and cirrhosis (n=28). Of all protein-biomarkers, 14-3-3Sigma (14-3-3S) exhibited the most pronounced up-regulation (58.8×) in proteomics and superior diagnostic accuracy (73.0%) in the differentiation of HCC from non-tumorous hepatocytes also compared to established biomarkers as GPC3 (64.7%) and GS (45.4%). 14-3-3S was part of the best diagnostic three-biomarker panel (GPC3, HSP70, 14-3-3S) for the differentiation of HCC and HCA which is of most important significance. Exclusion of GS and inclusion of 14-3-3S in the panel (>1 marker positive) resulted in a profound increase in specificity (+44.0%) and accuracy (+11.0%) while sensitivity remained stable (96.0%). 14-3-3S is an interesting protein biomarker with the potential to further improve the accuracy of differential diagnostic process of hepatocellular tumors. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

DEFF Research Database (Denmark)

Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ

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Moeller, Hanne B; Slengerik-Hansen, Joachim; Aroankins, Takwa

2016-01-01

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With t...... levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3 θ and ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation and degradation....

Role of 14-3-3η protein on cardiac fatty acid metabolism and macrophage polarization after high fat diet induced type 2 diabetes mellitus.

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Sreedhar, Remya; Arumugam, Somasundaram; Thandavarayan, Rajarajan A; Karuppagounder, Vengadeshprabhu; Koga, Yusuke; Nakamura, Takashi; Harima, Meilei; Watanabe, Kenichi

2017-07-01

Diabetic cardiomyopathy (DCM), a metabolic disorder, is one of the leading causes of mortality around the world and its pathogenesis involves cardiac inflammation and altered metabolic profile. Altered fatty acid metabolism during DCM can cause macrophage polarization in which inflammatory M1 phenotype dominates over the anti-inflammatory M2 phenotype. Hence, it is essential to identify a specific target, which could revert the metabolic profile and thereby reducing the M1 macrophage polarization. 14-3-3η protein has several cellular protective functions especially in the heart as plenty of reports available in various animal models of heart failure including diabetes mellitus. However, its role in the cardiac fatty acid metabolism and macrophage polarization remains unidentified. The present study has been designed to delineate the effect of cardiospecific dominant negative mutation of 14-3-3η protein (DN14-3-3) on various lipid metabolism related marker proteins expressions and cardiac macrophage phenotype in high fat diet (HFD) fed mice. Feeding HFD for 12 weeks has produced significant increase in body weight in the DN14-3-3 (TG) mice than C57BL6/J (WT) mice. Western blotting and immunohistochemical staining analysis of the heart tissue has revealed an increase in the expression of markers of cardiac fatty acid synthesis related proteins in addition to the reduced expression of fatty acid oxidation related proteins in TG mice fed HFD than WT mice fed HFD. Furthermore, the M1 macrophage marker proteins were increasingly expressed while M2 markers expressions were reduced in the hearts of TG mice fed HFD. In conclusion, our current study has identified that there is a definite role for the 14-3-3η protein against the pathogenesis of heart failure via regulation of cardiac fatty acid metabolism and macrophage polarization. Copyright © 2017. Published by Elsevier Ltd.

Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis.

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Sun, Zhongfeng; Song, Jiabin; Xin, Xi'an; Xie, Xianan; Zhao, Bin

2018-01-01

Arbuscular mycorrhizal (AM) fungi are soil-borne fungi belonging to the ancient phylum Glomeromycota and are important symbionts of the arbuscular mycorrhiza, enhancing plant nutrient acquisition and resistance to various abiotic stresses. In contrast to their significant physiological implications, the molecular basis involved is poorly understood, largely due to their obligate biotrophism and complicated genetics. Here, we identify and characterize three genes termed Fm201 , Ri14-3-3 and RiBMH2 that encode 14-3-3-like proteins in the AM fungi Funneliformis mosseae and Rhizophagus irregularis , respectively. The transcriptional levels of Fm201 , Ri14-3-3 and RiBMH2 are strongly induced in the pre-symbiotic and symbiotic phases, including germinating spores, intraradical hyphae- and arbuscules-enriched roots. To functionally characterize the Fm201 , Ri14-3-3 and RiBMH2 genes, we took advantage of a yeast heterologous system owing to the lack of AM fungal transformation systems. Our data suggest that all three genes can restore the lethal Saccharomyces cerevisiae bmh1 bmh2 double mutant on galactose-containing media. Importantly, yeast one-hybrid analysis suggests that the transcription factor RiMsn2 is able to recognize the STRE (CCCCT/AGGGG) element present in the promoter region of Fm201 gene. More importantly, Host-Induced Gene Silencing of both Ri14-3-3 and RiBMH2 in Rhizophagus irregularis impairs the arbuscule formation in AM symbiosis and inhibits the expression of symbiotic PT4 and MST2 genes from plant and fungal partners, respectively. We further subjected the AM fungus- Medicago truncatula association system to drought or salinity stress. Accordingly, the expression profiles in both mycorrhizal roots and extraradical hyphae reveal that these three 14-3-3-like genes are involved in response to drought or salinity stress. Collectively, our results provide new insights into molecular functions of the AM fungal 14-3-3 proteins in abiotic stress responses and

Accuracy of diagnosis criteria in patients with suspected diagnosis of sporadic Creutzfeldt-Jakob disease and detection of 14-3-3 protein, France, 1992 to 2009.

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Peckeu, Laurene; Delasnerie-Lauprètre, Nicole; Brandel, Jean-Philippe; Salomon, Dominique; Sazdovitch, Véronique; Laplanche, Jean-Louis; Duyckaerts, Charles; Seilhean, Danielle; Haïk, Stéphane; Hauw, Jean-Jacques

2017-10-01

Diagnostic criteria of Creutzfeldt-Jakob disease (CJD), a rare and fatal transmissible nervous system disease with public health implications, are determined by clinical data, electroencephalogram (EEG), detection of 14-3-3 protein in cerebrospinal fluid (CSF), brain magnetic resonance imaging and prion protein gene examination. The specificity of protein 14-3-3 has been questioned. We reviewed data from 1,572 autopsied patients collected over an 18-year period (1992-2009) and assessed whether and how 14-3-3 detection impacted the diagnosis of sporadic CJD in France, and whether this led to the misdiagnosis of treatable disorders. 14-3-3 detection was introduced into diagnostic criteria for CJD in 1998. Diagnostic accuracy decreased from 92% for the 1992-1997 period to 85% for the 1998-2009 period. This was associated with positive detections of 14-3-3 in cases with negative EEG and alternative diagnosis at autopsy. Potentially treatable diseases were found in 163 patients (10.5%). This study confirms the usefulness of the recent modification of diagnosis criteria by the addition of the results of CSF real-time quaking-induced conversion, a method based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate that has proven to be a highly specific test for diagnosis of sporadic CJD.

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

International Nuclear Information System (INIS)

Wang, Zhihui; Tropè, Claes G; Suo, Zhenhe; Trøen, Gunhild; Yang, Guanrui; Nesland, Jahn M; Holm, Ruth

2008-01-01

14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown. We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome. In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (p = 0.001, p = 0.002 and p = 0.001, respectively) and deep invasion (p = 0.01, p = 0.001 and p = 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival. Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

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Tsukamoto, Hiroki; Takeuchi, Shino; Kubota, Kanae; Kobayashi, Yohei; Kozakai, Sao; Ukai, Ippo; Shichiku, Ayumi; Okubo, Misaki; Numasaki, Muneo; Kanemitsu, Yoshitomi; Matsumoto, Yotaro; Nochi, Tomonori; Watanabe, Kouichi; Aso, Hisashi; Tomioka, Yoshihisa

2018-05-14

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NFκB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

The Prognostic Value of 14-3-3 Isoforms in Vulvar Squamous Cell Carcinoma Cases: 14-3-3β and ε Are Independent Prognostic Factors for These Tumors

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Wang, Zhihui; Nesland, Jahn M.; Suo, Zhenhe; Trope, Claes G.; Holm, Ruth

2011-01-01

Background The 14-3-3 family is comprised of highly conserved proteins that are functionally important in the maintenance of homeostasis. Their involvement with the cell cycle, their association with proto-oncogenes and oncogenes, and their abnormal expression in various tumors has linked this family of proteins to the etiology of human cancer. Mounting evidence now indicates that 14-3-3σ is a cancer suppressor gene but the roles of the other 14-3-3 isoforms and their interactions in tumorigenesis have not yet been elucidated. In our current study, we examined the expression of 14-3-3β, γ, ε, ζ, η and τ in a large series of vulvar squamous cell carcinomas to evaluate any clinical significance. Methods Tumor biopsies from 298 vulvar carcinomas were examined by immunohistochemistry for the expression of 14-3-3β, γ, ε, ζ, η and τ. Statistical analyses were employed to validate any associations between the expression of any 14-3-3 isoform and clinicopathologic variables for this disease. Results High cytoplasmic levels of 14-3-3β, γ, ζ, ε and η were observed in 79%, 58%, 50%, 86% and 54% of the vulvar carcinomas analyzed, respectively, whereas a low nuclear expression of 14-3-3τ was present in 80% of these cases. The elevated cytoplasmic expression of 14-3-3β, γ, ε, ζ and η was further found to be associated with advanced disease and aggressive features of these cancers. The overexpression of cytoplasmic 14-3-3β and ε significantly correlated with a poor disease-specific survival by univariate analysis (P = 0.007 and P = 0.04, respectively). The independent prognostic significance of these factors was confirmed by multivariate analysis (P = 0.007 and P = 0.009, respectively). Conclusions We reveal for the first time that the 14-3-3β, γ, ε, ζ, η and τ isoforms may be involved in the progression of vulvar carcinomas. Furthermore, our analyses show that high cytoplasmic levels of 14-3-3β and ε independently correlate with

The prognostic value of 14-3-3 isoforms in vulvar squamous cell carcinoma cases: 14-3-3β and ε are independent prognostic factors for these tumors.

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Zhihui Wang

Full Text Available BACKGROUND: The 14-3-3 family is comprised of highly conserved proteins that are functionally important in the maintenance of homeostasis. Their involvement with the cell cycle, their association with proto-oncogenes and oncogenes, and their abnormal expression in various tumors has linked this family of proteins to the etiology of human cancer. Mounting evidence now indicates that 14-3-3σ is a cancer suppressor gene but the roles of the other 14-3-3 isoforms and their interactions in tumorigenesis have not yet been elucidated. In our current study, we examined the expression of 14-3-3β, γ, ε, ζ, η and τ in a large series of vulvar squamous cell carcinomas to evaluate any clinical significance. METHODS: Tumor biopsies from 298 vulvar carcinomas were examined by immunohistochemistry for the expression of 14-3-3β, γ, ε, ζ, η and τ. Statistical analyses were employed to validate any associations between the expression of any 14-3-3 isoform and clinicopathologic variables for this disease. RESULTS: High cytoplasmic levels of 14-3-3β, γ, ζ, ε and η were observed in 79%, 58%, 50%, 86% and 54% of the vulvar carcinomas analyzed, respectively, whereas a low nuclear expression of 14-3-3τ was present in 80% of these cases. The elevated cytoplasmic expression of 14-3-3β, γ, ε, ζ and η was further found to be associated with advanced disease and aggressive features of these cancers. The overexpression of cytoplasmic 14-3-3β and ε significantly correlated with a poor disease-specific survival by univariate analysis (P = 0.007 and P = 0.04, respectively. The independent prognostic significance of these factors was confirmed by multivariate analysis (P = 0.007 and P = 0.009, respectively. CONCLUSIONS: We reveal for the first time that the 14-3-3β, γ, ε, ζ, η and τ isoforms may be involved in the progression of vulvar carcinomas. Furthermore, our analyses show that high cytoplasmic levels of 14-3-3β and ε

Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat (Triticum aestivum L.

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Xinguo Wang

2016-06-01

Full Text Available The purpose of this study was to characterize Ta14S homoeologs and assess their functions in wheat seed development. The genomic and cDNA sequences of three Ta14S homoeologous genes encoding 14-3-3 proteins were isolated. Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure, whereas the cDNA sequences were variable in the 5′ and 3′-UTR. The three genes, designated as Ta14S-2A, Ta14S-2B and Ta14S-2D, were located in homoeologous group 2 chromosomes. The polypeptide chains of the three Ta14S genes were highly similar. These genes were most homologous to Hv14A from barley. Real-time quantitative PCR indicated that the three Ta14S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues. Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated. The transcription of Ta14S-2B was clearly higher during seed germination, whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition (0–12 h, but declined thereafter. The results suggested that the three Ta14S homoeologous genes have regulatory roles in seed development and germination.

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

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Wang, Zhihui; Tropè, Claes G; Suo, Zhenhe; Trøen, Gunhild; Yang, Guanrui; Nesland, Jahn M; Holm, Ruth

2008-01-01

Background 14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown. Methods We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome. Results In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (p = 0.001, p = 0.002 and p = 0.001, respectively) and deep invasion (p = 0.01, p = 0.001 and p = 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival. Conclusion Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis. PMID:18950492

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

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Suo Zhenhe

2008-10-01

Full Text Available Abstract Background 14-3-3 sigma (σ promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown. Methods We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome. Results In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (p = 0.001, p = 0.002 and p = 0.001, respectively and deep invasion (p = 0.01, p = 0.001 and p = 0.007, respectively. Variations of 14-3-3σ protein expression were not associated to disease-specific survival. Conclusion Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis.

Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress.

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Reynolds K Brobey

Full Text Available The reactive oxygen species (ROS-sensitive apoptosis signal-regulating kinase 1 (ASK1 signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1 covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2 in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx proteins simultaneously engage in a tripartite complex formation; 3 Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

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Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

2016-01-01

ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

Impaired binding of 14-3-3 to C-RAF in noonan syndrome suggests new approaches in diseases with increased ras signaling

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Molzan, M.; Schumacher, B.; Ottmann, C.; Baljuls, A.; Polzien, L.; Weyand, M.; Thiel, P.; Rose, R.; Rose, M.; Kuhenne, P.; Kaiser, M.; Rapp, U.R.; Kuhlmann, J.; Ottmann, C.

2010-01-01

The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser(259), a regulatory site for inhibition by 14-3-3 proteins. We

N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

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Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

2007-01-01

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies

Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

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Chiu-Ping Fang

Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

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Rha, Jennifer; Jones, Stephanie K; Fidler, Jonathan; Banerjee, Ayan; Leung, Sara W; Morris, Kevin J; Wong, Jennifer C; Inglis, George Andrew S; Shapiro, Lindsey; Deng, Qiudong; Cutler, Alicia A; Hanif, Adam M; Pardue, Machelle T; Schaffer, Ashleigh; Seyfried, Nicholas T; Moberg, Kenneth H; Bassell, Gary J; Escayg, Andrew; García, Paul S; Corbett, Anita H

2017-10-01

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

14-3-3σ regulates β-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3β.

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Tzu-Ching Chang

Full Text Available Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear.In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding.Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

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Chen, Yixing; Zhou, Xiaojin; Chang, Shu; Chu, Zhilin; Wang, Hanmeng; Han, Shengcheng; Wang, Yingdian

2017-12-02

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca 2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Prenatal Protein Malnutrition Decreases KCNJ3 and 2DG Activity in Rat Prefrontal Cortex

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Amaral, A.C.; Jakovcevski, M.; McGaughy, J.A.; Calderwood, S.K.; Mokler, D.J.; Rushmore, R.J.; Galler, J.R.; Akbarian, S.A.; Rosene, D.L.

2014-01-01

Prenatal protein malnutrition (PPM) in rats causes enduring changes in brain and behavior including increased cognitive rigidity and decreased inhibitory control. A preliminary gene microarray screen of PPM rat prefrontal cortex (PFC) identified alterations in KCNJ3 (GIRK1/Kir3.1), a gene important for regulating neuronal excitability. Follow-up with polymerase chain reaction and Western blot showed decreased KCNJ3 expression in PFC, but not hippocampus or brainstem. To verify localization of the effect to the PFC, baseline regional brain activity was assessed with 14C-2-deoxyglucose. Results showed decreased activation in PFC but not hippocampus. Together these findings point to the unique vulnerability of the PFC to the nutritional insult during early brain development, with enduring effects in adulthood on KCNJ3 expression and baseline metabolic activity. PMID:25446346

14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in Head and Neck cancer cells

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Macha, Muzafar A; Matta, Ajay; Chauhan, SS; Siu, KW Michael; Ralhan, Ranju

2010-01-01

The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells. Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies. Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP). GS targets 14-3-3 zeta associated cellular pathways for reducing

Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

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Hawdon John M

2009-04-01

Full Text Available Abstract Background Third-stage infective larvae (L3 of hookworms are in an obligatory state of developmental arrest that ends upon entering the definitive host, where they receive a signal that re-activates development. Recovery from the developmentally arrested dauer stage of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development. Results To determine if hookworm 14-3-3 proteins play a similar role in L3 activation, hookworm FTT-2 was identified and tested for its ability to interact with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-FTT-2, and was most closely related to FTT-2 from other nematodes. Ac-FTT-2 was expressed in HEK 293T cells, and was recognized by an antibody against human 14-3-3β isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-DAF-16 when co-expressed in serum-stimulated HEK 293T cells. This interaction requires intact Akt consensus phosphorylation sites at serine107 and threonine312, but not serine381. Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated L3 or adult A. caninum. Conclusion The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

International Nuclear Information System (INIS)

Liffers, Sven-T; Schwarte-Waldhoff, Irmgard; Meyer, Helmut E; Stühler, Kai; Hahn, Stephan A; Maghnouj, Abdelouahid; Munding, Johanna B; Jackstadt, René; Herbrand, Ulrike; Schulenborg, Thomas; Marcus, Katrin; Klein-Scory, Susanne; Schmiegel, Wolff

2011-01-01

Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells. High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction. We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization. Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

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Daniel Thomas

Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer

International Nuclear Information System (INIS)

Young, Gavin M.; Radhakrishnan, Vijayababu M.; Centuori, Sara M.; Gomes, Cecil J.; Martinez, Jesse D.

2015-01-01

The 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers. DNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression. Here we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells. To

Activity of vegetative insecticidal proteins Vip3Aa58 and Vip3Aa59 of Bacillus thuringiensis against lepidopteran pests.

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Baranek, Jakub; Kaznowski, Adam; Konecka, Edyta; Naimov, Samir

2015-09-01

Vegetative insecticidal proteins (Vips) secreted by some isolates of Bacillus thuringiensis show activity against insects and are regarded as insecticides against pests. A number of B. thuringiensis strains harbouring vip3A genes were isolated from different sources and identified by using a PCR based approach. The isolates with the highest insecticidal activity were indicated in screening tests, and their vip genes were cloned and sequenced. The analysis revealed two polymorphic Vip protein forms, which were classified as Vip3Aa58 and Vip3Aa59. After expression of the vip genes, the proteins were isolated and characterized. The activity of both toxins was estimated against economically important lepidopteran pests of woodlands (Dendrolimus pini), orchards (Cydia pomonella) and field crops (Spodoptera exigua). Vip3Aa58 and Vip3Aa59 were highly toxic and their potency surpassed those of many Cry proteins used in commercial bioinsecticides. Vip3Aa59 revealed similar larvicidal activity as Vip3Aa58 against S. exigua and C. pomonella. Despite 98% similarity of amino acid sequences of both proteins, Vip3Aa59 was significantly more active against D. pini. Additionally the effect of proteolytic activation of Vip58Aa and Vip3Aa59 on toxicity of D. pini and S. exigua was studied. Both Vip3Aa proteins did not show any activity against Tenebrio molitor (Coleoptera) larvae. The results suggest that the Vip3Aa58 and Vip3Aa59 toxins might be useful for controlling populations of insect pests of crops and forests. Copyright © 2015 Elsevier Inc. All rights reserved.

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

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Lixin Liu

2015-07-01

Full Text Available As a protective factor for lipopolysaccharide (LPS-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6, interleukin-1β (IL-1β and inducible nitric oxide synthase (iNOS. Enzyme-linked immunosorbent assay (ELISA analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB and mitogen-activated protein kinase (MAPKs and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK, p38 mitogen-activated protein kinase (p38MAPK and inhibitor of NF-κB (IκB phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR, ribosomal protein S6 kinase 1 (S6K1, serine/threonine protein kinase Akt 1 (AKT1, sterol regulatory element binding protein 1 (SREBP1 and peroxisome proliferator-activated receptor gamma

Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

Czech Academy of Sciences Publication Activity Database

Dowling, P.; Hughes, D. J.; Larkin, A.M.; Meiller, J.; Henry, M.; Meleady, P.; Lynch, V.; Pardini, B.; Naccarati, A.; Levý, M.; Vodička, Pavel; Neary, P.; Clynes, M.

2015-01-01

Roč. 441, feb. (2015), s. 133-141 ISSN 0009-8981 Institutional support: RVO:68378041 Keywords : biomarkers * colorectal cancer * proteomics * mass spectrometry * 14-3-3 proteins * pyruvate kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.799, year: 2015

Protective immunity against Eimeria maxima induced by vaccines of Em14-3-3 antigen.

Science.gov (United States)

Liu, Tingqi; Huang, Jingwei; Ehsan, Muhammad; Wang, Shuai; Fei, Hong; Zhou, Zhouyang; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2018-04-15

Eimeria maxima 14-3-3 (Em14-3-3) open reading frame (ORF) which consisted of 861 bp encoding a protein of 286 amino acids was successfully amplified and sequenced. Subsequently, the Em14-3-3 ORF was subcloned into pET-32a (+) and pVAX1, respectively. RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo. Immunofluorescence analysis showed that Em14-3-3 was expressed in both the sporozoites and merozoites. The animal experiments demonstrated that both rEm14-3-3 and pVAX1-14-3-3 could clearly alleviate jejunum lesions and body weight loss. The Em14-3-3 vaccines could increase oocyst decrease ratio, as well as produce an anticoccidial index of more than 165. The percentages of CD4 + in both the Em14-3-3 immunized groups were much higher, when compared with those of PBS, pET32a (+), and pVAX1 controls (P maxima. Copyright © 2018 Elsevier B.V. All rights reserved.

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

OpenAIRE

Wang, Zhihui; Tropè, Claes G; Suo, Zhenhe; Trøen, Gunhild; Yang, Guanrui; Nesland, Jahn M; Holm, Ruth

2008-01-01

Background 14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown. Methods ...

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Science.gov (United States)

Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

1995-08-04

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

14-3-3epsilon contributes to tumour suppression in laryngeal carcinoma by affecting apoptosis and invasion

International Nuclear Information System (INIS)

Che, Xing-Hua; Chen, Hong; Xu, Zhen-Ming; Shang, Chao; Sun, Kai-Lai; Fu, Wei-Neng

2010-01-01

14-3-3epsilon regulates a wide range of biological processes, including cell cycle control, proliferation, and apoptosis, and plays a significant role in neurogenesis and the formation of malignant tumours. However, the exact function and regulatory mechanism of 14-3-3epsilon in carcinogenesis have not been elucidated. The expression of 14-3-3epsilon was assessed by RT-PCR and western blotting. The invasiveness and viability of Hep-2 cells were determined by the transwell migration assay and MTT assay, respectively. Cell cycle and apoptosis of Hep-2 cells were detected by flow cytometry. The mRNA and protein expression of 14-3-3epsilon in larynx squamous cell carcinoma (LSCC) tissues were significantly lower than those in clear surgical margin tissues. Statistical analysis showed that the 14-3-3epsilon protein level in metastatic lymph nodes was lower than that in paired tumour tissues. In addition, the protein level of 14-3-3epsilon in stage III or IV tumours was significantly lower than that in stage I or II tumours. Compared with control Hep-2 cells, the percentages of viable cells in the 14-3-3epsilon-GFP and negative control GFP groups were 36.68 ± 14.09% and 71.68 ± 12.10%, respectively. The proportions of S phase were 22.47 ± 3.36%, 28.17 ± 3.97% and 46.15 ± 6.82%, and the apoptotic sub-G1 populations were 1.23 ± 1.02%, 2.92 ± 1.59% and 13.72 ± 3.89% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The percentages of the apoptotic cells were 0.84 ± 0.25%, 1.08 ± 0.24% and 2.93 ± 0.13% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The numbers of cells that penetrated the filter membrane in the control, negative control GFP and 14-3-3epsilon-GFP groups were 20.65 ± 1.94, 17.63 ± 1.04 and 9.1 ± 0.24, respectively, indicating significant differences among the different groups. Decreased expression of 14-3-3epsilon in LSCC tissues contributes to the initiation and progression of LSCC

Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (Musa acuminata L. AAA group, cv. Brazilian).

Science.gov (United States)

Li, Mei-Ying; Xu, Bi-Yu; Liu, Ju-Hua; Yang, Xiao-Liang; Zhang, Jian-Bin; Jia, Cai-Hong; Ren, Li-Cheng; Jin, Zhi-Qiang

2012-02-01

To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening.

Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells

International Nuclear Information System (INIS)

Vara, F.; Schneider, J.A.; Rozengurt, E.

1985-01-01

Diacylglycerol and phorbol esters activate protein kinase C in intact cells. The authors report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive 86 Rb + uptake, a measure of the activity of the Na + /K + pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na + influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt 2 ) also enhanced ouabain sensitive 86 Rb + uptake and amiloride-sensitive 22 Na + influx. Prolonged treatment (40 hr) of 3T3 cells with PBt 2 at a saturating dose, which reduces the number of PBt 2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt 2 . They suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na + /H + antiport activity, which, in turn, leads to Na + influx, intracellular pH modulation, and stimulation of the Na + /K + pump

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

International Nuclear Information System (INIS)

Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

2008-01-01

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

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Helene J Bustad

Full Text Available Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers

International Nuclear Information System (INIS)

Li, Zhaomin; Dong, Zizheng; Myer, David; Yip-Schneider, Michele; Liu, Jianguo; Cui, Ping; Schmidt, C Max; Zhang, Jian-Ting

2010-01-01

Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis. Western blot analysis was used to determine 14-3-3σ protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays. We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3σ in a pancreatic cancer cell line caused resistance to γ-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest. The increased level of 14-3-3σ protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3σ may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients

Vitamin K3-2,3-epoxide induction of apoptosis with activation of ROS-dependent ERK and JNK protein phosphorylation in human glioma cells.

Science.gov (United States)

Wu, Jender; Chien, Chih-Chiang; Yang, Liang-Yo; Huang, Guan-Cheng; Cheng, Min-Chi; Lin, Che-Tong; Shen, Shing-Chuan; Chen, Yen-Chou

2011-08-15

2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC(50) values of EPO and EPO1 in U87 cells were 37.5 and 15.7μM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells. Copyright © 2011. Published by Elsevier Ireland Ltd.

Yeast 14-3-3 proteins participate in the regulation of cell cation homeostasis via interaction with Nha1 alkali-metal-cation/proton antiporter

Czech Academy of Sciences Publication Activity Database

Zahrádka, Jaromír; Van Heusden, G.P.H.; Sychrová, Hana

2012-01-01

Roč. 1820, č. 7 (2012), s. 849-858 ISSN 0304-4165 R&D Projects: GA MŠk(CZ) LC531; GA MŠk(CZ) OC10012; GA AV ČR(CZ) IAA500110801 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : yeast * 14-3-3 proteins * ion homeostasis * Nha1 antiporter Subject RIV: CE - Biochemistry Impact factor: 3.848, year: 2012

Aberrant upregulation of 14-3-3ơ expression serves as an inferior prognostic biomarker for gastric cancer

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Li Hai-gang

2011-09-01

Full Text Available Abstract Background 14-3-3ơ is an intracellular, phosphoserine binding protein and proposed to be involved in tumorigenesis. However, the expression dynamics of 14-3-3ơ and its clinicopathological/prognostic significance in human tumors are still controversial. Methods The method of immunohistochemistry (IHC and Western blot were utilized to examine the protein expression of 14-3-3ơ in gastric cancer and paired normal adjacent gastric mucosal tissues. Receive operating characteristic (ROC curve analysis was employed to determine a cutoff score for 14-3-3ơ expression in a training set (n = 66. For validation, the ROC-derived cutoff score was subjected to analysis of the association of 14-3-3ơ expression with patient outcome and clinical characteristics in a testing set (n = 86 and overall patients (n = 152. Results The expression frequency and expression levels of 14-3-3ơ were significantly higher in gastric cancer than in normal gastric mucosal tissues. Correlation analysis demonstrated that high expression of 14-3-3ơ in gastric cancer was significantly correlated with clinical stage and tumor invasion. Furthermore, in the testing set and overall patients, Kaplan-Meier analysis showed that elevated 14-3-3ơ expression predicted poorer overall survival (OS and progression-free survival (PFS. Importantly, high 14-3-3ơ expression was also associated with shortened survival time in stage III and stage IV gastric cancer patients. Multivariate analyses revealed that 14-3-3ơ expression was an independent prognostic parameter in gastric cancer. Conclusions These findings provide evidence that high expression of 14-3-3ơ may be important in the tumor progression and servers as an independent molecular marker for poor prognosis of gastric cancer. Thus, overexpression of 14-3-3ơ identifies patients at high risk and is a novel therapeutic molecular target for this tumor.

Aberrant upregulation of 14-3-3ơ expression serves as an inferior prognostic biomarker for gastric cancer

International Nuclear Information System (INIS)

Zhou, Wei-hua; Tang, Fang; Xu, Jie; Wu, Xing; Feng, Zhi-ying; Li, Hai-gang; Lin, Dong-jun; Shao, Chun-kui; Liu, Quentin

2011-01-01

14-3-3ơ is an intracellular, phosphoserine binding protein and proposed to be involved in tumorigenesis. However, the expression dynamics of 14-3-3ơ and its clinicopathological/prognostic significance in human tumors are still controversial. The method of immunohistochemistry (IHC) and Western blot were utilized to examine the protein expression of 14-3-3ơ in gastric cancer and paired normal adjacent gastric mucosal tissues. Receive operating characteristic (ROC) curve analysis was employed to determine a cutoff score for 14-3-3ơ expression in a training set (n = 66). For validation, the ROC-derived cutoff score was subjected to analysis of the association of 14-3-3ơ expression with patient outcome and clinical characteristics in a testing set (n = 86) and overall patients (n = 152). The expression frequency and expression levels of 14-3-3ơ were significantly higher in gastric cancer than in normal gastric mucosal tissues. Correlation analysis demonstrated that high expression of 14-3-3ơ in gastric cancer was significantly correlated with clinical stage and tumor invasion. Furthermore, in the testing set and overall patients, Kaplan-Meier analysis showed that elevated 14-3-3ơ expression predicted poorer overall survival (OS) and progression-free survival (PFS). Importantly, high 14-3-3ơ expression was also associated with shortened survival time in stage III and stage IV gastric cancer patients. Multivariate analyses revealed that 14-3-3ơ expression was an independent prognostic parameter in gastric cancer. These findings provide evidence that high expression of 14-3-3ơ may be important in the tumor progression and servers as an independent molecular marker for poor prognosis of gastric cancer. Thus, overexpression of 14-3-3ơ identifies patients at high risk and is a novel therapeutic molecular target for this tumor

Activation of AMP-Activated Protein Kinase Attenuates Tumor Necrosis Factor-α-Induced Lipolysis via Protection of Perilipin in 3T3-L1 Adipocytes

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Seok-Woo Hong

2014-12-01

Full Text Available BackgroundTumor necrosis factor (TNF-α and AMP-activated protein kinase (AMPK are known to stimulate and repress lipolysis in adipocytes, respectively; however, the mechanisms regulating these processes have not been completely elucidated.MethodsThe key factors and mechanism of action of TNF-α and AMPK in lipolysis were investigated by evaluating perilipin expression and activity of protein kinase RNA-like endoplasmic reticulum kinase (PERK/eukaryotic initiation factor 2 α (eIF2α by Western blot and an immunofluorescence assay in 24-hour TNF-α-treated 3T3-L1 adipocytes with artificial manipulation of AMPK activation.ResultsEnhancement of AMPK activity by the addition of activator minoimidazole carboxamide ribonucleotide (AICAR suppressed TNF-α-induced lipolysis, whereas the addition of compound C, an inhibitor of AMPK phosphorylation, enhanced lipolysis. Perilipin, a lipid droplet-associated protein, was decreased by TNF-α and recovered following treatment with AICAR, showing a correlation with the antilipolytic effect of AICAR. Significant activation of PERK/eIF2α, a component of the unfolded protein response signaling pathway, was observed in TNF-α or vesicle-treated 3T3-L1 adipocytes. The antilipolytic effect and recovery of perilipin expression by AICAR in TNF-α-treated 3T3-L1 adipocytes were significantly diminished by treatment with 2-aminopurine, a specific inhibitor of eIF2α.ConclusionThese data indicated that AICAR-induced AMPK activation attenuates TNF-α-induced lipolysis via preservation of perilipin in 3T3-L1 adipocytes. In addition, PERK/eIF2α activity is a novel mechanism of the anti-lipolytic effect of AICAR.

Genetic polymorphism of horse serum protein 3 (SP3).

Science.gov (United States)

Juneja, R K; Sandberg, K; Kuryl, J; Gahne, B

1989-01-01

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles (D, F, I, S). Evidence was provided that the F allele can be further divided into two alleles (F1 and F2); the mobilities of F1 and F2 variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.

Synthesis of [3-14C]- and [phenyl-U-14C] olaquindox

International Nuclear Information System (INIS)

Maul, W.; Scherling, D.; Seng, F.

1981-01-01

Olaquindox is a new feed additive. [ 14 C]Olaquindox, labelled in different positions, was needed for tracer-studies of pharmacokinetics, biotransformation and residues in several species of animals. 2-[N-(2-hydroxethyl)-carbamoyl]-3-methyl-[3- 14 C]quinoxaline-1,4-dioxide([3- 14 C]Olaquindox) was synthesized from barium[ 14 C]carbonate (22 mmoles; 1.15 Ci) via [1- 14 C]acetic acid, sodium[1- 14 C]acetate, [1- 14 C]acetylchloride, ethyl[3- 14 C]acetoacetate and 2-carbethoxy-3-methyl-[3- 14 C]quinoxaline-1,4-dioxide with an overall yield of 10%, based on barium[ 14 C]carbonate. The radiochemical purity was better than 98% (tlc). The specific activities of three preparations were 10.5, 8.4 and 5.45 μCi/mg respectively. [phenyl-U- 14 C]Olaquindox was synthesized starting from [U- 14 C]aniline (19.8 mmoles; 284.4 mCi). Intermediate products were N-acetyl[U- 14 C]aniline, 2-nitro-N-acetyl[U- 14 C]aniline, 2-nitro[U- 14 C]aniline and [U- 14 C]benzofurazanoxide. The total yield was 50% as calculated for [U- 14 C]aniline. At calibration samples of two preparations showed specific activities of 49.5 and 11.1 μCi/mg respectively. The radiochemical purity was checked by tlc and exceeded 98%. (author)

Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14

NARCIS (Netherlands)

Meijers, J. C.; Chung, D. W.

1991-01-01

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts

Nucleocytoplasmic shuttling activity of ataxin-3.

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Sandra Macedo-Ribeiro

2009-06-01

Full Text Available Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease (MJD, is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated genes. Disease protein misfolding and aggregation, often within the nucleus of affected neurons, characterize polyglutamine disorders. Several evidences have implicated the nucleus as the primary site of pathogenesis for MJD. However, the molecular determinants for the nucleocytoplasmic transport of human ataxin-3 (Atx3, the protein which is mutated in patients with MJD, are not characterized. In order to characterize the nuclear shuttling activity of Atx3, we performed yeast nuclear import assays and found that Atx3 is actively imported into the nucleus, by means of a classical nuclear localizing sequence formed by a cluster of lysine and arginine residues. On the other hand, when active nuclear export was inhibited using leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, both endogenous Atx3 and transfected GFP-Atx3 accumulated inside the nucleus of a subpopulation of COS-7 cells, whereas both proteins are normally predominant in the cytoplasm. Additionally, using a Rev(1.4-GFP nuclear export assay, we performed an extensive analysis of six putative aliphatic nuclear export motifs identified in Atx3 amino acid sequence. Although none of the tested peptide sequences were found to drive nuclear export when isolated, we have successfully mapped the region of Atx3 responsible for its CRM1-independent nuclear export activity. Curiously, the N-terminal Josephin domain alone is exported into the cytoplasm, but the nuclear export activity of Atx3 is significantly enhanced in a longer construct that is truncated after the two ubiquitin interaction motifs, upstream from the polyQ tract. Our data show that Atx3 is actively imported to and exported from the cell nucleus, and that its nuclear export activity is dependent on a motif

Environmental 14C and 3H activities: global trends and local contamination

International Nuclear Information System (INIS)

Krajcar Bronic, I.; Obelic, B.; Horvatincic, N.

2000-01-01

The anthropogenic disturbance of natural distributions of radiocarbon ( 14 C) and tritium ( 3 H) due to the release of bomb-produced isotopes occurred after the World War II and at the same time the monitoring of these isotopes started at several stations in the world. Radioactive isotopes 14 C and 3 H, together with the stable isotopes 2 H and 18 O, are very important tracers in environmental, climatological and hydrological studies. Monitoring of environmental levels of 14 C and 3 H in Croatia started more then 20 years ago, while that of the stable isotopes somewhat later. The monitoring was performed at the three types of stations: a) 'clean-air' sites, which are supposed to reflect only the global disturbance of the atmospheric isotope concentrations, b) in a densely populated industrial center, where the effect of intense fossil-fuel combustion is expected, and local contamination from institutions using radioactive-labeled material is also possible, and c) at locations around the Nuclear Power Plant Krsko. The mean yearly 3 H activities in precipitation continuously decrease since the beginning of monitoring approaching slowly the natural equilibrium. The monthly 3 H activities show seasonal variations, with maximum in early summer and minimum in early winter. Both seasonal variations and the decrease of the mean yearly values are typical for continental stations of the Northern Hemisphere. At the sampling site located at the Institute, several periods of higher 3 H activities were observed, due to the local contamination with the tritium-labeled material. The 14 C concentration in the atmosphere shows also the continuous decrease of the mean yearly values and superposed seasonal fluctuations, with higher activity during summer. Seasonal peak-to-peak variations are higher in the area of the city of Zagreb than at the clean-air site on the mountain (about 1000 m a.s.l.). This difference is caused by the introduction of CO 2 (containing no 14 C isotope

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Science.gov (United States)

Mangin, P H; Receveur, N; Wurtz, V; David, T; Gachet, C; Lanza, F

2009-09-01

Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Generation of 3D templates of active sites of proteins with rigid prosthetic groups.

Science.gov (United States)

Nebel, Jean-Christophe

2006-05-15

With the increasing availability of protein structures, the generation of biologically meaningful 3D patterns from the simultaneous alignment of several protein structures is an exciting prospect: active sites could be better understood, protein functions and protein 3D structures could be predicted more accurately. Although patterns can already be generated at the fold and topological levels, no system produces high-resolution 3D patterns including atom and cavity positions. To address this challenge, our research focuses on generating patterns from proteins with rigid prosthetic groups. Since these groups are key elements of protein active sites, the generated 3D patterns are expected to be biologically meaningful. In this paper, we present a new approach which allows the generation of 3D patterns from proteins with rigid prosthetic groups. Using 237 protein chains representing proteins containing porphyrin rings, our method was validated by comparing 3D templates generated from homologues with the 3D structure of the proteins they model. Atom positions were predicted reliably: 93% of them had an accuracy of 1.00 A or less. Moreover, similar results were obtained regarding chemical group and cavity positions. Results also suggested our system could contribute to the validation of 3D protein models. Finally, a 3D template was generated for the active site of human cytochrome P450 CYP17, the 3D structure of which is unknown. Its analysis showed that it is biologically meaningful: our method detected the main patterns of the cytochrome P450 superfamily and the motifs linked to catalytic reactions. The 3D template also suggested the position of a residue, which could be involved in a hydrogen bond with CYP17 substrates and the shape and location of a cavity. Comparisons with independently generated 3D models comforted these hypotheses. Alignment software (Nestor3D) is available at http://www.kingston.ac.uk/~ku33185/Nestor3D.html

Quantitation of some amino-terminal residues in proteins using 3H-labelled dansyl chloride and 14C labelled amino acids

International Nuclear Information System (INIS)

Flengsrud, R.

1979-01-01

A method for quantitation of amino-terminal residues in proteins is presented. The method is a modification of a double isotope-labelling technique, using 3 H-labelled dansyl chloride and 14 C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3 H/ 14 C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations of +- 7.4%, +-3.4% and +-10.3%, respectively. An application of the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used. (author)

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As+3- and MMA+3-induced apoptosis through inhibition of telomerase activity via JNK activation

International Nuclear Information System (INIS)

Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

2008-01-01

The effects of six arsenic compounds including As +3 , MMA +3 , DMA +3 , As +5 , MMA +5 , and DMA +5 on the viability of NIH3T3 cells were examined. As +3 and MMA +3 , but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As +3 and MMA +3 were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As +3 and MMA +3 treatments. An increase in the intracellular peroxide level was examined in As +3 - and MMA +3 -treated NIH3T3 cells, and As +3 - and MMA +3 -induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As +3 - and MMA +3 -induced cytotoxicity. Suppression of JNKs significantly inhibited As +3 - and MMA +3 -induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As +3 - and MMA +3 -induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As +3 or MMA +3 . These data provide the first evidence to indicate that apoptosis induced by As +3 and MMA +3 is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved

Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line

International Nuclear Information System (INIS)

Vaskivuo, Liisa; Rysae, Jaana; Koivuperae, Johanna; Myllynen, Paeivi; Vaskivuo, Tommi; Chvalova, Katerina; Serpi, Raisa; Savolainen, Eeva-Riitta; Puistola, Ulla; Vaehaekangas, Kirsi

2006-01-01

p14 ARF tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14 ARF is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14 ARF to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14 ARF protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14 ARF protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14 ARF can respond to DNA damage without oncogene activation in cell lines without functional p53

Nuclear protein IκB-ζ inhibits the activity of STAT3

International Nuclear Information System (INIS)

Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

2009-01-01

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+-ATPase by combining X-ray crystallography and electron cryomicroscopy

NARCIS (Netherlands)

Ottmann, C.; Marco, S.; Jaspert, N.; Marcon, C.; Schauer, N.; Weyand, M.; Vandermeeren, C.; Duby, G.; Boutry, M.; Wittinghofer, A.; Rigaud, J.-L.; Oecking, C.

2007-01-01

Regulatory 14-3-3 proteins activate the plant plasma membrane H+-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

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Ryunosuke Muro

Full Text Available The Ras-mitogen-activated protein kinase (MAPK pathway is crucial for T cell receptor (TCR signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3 is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling.

Science.gov (United States)

Guo, Xing; Ramirez, Alejandro; Waddell, David S; Li, Zhizhong; Liu, Xuedong; Wang, Xiao-Fan

2008-01-01

The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.

Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

Science.gov (United States)

De Groene, E M; Nijmeijer, S M; Horbach, G J; Witkamp, R F

1995-09-07

Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly

Directory of Open Access Journals (Sweden)

Williams David E

2011-05-01

Full Text Available Abstract Background Histone deacetylase (HDAC inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN, an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells. Results Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1. Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest. Conclusion The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor

Generation of 3D templates of active sites of proteins with rigid prosthetic groups

OpenAIRE

Nebel, Jean-Christophe

2006-01-01

MOTIVATION: With the increasing availability of protein structures, the generation of biologically meaningful 3D patterns from the simultaneous alignment of several protein structures is an exciting prospect: active sites could be better understood, protein functions and protein 3D structures could be predicted more accurately. Although patterns can already be generated at the fold and topological levels, no system produces high-resolution 3D patterns including atom and cavity positions. To a...

Crystal structure and magnetic properties of the Ba3TeCo3P2O14, Pb3TeCo3P2O14, and Pb3TeCo3V2O14 langasites

DEFF Research Database (Denmark)

Krizan, J.W.; de la Cruz, C.; Andersen, Niels Hessel

2013-01-01

We report the structural and magnetic characterizations of Ba3TeCo3P2O14, Pb3TeCo3P2O14, and Pb3TeCo3V2O14, compounds that are based on the mineral dugganite, which is isostructural to langasites. The magnetic part of the structure consists of layers of Co2+ triangles. Nuclear and magnetic...... structures were determined through a co-refinement of synchrotron and neutron powder diffraction data. In contrast to the undistorted P321 langasite structure of Ba3TeCo3P2O14, a complex structural distortion yielding a large supercell is found for both Pb3TeCo3P2O14 and Pb3TeCo3V2O14. Comparison...... of the three compounds studied along with the zinc analog Pb3TeZn3P2O14, also characterized here, suggests that the distortion is driven by Pb2+ lone pairs; as such, the Pb compounds crystallize in a pyroelectric space group, P2. Magnetic susceptibility, magnetization, and heat capacity measurements were...

THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

Directory of Open Access Journals (Sweden)

O. L. Nosareva

2015-01-01

Full Text Available The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro.Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N-ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method.Results. Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study.Conclusion. The carried out research shows that caspase-3 and protein

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

Science.gov (United States)

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

Czech Academy of Sciences Publication Activity Database

Procházka, Radek; Blaha, Milan

2015-01-01

Roč. 61, č. 6 (2015), s. 495-502 ISSN 0916-8818 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : cumulus oocyte complexes * meiosis resumption * mitogen-activated protein kinase 3/1 (MAPK3/1) Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.453, year: 2015

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

International Nuclear Information System (INIS)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-01-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [ 3 H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating

Problems associated with scintillation counting of NaH14CO3 and gel suspension counting of Ba14CO3

International Nuclear Information System (INIS)

MacRae, J.C.; Wilson, S.

1978-01-01

Liquid NaH 14 CO 3 was assayed in emulsion-type (NE260 and Unisolve) and dioxan-based (NE250) scintillation cocktails contained in glass or polyethylene vials kept at 2 0 or 24-30 0 C. Different particle size ranges of standard Ba 14 CO 3 were assayed by gel suspension counting in Cabosil scintillation cocktail and in NE260 following solubilisation in EDTA-tetrasodium salt. Initial detectable activities of NaH 14 CO 3 in glass and polyethylene vials in NE250, NE260 and Unisolve were 97 and 96, 68 and 83, 71 and 89% of the true value respectively. Subsequent losses of activity over 7 days with the emulsion-type scintillators was greater from the polyethylene vials. Addition of phenylethylamine to the NE260 and Unisolve cocktails gave the true activity levels for all vials with no loss of activity over 6 days. When different particle size ranges of Standard Ba 14 CO 3 were suspended in Cabosil scintillation cocktail there was considerable variation in counting efficiency (77-88%) with little relationship between particle size and counting efficiency. The relationship between counting efficiency and channels ratio was not sufficiently precise for predictive purposes. Solubilisation of Ba 14 CO 3 in EDTA-tetrasodium salt gave similar counting efficiency and channels ratio values for all samples. (U.K.)

UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation.

Science.gov (United States)

Kim, Mihwa; Morales, Liza D; Baek, Minwoo; Slaga, Thomas J; DiGiovanni, John; Kim, Dae Joon

2017-10-31

Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation.

Differentially expressed proteins on postoperative 3

Directory of Open Access Journals (Sweden)

Jialili Ainuer

2011-04-01

Full Text Available 【Abstract】Objectives: Surgical repair of Achilles tendon (AT rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods: Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n=16 received postoperative cast immobilization; Group B (early motion group, n=16 received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing twodimensional polyacrylamide gel electrophoresis (2D-PAGE. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI protein database retrieval and then for bioinformatics analysis. Results: A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

Energy Technology Data Exchange (ETDEWEB)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-12-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with (/sup 3/H)DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating.

Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation.

Science.gov (United States)

Zhou, Haoming; Wang, Han; Ni, Ming; Yue, Shi; Xia, Yongxiang; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Lu, Ling; Wang, Xuehao; Zhai, Yuan

2018-02-13

Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection

A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response

DEFF Research Database (Denmark)

Blasius, Melanie; Wagner, Sebastian A; Choudhary, Chuna Ram

2014-01-01

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome...

Radiobiological half-lives for carbon-14 and hydrogen-3 leucine in man

International Nuclear Information System (INIS)

Classic, K.L.; Schwenk, W.F.; Haymond, M.W.

1986-01-01

In vivo estimates of protein metabolism in many are often made by oral or intravenous administration of leucine or its ∼-ketoacid, ∼-ketoisocaproate, labeled with 14 C or 3 H. Previous estimates of radiation dose from such tracers have been based on the measurement of 14 CO 2 in breath. Using measurements of the decay of 3 H or 14 C leucine from plasma proteins, longer biological half-lives for these compounds were obtained. The estimated total-body radiation absorbed dose is 0.97 mrad/uCi for [1- 14 C]KIC (or [1- 14 C]leucine) and 0.11 mrad/ + Ci for ]4,5- 3 H]leucine (or [ 3 H]KIC). Assuming administered doses of 100 μCi each, the total-body radiation absorbed dose is still well within the limits set by the FDA for Radioactive Drug Research Committees. 12 references, 3 figures, 3 tables

The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: Involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end

DEFF Research Database (Denmark)

Fuglsang, Anja T; Borch, Jonas; Bych, Katrine

2003-01-01

14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine...... of the Arabidopsis plasma membrane H+-ATPase isoform 2 (AHA2). Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3. Among these...

SH2/SH3 signaling proteins.

Science.gov (United States)

Schlessinger, J

1994-02-01

SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that are activated by protein tyrosine kinases. SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins. SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis, respectively. The three-dimensional structures of several SH2 and SH3 domains have been determined by NMR and X-ray crystallography, and the molecular basis of their specificity is beginning to be unveiled.

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

International Nuclear Information System (INIS)

Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

1987-01-01

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3

Icaritin induces MC3T3-E1 subclone14 cell differentiation through estrogen receptor-mediated ERK1/2 and p38 signaling activation.

Science.gov (United States)

Wu, Zhidi; Ou, Ling; Wang, Chaopeng; Yang, Li; Wang, Panpan; Liu, Hengrui; Xiong, Yingquan; Sun, Kehuan; Zhang, Ronghua; Zhu, Xiaofeng

2017-10-01

Icaritin (ICT), a hydrolytic product of icariin from the genus Epimedium, has many indicated pharmacological and biological activities. Several studies have shown that ICT has potential osteoprotective effects, including stimulation of osteoblast differentiation and inhibition of osteoclast differentiation. However, the molecular mechanism for this anabolic action of ICT remains largely unknown. Here, we found that ICT could enhance MC3T3-E1 subclone 14 preosteoblastic cell differentiation associated with increased mRNA levels and protein expression of the differentiation markers alkaline phosphatase (ALP), type 1 collagen (COL1), osteocalcin (OC), osteoponin (OPN) and runt-related transcription factor 2 (RUNX2), and improved mineralization, confirmed by bone nodule formation and collagen synthesis. To characterize the underlying mechanisms, we examined the effect of ICT on estrogen receptor (ER) and mitogen-activated protein kinase (MAPK) signaling. ICT treatment induced p38 kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, but it demonstrated at the same time point no effect on activation of c-Jun N-terminal kinase (JNK). ER antagonist ICI182780, p38 antagonist SB203580 and ERK1/2 antagonist PD98059 markedly inhibited the ICT-induced the mRNA expression of ALP, COL1, OC and OPN. ICI182780 attenuated the ICT-induced phosphorylation of p38 and ERK1/2. These observations indicate a potential mechanism of osteogenic effects of ICT involving the ERK1/2 and p38 pathway activation through the ER. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

AMP-activated protein kinase couples 3-bromopyruvate-induced energy depletion to apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins.

Science.gov (United States)

Bodur, Cagri; Karakas, Bahriye; Timucin, Ahmet Can; Tezil, Tugsan; Basaga, Huveyda

2016-11-01

Most tumors primarily rely on glycolysis rather than mitochondrial respiration for ATP production. This phenomenon, also known as Warburg effect, renders tumors more sensitive to glycolytic disturbances compared to normal cells. 3-bromopyruvate is a potent inhibitor of glycolysis that shows promise as an anticancer drug candidate. Although investigations revealed that 3-BP triggers apoptosis through ATP depletion and subsequent AMPK activation, the underlying molecular mechanisms coupling AMPK to apoptosis are poorly understood. We showed that 3-BP leads to a rapid ATP depletion which was followed by growth inhibition and Bax-dependent apoptosis in HCT116 cells. Apoptosis was accompanied with activation of caspase-9 and -3 while pretreatment with a general caspase inhibitor attenuated cell death. AMPK, p38, JNK, and Akt were phosphorylated immediately upon treatment. Pharmacological inhibition and silencing of AMPK largely inhibited 3-BP-induced apoptosis and reversed phosphorylation of JNK. Transcriptional activity of FoxO3a was dramatically increased subsequent to AMPK-mediated phosphorylation of FoxO3a at Ser413. Cell death analysis of cells transiently transfected with wt or AMPK-phosphorylation-deficient FoxO3 expression plasmids verified the contributory role of AMPK-FoxO3a axis in 3-BP-induced apoptosis. In addition, expression of proapoptotic Bcl-2 proteins Bim and Bax were upregulated in an AMPK-dependent manner. Bim was transcriptionally activated in association with FoxO3a activity, while Bax upregulation was abolished in p53-null cells. Together, these data suggest that AMPK couples 3-BP-induced metabolic disruption to intrinsic apoptosis via modulation of FoxO3a-Bim axis and Bax expression. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

OpenAIRE

Halfter, Ursula; Ishitani, Manabu; Zhu, Jian-Kang

2000-01-01

The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na+ and K+ homeostasis and plant tolerance to high Na+ and low K+ environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis ...

Effects of short-term caloric restriction on circulating free IGF-I, acid-labile subunit, IGF-binding proteins (IGFBPs)-1-4, and IGFBPs-1-3 protease activity in obese subjects

DEFF Research Database (Denmark)

Rasmussen, Michael Højby; Juul, Anders; Kjems, Lise Lund

2006-01-01

, no published data exist on free IGF-I levels, acid labile subunit (ALS), or IGFBP protease activity in relation to GH release during a hypocaloric diet. The main purpose of this study was to determine free IGF-I, ALS, IGFBPs-1-4, and IGFBPs-1-3 protease activity in relation to 24-h GH release before and after......Decreased levels of GH and total IGF-I have been reported in obesity. It has been hypothesized that increased free (biologically active) IGF-I levels generated from IGF-binding protein (IGFBP) protease activity could be the mechanism for the low GH release in dieting obese subjects. However...... a short-term very low-calorie diet (VLCD)....

Effects of short-term caloric restriction on circulating free IGF-I, acid-labile subunit, IGF-binding proteins (IGFBPs)-1-4, and IGFBPs-1-3 protease activity in obese subjects

DEFF Research Database (Denmark)

Rasmussen, Michael Højby; Juul, Anders; Kjems, Lise Lund

2006-01-01

Decreased levels of GH and total IGF-I have been reported in obesity. It has been hypothesized that increased free (biologically active) IGF-I levels generated from IGF-binding protein (IGFBP) protease activity could be the mechanism for the low GH release in dieting obese subjects. However......, no published data exist on free IGF-I levels, acid labile subunit (ALS), or IGFBP protease activity in relation to GH release during a hypocaloric diet. The main purpose of this study was to determine free IGF-I, ALS, IGFBPs-1-4, and IGFBPs-1-3 protease activity in relation to 24-h GH release before and after...... a short-term very low-calorie diet (VLCD)....

Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

Energy Technology Data Exchange (ETDEWEB)

Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin [Department of Neurological Disease, Xi' an Central Hospital, Xi' an Jiaotong University, Xi' an, Shaanxi 710000 (China); Bie, Xiao-Hua, E-mail: biexiaohua_xjtu@126.com [Department of Functional Neurosurgery, Xi' an Red Cross Hospital, Xi' an Jiaotong University, Xi' an, Shaanxi 710054 (China)

2015-07-10

We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation.

Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

International Nuclear Information System (INIS)

Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin; Bie, Xiao-Hua

2015-01-01

We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation

The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.

Science.gov (United States)

Tang, Fenfen; Xia, Hongjie; Wang, Peipei; Yang, Jie; Zhao, Tianyong; Zhang, Qi; Hu, Yuanyang; Zhou, Xi

2014-09-01

Human enterovirus 71 (EV71) belongs to the genus Enterovirus in the family Picornaviridae and has been recognized as one of the most important pathogens that cause emerging infectious disease. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication. Here we expressed EV71 3AB protein as recombinant protein in a eukaryotic expression system and uncovered that this protein possesses a nucleic acid helix-destabilizing and strand annealing acceleration activity in a dose-dependent manner, indicating that EV71 3AB is a nucleic acid chaperone protein. Moreover, we characterized the RNA chaperone activity of EV71 3AB, and revealed that divalent metal ions, such as Mg(2+) and Zn(2+), were able to inhibit the RNA helix-destabilizing activity of 3AB to different extents. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103, are critical and probably the active sites of 3AB for its RNA chaperone activity. This report reveals that EV71 3AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones, and provides novel knowledge about the virology of EV71. Copyright © 2014 Elsevier Inc. All rights reserved.

Suppression of STAT3 NH2 -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

Science.gov (United States)

Ray, Sutapa; Coulter, Don W; Gray, Shawn D; Sughroue, Jason A; Roychoudhury, Shrabasti; McIntyre, Erin M; Chaturvedi, Nagendra K; Bhakat, Kishor K; Joshi, Shantaram S; McGuire, Timothy R; Sharp, John G

2018-04-01

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH 2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients. © 2017 Wiley Periodicals, Inc.

Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart.

Science.gov (United States)

Inomata, Yui; Nagasaka, Shouta; Miyate, Kazuki; Goto, Yuta; Hino, Chizuru; Toukairin, Chihiro; Higashio, Rieko; Ishida, Kinji; Saino, Tomoyuki; Hirose, Masamichi; Tsumura, Hideki; Sanbe, Atsushi

2018-02-19

Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart. Copyright © 2018 Elsevier Inc. All rights reserved.

Radiolysis of Ca14CO3

International Nuclear Information System (INIS)

Sanchez, M.G.A.

1986-01-01

The partition-ion exclusion chromatography is evaluated to analyse non-ionic organic compounds obtained from radiolysis of high specific activity Ca 14 CO 3 . The Ca 14 CO 3 was irradiated by β - decay of carbon-14 or by γ rays from a cobalt-60 source. The crystals were dissolved for qualitative and quantitative analysis of the radiolytic products. Formic and oxalic acids were produced in high yields. Glyoxylic, acetic and glycolic acids, formaldehyde and methanol were produced in low yields. Quantitative determination was carried out by liquid scintillation spectroscopy and the chemical yields (G-values) were calculated for the products. Mechanisms of product formation are proposed based on thermal annealing experiments. (Author) [pt

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

DEFF Research Database (Denmark)

Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline

2016-01-01

implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1...

Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

Science.gov (United States)

Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

2016-02-24

Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country.

AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

Science.gov (United States)

2013-01-01

Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

The potency of STAT (signal transducers and activators of transcription) 3 protein as growth promoter for chicken

Science.gov (United States)

Ma'ruf, Anwar; Iswati, Sri; Hidajati, Nove; Damayanti, Ratna

2017-09-01

The long-term objective of this study was to produce STAT synthetic protein in chicken during growth period resulting from the increase of growth hormone (GH) as growth promoter. This study used ten male chicken Lohman from PT. Multibreeder Indonesia. The chicken were kept within batteried cage, with a capacity of one chicken in each cage. The chickens were fed twice a day, at 6 a.m. and 6 p.m. with the amount of feed 10% less than standard. On day 21 the chicken were slaughtered to obtain the samples, i.e., adipose, liver and muscles for the following examinations (1) isolation of STAT-3 signaling protein from adipose, liver and muscles of the chicken, (2) analysis of STAT-3 signaling protein using SDS-PAGE method, and (3) identification of STAT-3 signaling protein using Western blot method by means of protein detection using electrophoresis with polyacrylamide gels. Results of examination on protein in hepatic, muscle and adipose of chickens in growth period revealed that STAT protein was positively present in those tissues. This finding was followed-up with SDS-PAGE examination, from which we found the presence of protein band between the markers of 116 kDa and 14.4 kDa. The protein band was supposedly the STAT-3 protein. To prove that protein band formed was the STAT-3, Western blot examination was conducted using rabbit polyclonal antibody STAT-3. The result showed the formation of the protein band, indicating the presence of reaction between antigen (STAT-3 protein) and STAT-3 protein antibody. In conclusion, STAT-3 protein is present in hepatic, muscular, and adipose tissues, with molecular weight of 59.4 kDa.

Synthesis of ethyl [14CH3]methylmalonyl thioglycolate as a possible substrate analogue of [14CH3]methylmalonyl coenzyme-A

International Nuclear Information System (INIS)

Kovacs, I.; Kovacs, Z.

1991-01-01

Ethyl methylmalonyl thioglycolate is a potential substrate analogue of methylmalonyl-coenzyme-A (methylmalonyl-CoA) in the investigation of propionic acid metabolism. To prove this hypothesis, the tracer ethyl [ 14 CH 3 ] methylmalonyl thioglycolate was synthesized via methyl-Meldrum's acid to carry out the biochemical examinations. The method described can also be used to synthesize [ 14 CH 3 ] methylmalonyl-CoA by transesterification of active labelled methylmalonyl thiophenyl ester. This latter intermediate is chemically stable when stored at room temperature, and the unstable [ 14 CH 3 ]methylmalonyl-CoA can be prepared in one step just preceeding the biochemical experiments. (author)

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

Science.gov (United States)

Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

2017-12-01

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

Science.gov (United States)

Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

2016-07-08

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

14-3-3theta protects against neurotoxicity in a cellular Parkinson's disease model through inhibition of the apoptotic factor Bax.

Directory of Open Access Journals (Sweden)

Sunny R Slone

Full Text Available Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP(+ in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

Synthesis of C-9-14C-1,8-dihydroxy-3-carboxyanthraquinone

International Nuclear Information System (INIS)

De Witte, P.; Lemli, J.

1988-01-01

The synthesis of C-9- 14 C-rhein is reported using 14 CO 2 as a 14 C-source. After preparing 14 C-1, 8-dimethoxy-3-methylanthraquinone by a condensation reaction, the product is demethylated and the 3-methyl group converted to the corresponding 3-carboxy group. The radio-active yield of the total synthesis, starting with 1 Ci 14 CO 2 is 6,9% (6, 9 mCi); 352 mg 14 rhein is produced with a specific activity of 55,7 mCi/mmol. (author)

Royal Jelly-Mediated Prolongevity and Stress Resistance in Caenorhabditis elegans Is Possibly Modulated by the Interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 Proteins.

Science.gov (United States)

Wang, Xiaoxia; Cook, Lauren F; Grasso, Lindsay M; Cao, Min; Dong, Yuqing

2015-07-01

Recent studies suggest that royal jelly (RJ) and its related substances may have antiaging properties. However, the molecular mechanisms underlying the beneficial effects remain elusive. We report that the effects of RJ and enzyme-treated RJ (eRJ) on life span and health span in Caenorhabditis elegans (C elegans) are modulated by the sophisticated interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. Dietary supplementation with RJ or eRJ increased C. elegans life span in a dose-dependent manner. The RJ and eRJ consumption increased the tolerance of C elegans to oxidative stress, ultraviolet irradiation, and heat shock stress. Our genetic analyses showed that RJ/eRJ-mediated life-span extension requires insulin/IGF-1 signaling and the activities of DAF-16, SIR-2.1, HCF-1, and FTT-2, a 14-3-3 protein. Earlier studies reported that DAF-16/FOXO, SIR-2.1/SIRT1, FTT-2, and HCF-1 have extensive interplays in worms and mammals. Our present findings suggest that RJ/eRJ-mediated promotion of longevity and stress resistance in C elegans is dependent on these conserved interplays. From an evolutionary point of view, this study not only provides new insights into the molecular mechanisms of RJ's action on health span promotion in C elegans, but also has imperative implications in using RJ/eRJ as nutraceuticals to delay aging and age-related disorders. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression and Prognostic Significance of 14-3-3sigma and ERM Family Protein Expression in Periampullary Neoplasms

NARCIS (Netherlands)

Hustinx, Steven R.; Fukushima, Noriyoshi; Zahurak, Marianna L.; Riall, Taylor Sohn; Maitra, Anirban; Brosens, Lodewijk; Cameron, John L.; Yeo, Charles J.; Offerhaus, G. Johan A.; Hruban, Ralph H.; Goggins, Michael

2005-01-01

Aberrant gene expression in pancreatic ductal adenocarcinomas contributes to the dismal outcome of patients who develop this disease. The 5' region of 14-3-3sigma (stratifin) is hypomethylated in pancreatic adenocarcinomas and is associated with gene overexpression. In multiple experimental systems,

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

Science.gov (United States)

Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

2015-03-27

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Genome-wide identification, phylogeny, and expression analyses of the 14-3-3 family reveal their involvement in the development, ripening and abiotic stress response in banana

Directory of Open Access Journals (Sweden)

meiying li

2016-09-01

Full Text Available Plant 14-3-3 proteins act as critical components of various cellular signaling processes and play an important role in regulating multiple physiological processes. However, less information is known about the 14-3-3 gene family in banana. In this study, 25 14-3-3 genes were identified from the banana genome. Based on the evolutionary analysis, banana 14-3-3 proteins were clustered into ε and non-ε groups. Conserved motif analysis showed that all identified banana 14-3-3 genes had the typical 14-3-3 motif. The gene structure of banana 14-3-3 genes showed distinct class-specific divergence between the ε group and the non-ε group. Most banana 14-3-3 genes showed strong transcript accumulation changes during fruit development and postharvest ripening in two banana varieties, indicating that they might be involved in regulating fruit development and ripening. Moreover, some 14-3-3 genes also showed great changes after osmotic, cold, and salt treatments in two banana varieties, suggested their potential role in regulating banana response to abiotic stress. Taken together, this systemic analysis reveals the involvement of banana 14-3-3 genes in fruit development, postharvest ripening, and response to abiotic stress and provides useful information for understanding the functions of 14-3-3 genes in banana.

Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

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Valsala Haridas

Full Text Available Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a the cross-talk that occurs between redox and phosphorylation processes, and (b the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

RKIP phosphorylation–dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3

Energy Technology Data Exchange (ETDEWEB)

Hahm, Jong Ryeal [Department of Internal Medicine, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Ahmed, Mahmoud [Department of Biochemistry and Convergence Medical Science, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr [Department of Biochemistry and Convergence Medical Science, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of)

2016-09-09

3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-L-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-L-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity. - Highlights: • Overexpression of GFP-LC3 in 3T3-L1 cells produces intracellular lipid droplets. • SREBP2 is highly activated in preadipocytes transfected with adenoviral GFP-LC3. • RKIP phosphorylation at serine 153 is significantly increased during adipogenesis. • RKIP knockdown promotes ERK1 and PPARγ activation during adipogenesis. • RKIP-dependent ERK1 activation increases triacylglycerides in

Synthesis of [14α, 15α-3H]-norgestrel

International Nuclear Information System (INIS)

Wagner, H.

1988-01-01

The preparation of the well-known gestogen norgestrel in a tritium labelled form is described. Starting material was 18-methyl-3-methoxy-estra-1,3,5(10),8,14-pentaen-17β-o1 acetate. In a four step synthesis 17α-ethinyl-17β-hydroxy-18-methyl-[14α,15α- 3 H]-estr-4-en-3-one was obtained with a specific activity of 52 Ci/mmol and a radiochemical purity > 99%. (author)

Radioactive levels and doses of 3H and 14C in white spirits

International Nuclear Information System (INIS)

Deng, G.

1992-01-01

'Full Text:' White (and yeast) spirits is a general name for strong alcoholic beverages in China. The paper reports levels and doses of 3 H and 14 C in 65 spirits samples between 1986 and 1987. Experiments were made by measuring end analyzing each sample, using a low background liquid scintillation spectrometer. Radioactive levels of 65 spirits samples are as follows: Variant range of 3 H activity is 98.2 - 170.6Bq.dm -3 and its average is 149.2 ± 17.3Bq.dm -3 ; Variant range of 14 C activity is 38.8-80.2Bq.dm -3 and its average is 57.4±8.2Bq.dm -3 . If the man drinks 200cm 3 of spirits daily, the annual dose equivalents will be 0.19uSv of 3 H and 2.5uSv of 14 C . In ordinary strong alcoholic beverages that contain 57-60% alcohol, the mean 3 H and 14 C activities are 153.8Bq.dm -3 and 60.3Bq.dm -3 , respectively, but in spirits of lower alcoholic content (38-40%), the mean 3H activity is 114.6Bq.dm -3 , that is 25.5% less than ordinary spirits, and the mean 14 C activity is 46.1Bq.dm -3 , that is 23.5% less than ordinary spirits. We compared the 3 H and 14 C contents of five kinds of staple grains from both Sichuan and Guangdong provinces. We learned that the level of activity in spirits is ten times higher than in grains and water, and the level of 14 C activity in spirits is equivalent to that in grains. White spirits has fully concentrated 3 H and 14 C from both grain and water, and activities increase with increasing alcoholic content. 3 H in spirits probably is averaged from both water and grain, and 14 C is averaged mostly from grain. (author)

Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

Science.gov (United States)

Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

2004-07-01

The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells

International Nuclear Information System (INIS)

Nagel, Stefan; Scherr, Michaela; MacLeod, Roderick AF; Venturini, Letizia; Przybylski, Grzegorz K; Grabarczyk, Piotr; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; Schmidt, Christian A; Drexler, Hans G

2009-01-01

Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs. Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses. Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

Science.gov (United States)

Ursini-Siegel, J; Hardy, W R; Zheng, Y; Ling, C; Zuo, D; Zhang, C; Podmore, L; Pawson, T; Muller, W J

2012-11-29

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

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Carly St. Germain

2010-07-01

Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

Cancer3D: understanding cancer mutations through protein structures.

Science.gov (United States)

Porta-Pardo, Eduard; Hrabe, Thomas; Godzik, Adam

2015-01-01

The new era of cancer genomics is providing us with extensive knowledge of mutations and other alterations in cancer. The Cancer3D database at http://www.cancer3d.org gives an open and user-friendly way to analyze cancer missense mutations in the context of structures of proteins in which they are found. The database also helps users analyze the distribution patterns of the mutations as well as their relationship to changes in drug activity through two algorithms: e-Driver and e-Drug. These algorithms use knowledge of modular structure of genes and proteins to separately study each region. This approach allows users to find novel candidate driver regions or drug biomarkers that cannot be found when similar analyses are done on the whole-gene level. The Cancer3D database provides access to the results of such analyses based on data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). In addition, it displays mutations from over 14,700 proteins mapped to more than 24,300 structures from PDB. This helps users visualize the distribution of mutations and identify novel three-dimensional patterns in their distribution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The C-Terminal Segment of Yeast BMH Proteins Exhibits Different Structure Compared to Other 14-3-3 Protein Isoforms

Czech Academy of Sciences Publication Activity Database

Veisová, Dana; Řežábková, L.; Štěpánek, M.; Novotná, P.; Herman, P.; Večeř, J.; Obšil, T.; Obšilová, Veronika

2010-01-01

Roč. 49, č. 18 (2010), s. 3853-3861 ISSN 0006-2960 R&D Projects: GA AV ČR(CZ) IAA501110801; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : yeast BMH proteins * sedimentation equilibrium and velocity measurements * dynamic light scattering Subject RIV: BO - Biophysics Impact factor: 3.226, year: 2010

DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

DEFF Research Database (Denmark)

Nguyen, Thanh Hung; Kugler, Jan-Michael; Cohen, Stephen Michael

2017-01-01

/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family...... deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor...

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

Science.gov (United States)

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.

IP3 production in the hypersensitive response of lemon seedlings against Alternaria alternata involves active protein tyrosine kinases but not a G-protein

Directory of Open Access Journals (Sweden)

XIMENA ORTEGA

2005-01-01

Full Text Available IP3 increase and de novo synthesis of scoparone are produced in the hypersensitive response (HR of lemon seedlings against the fungus Alternaria alternata. To elucidate whether a G-protein and/or a protein tyrosine kinase (PTK are involved in signal transduction leading to the production of such a defensive response, we studied the HR in this plant system after treatment with G-protein activators alone and PTK inhibitors in the presence of fungal conidia. No changes in the level of IP3 were detected in response to the treatment with the G-protein activators cholera toxin or mastoparan, although the HR was observed in response to these compounds as determined by the scoparone synthesis. On the contrary, the PTK inhibitors lavendustin A and 2,5-dihidroxy methyl cinnamate (DHMC not only prevented the IP3 changes observed in response to the fungal inoculation of lemon seedlings but also blocked the development of the HR. These results suggest that the IP3 changes observed in response to A. alternata require a PTK activity and are the result of a G-protein independent Phospholipase C activity, even though the activation of a G-protein can also lead to the development of a HR. Therefore, it appears that more than one signaling pathway may be activated for the development of HR in lemon seedlings: one involving a G-protein and the other involving a PTK-dependent PLC.

Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt.

Science.gov (United States)

Zhang, Q-G; Han, D; Xu, J; Lv, Q; Wang, R; Yin, X-H; Xu, T-L; Zhang, G-Y

2006-12-01

Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)-MLK3-Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

Science.gov (United States)

Liu, Xiao; Pichulik, Tica; Wolz, Olaf-Oliver; Dang, Truong-Minh; Stutz, Andrea; Dillen, Carly; Delmiro Garcia, Magno; Kraus, Helene; Dickhöfer, Sabine; Daiber, Ellen; Münzenmayer, Lisa; Wahl, Silke; Rieber, Nikolaus; Kümmerle-Deschner, Jasmin; Yazdi, Amir; Franz-Wachtel, Mirita; Macek, Boris; Radsak, Markus; Vogel, Sebastian; Schulte, Berit; Walz, Juliane Sarah; Hartl, Dominik; Latz, Eicke; Stilgenbauer, Stephan; Grimbacher, Bodo; Miller, Lloyd; Brunner, Cornelia; Wolz, Christiane; Weber, Alexander N R

2017-10-01

The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK. Copyright © 2017 American Academy of Allergy

Crystal structures of a yeast 14-3-3 protein from Lachancea thermotolerans in the unliganded form and bound to a human lipid kinase PI4KB-derived peptide reveal high evolutionary conservation

Czech Academy of Sciences Publication Activity Database

Eisenreichová, Andrea; Klíma, Martin; Bouřa, Evžen

2016-01-01

Roč. 72, č. 11 (2016), s. 799-803 ISSN 2053-230X R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : 14-3-3 proteins * Bmh1 * Bmh2 * crystal structure * phosphopeptide Subject RIV: CE - Biochemistry Impact factor: 0.799, year: 2016

Activated Protein C Attenuates Severe Inflammation by Targeting VLA-3high Neutrophil Subpopulation in Mice.

Science.gov (United States)

Sarangi, Pranita P; Lee, Hyun-Wook; Lerman, Yelena V; Trzeciak, Alissa; Harrower, Eric J; Rezaie, Alireza R; Kim, Minsoo

2017-10-15

The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3 high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1 high CD11b high VLA-3 high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities. Copyright © 2017 by The American Association of Immunologists, Inc.

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast.

Science.gov (United States)

Juhásová, Barbora; Mentel, Marek; Bhatia-Kiššová, Ingrid; Zeman, Igor; Kolarov, Jordan; Forte, Michael; Polčic, Peter

2011-09-02

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Human procaspase-2 phosphorylation at both S139 and S164 is required for 14-3-3 binding

Czech Academy of Sciences Publication Activity Database

Kalábová, Dana; Šmídová, Aneta; Petrvalská, Olivia; Alblová, Miroslava; Košek, Dalibor; Man, Petr; Obšil, Tomáš; Obšilová, Veronika

2017-01-01

Roč. 493, č. 2 (2017), s. 940-945 ISSN 0006-291X R&D Projects: GA ČR(CZ) GA17-00726S; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : procaspase-2 * 14-3-3 * protein-protein interaction * phosphorylation * caspase-2 Subject RIV: EB - Genetics ; Molecular Biology; CE - Biochemistry (MBU-M) OBOR OECD: Biochemistry and molecular biology; Biochemistry and molecular biology (MBU-M) Impact factor: 2.466, year: 2016

Identification of the 14-3-3 gene family in Rafflesia cantleyi

Science.gov (United States)

Rosli, Khadijah; Wan, Kiew-Lian

2018-04-01

Rafflesia is known to be the largest flower in the world. Due to its size and appearance, it is considered to be very unique. Little is known about the molecular biology of this rare parasitic flowering plant as it is very difficult to locate and has a short life-span as a flower. Physiological activities in plants are regulated by signalling regulators such as the members of the 14-3-3 gene family. The number of members of this gene family varies in plants and there are thirteen known members in Arabidopsis thaliana. Their role is to bind to phosphorylated targets to complete signal transduction processes. Sequence comparison using BLAST of transcriptome data from three different Rafflesia cantleyi floral bud stages against the Swissprot database revealed 27 transcripts annotated as members of this gene family. All of the transcripts were expressed during floral bud stage 1 (S1) while 14 and four transcripts were expressed during floral bud stages 2 (S2) and 3 (S3), respectively. Significant downregulation was recorded for six and nine transcripts at S1 vs. S2 and S2 vs. S3 respectively. This gene family may play a critical role as signalling regulators during the development of Rafflesia floral bud.

3,3′-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells

International Nuclear Information System (INIS)

Beaver, Laura M.; Yu, Tian-Wei; Sokolowski, Elizabeth I.; Williams, David E.; Dashwood, Roderick H.; Ho, Emily

2012-01-01

Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast, DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis. -- Highlights: ► DIM inhibits HDAC activity and decreases HDAC2 expression in prostate cancer cells. ► DIM is significantly more effective than I3C at inhibiting HDAC activity. ► I3C has no effect on HDAC protein expression. ► Inhibition of HDAC activity by DIM is associated with increased p21 expression. ► HDAC inhibition may be a novel epigenetic mechanism for cancer prevention with DIM.

3,3′-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Beaver, Laura M., E-mail: beaverl@onid.orst.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331 (United States); Yu, Tian-Wei, E-mail: david.yu@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); Sokolowski, Elizabeth I., E-mail: sokolowe@onid.orst.edu [School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331 (United States); Williams, David E., E-mail: david.williams@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); Department of Environmental and Molecular Toxicology, Oregon State University, 1007 Agriculture and Life Sciences Building, Corvallis, OR 97331 (United States); Dashwood, Roderick H., E-mail: rod.dashwood@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); Department of Environmental and Molecular Toxicology, Oregon State University, 1007 Agriculture and Life Sciences Building, Corvallis, OR 97331 (United States); Ho, Emily, E-mail: Emily.Ho@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331 (United States)

2012-09-15

Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast, DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis. -- Highlights: ► DIM inhibits HDAC activity and decreases HDAC2 expression in prostate cancer cells. ► DIM is significantly more effective than I3C at inhibiting HDAC activity. ► I3C has no effect on HDAC protein expression. ► Inhibition of HDAC activity by DIM is associated with increased p21 expression. ► HDAC inhibition may be a novel epigenetic mechanism for cancer prevention with DIM.

A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

Science.gov (United States)

Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

2011-01-01

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

DEFF Research Database (Denmark)

Brandauer, Josef; Andersen, Marianne A; Kellezi, Holti

2015-01-01

, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling......The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases...... in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p

14-3-3η Autoantibodies: Diagnostic Use in Early Rheumatoid Arthritis

NARCIS (Netherlands)

Maksymowych, Walter P.; Boire, Gilles; van Schaardenburg, Dirkjan; Wichuk, Stephanie; Turk, Samina; Boers, Maarten; Siminovitch, Katherine A.; Bykerk, Vivian; Keystone, Ed; Tak, Paul Peter; van Kuijk, Arno W.; Landewé, Robert; van der Heijde, Desiree; Murphy, Mairead; Marotta, Anthony

2015-01-01

To describe the expression and diagnostic use of 14-3-3η autoantibodies in early rheumatoid arthritis (RA). 14-3-3η autoantibody levels were measured using an electrochemiluminescent multiplexed assay in 500 subjects (114 disease-modifying antirheumatic drug-naive patients with early RA, 135 with

Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants

Energy Technology Data Exchange (ETDEWEB)

Phanchaisri, Boonrak [Science and Technology Research Institute, Chiang Mai University, Chiang Mai 50200 (Thailand); Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Samsang, Nuananong [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, Liang Deng; Singkarat, Somsorn [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Anuntalabhochai, Somboon, E-mail: soanu.1@gmail.com [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

2012-06-01

The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50-60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants

International Nuclear Information System (INIS)

Phanchaisri, Boonrak; Samsang, Nuananong; Yu, Liang Deng; Singkarat, Somsorn; Anuntalabhochai, Somboon

2012-01-01

The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50–60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

Protein: FBA3 [TP Atlas

Lifescience Database Archive (English)

Full Text Available FBA3 Ubiquitination CBLB RNF56 CBLB E3 ubiquitin-protein ligase CBL-B Casitas B-lineage lymphoma pr...oto-oncogene b, RING finger protein 56, SH3-binding protein CBL-B, Signal transduction prote

B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

Science.gov (United States)

Renault, Thibaud T; Elkholi, Rana; Bharti, Archana; Chipuk, Jerry E

2014-09-19

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhancing the Photocatalytic Activity of Sr4 Al14 O25 : Eu2+ , Dy3+ Persistent Phosphors by Codoping with Bi3+ Ions.

Science.gov (United States)

García, Carlos R; Oliva, Jorge; Romero, Maria Teresa; Diaz-Torres, Luis A

2016-03-01

The photocatalytic activity of Bismuth-codoped Sr 4 Al 14 O 25 : Eu 2+ , Dy 3+ persistent phosphors is studied by monitoring the degradation of the blue methylene dye UV light irradiation. Powder phosphors are obtained by a combustion synthesis method and a postannealing process in reductive atmosphere. The XRD patterns show a single orthorhombic phase Sr 4 Al 14 O 25 : Eu 2+ , Dy 3+ , Bi 3+ phosphors even at high Bismuth dopant concentrations of 12 mol%, suggesting that Bi ions are well incorporated into the host lattice. SEM micrographs show irregular micrograins with sizes in the range of 0.5-20 μm. The samples present an intense greenish-blue fluorescence and persistent emissions at 495 nm, attributed to the 5d-4f allowed transitions of Eu 2+ . The fluorescence decreases as Bi concentration increases; that suggest bismuth-induced traps formation that in turn quench the luminescence. The photocatalytic evaluation of the powders was studied under both 365 nm UV and solar irradiations. Sample with 12 mol% of Bi presented the best MB degradation activity; 310 min of solar irradiation allow 100% MB degradation, whereas only 62.49% MB degradation is achieved under UV irradiation. Our results suggest that codoping the persistent phosphors with Bi 3+ can be an alternative to enhance their photocatalytic activity. © 2016 The American Society of Photobiology.

Analysis of signal transducer and activator of transcription 3 (Stat 3) pathway in multiple myeloma: Stat 3 activation and cyclin D1 dysregulation are mutually exclusive events.

Science.gov (United States)

Quintanilla-Martinez, Leticia; Kremer, Marcus; Specht, Katja; Calzada-Wack, Julia; Nathrath, Michaela; Schaich, Robert; Höfler, Heinz; Fend, Falko

2003-05-01

The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three

Impairment of interferon regulatory factor-3 activation by hepatitis C virus core protein basic amino acid region 1.

Science.gov (United States)

Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori

2012-11-30

Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

Science.gov (United States)

Takahashi, Chika; Miyatake, Koichi; Kusakabe, Morioh; Nishida, Eisuke

2018-06-01

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Human NLRP3 inflammasome activation is Nox1-4 independent

NARCIS (Netherlands)

van Bruggen, Robin; Köker, M. Yavuz; Jansen, Machiel; van Houdt, Michel; Roos, Dirk; Kuijpers, Taco W.; van den Berg, Timo K.

2010-01-01

The NLRP3 inflammasome can be activated by pathogen-associated molecular patterns or endogenous danger-associated molecular patterns. The activation of the NLRP3 inflammasome results in proteolytic activation and secretion of cytokines of the interleukin-1 (IL-1) family. The precise mode of

Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

Science.gov (United States)

Yamamoto, N

1996-10-01

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

Science.gov (United States)

Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

1989-06-01

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

Detection of radioactively labeled proteins is quenched by silver staining methods: quenching is minimal for 14C and partially reversible for 3H with a photochemical stain

International Nuclear Information System (INIS)

Van Keuren, M.L.; Goldman, D.; Merril, C.R.

1981-01-01

Silver staining methods for protein detection in polyacrylamide gels have a quenching effect on autoradiography and fluorography. This effect was quantitated for proteins in two-dimensional gels by microdensitometry using a computer equipped with an image processor and by scintillation counting of proteins solubilized from the gels. The original histologically derived silver stain had a quenching effect that was severe and irreversible for 3 H detection and moderate for 14 C detection. A silver stain based on photochemical methods had minimal quenching of 14 C detection and less of a quenching effect than the histological stain for 3 H detection. The 3 H quenching effect was partially reversible for the photochemical stain

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

Science.gov (United States)

Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

2015-08-01

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

Science.gov (United States)

Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

2015-01-01

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

Synthesis of ethyl ( sup 14 CH sub 3 )methylmalonyl thioglycolate as a possible substrate analogue of ( sup 14 CH sub 3 )methylmalonyl coenzyme-A

Energy Technology Data Exchange (ETDEWEB)

Kovacs, I. (BIOGAL Pharmaceutical Works, Debrecen (Hungary)); Kovacs, Z. (Inst. of Nuclear Research, Debrecen (Hungary))

1991-11-01

Ethyl methylmalonyl thioglycolate is a potential substrate analogue of methylmalonyl-coenzyme-A (methylmalonyl-CoA) in the investigation of propionic acid metabolism. To prove this hypothesis, the tracer ethyl ({sup 14}CH{sub 3}) methylmalonyl thioglycolate was synthesized via methyl-Meldrum's acid to carry out the biochemical examinations. The method described can also be used to synthesize ({sup 14}CH{sub 3}) methylmalonyl-CoA by transesterification of active labelled methylmalonyl thiophenyl ester. This latter intermediate is chemically stable when stored at room temperature, and the unstable ({sup 14}CH{sub 3})methylmalonyl-CoA can be prepared in one step just preceeding the biochemical experiments. (author).

Effect of dietary and hormonal alterations on the activity of 3-methylcrotonyl-CoA carboylase in rat liver

International Nuclear Information System (INIS)

Paul, H.S.; Gleditsch, C.E.; Adibi, S.A.

1986-01-01

Previous studies have established that activities of leucine transaminase and branched-chain keto acid dehydrogenase (BCKDH) are under metabolic control. In the present experiment, the authors have investigated whether activity of 3-methylcrotonyl-CoA carboxylase (MCase), an enzyme distal to BCKDH in pathways of leucine oxidation, is also subjected to metabolic regulation. Initially, they developed optimal conditions to assay the activity of this enzyme. The assay was based on the incorporation of 14 CO 2 into 3-methylcrotonyl-CoA (MC-CoA) by liver mitochondria and required the presence of ATP and biotin. Subsequently, the authors determined the activity (nmol 14 CO 2 incorporated/mg protein/min, mean +/- SE, 6 rats) of MCase under normal (2.80 +/- 0.09) and altered conditions. The activity of MCase was not affected either by starvation (2.88 +/- 0.13), diabetes (2.95 +/- 0.11), low-protein (2.35 +/- 0.05), or high-protein diet (3.06 +/- 0.06). To further substantiate these results, they measured accumulation of MC-CoA by incubating liver mitochondria with α-ketoisocaproate (KIC). Of the KIC fraction that underwent flux through BCKDH (2.15 +/- 0.14 nmol/mg protein/min), only 0.26% accumulated as MC-CoA. Even if diabetes, when the flux through BCKDH was significantly increased (3.62 +/- 0.36), the accumulation of MC-CoA was negligible (0.33%). The authors conclude that a) MCase activity does not change in response to dietary and hormonal alterations, and b) MCase is not a rate-limiting reaction in pathways of leucine oxidation

Effect of dietary and hormonal alterations on the activity of 3-methylcrotonyl-CoA carboylase in rat liver

Energy Technology Data Exchange (ETDEWEB)

Paul, H.S.; Gleditsch, C.E.; Adibi, S.A.

1986-05-01

Previous studies have established that activities of leucine transaminase and branched-chain keto acid dehydrogenase (BCKDH) are under metabolic control. In the present experiment, the authors have investigated whether activity of 3-methylcrotonyl-CoA carboxylase (MCase), an enzyme distal to BCKDH in pathways of leucine oxidation, is also subjected to metabolic regulation. Initially, they developed optimal conditions to assay the activity of this enzyme. The assay was based on the incorporation of /sup 14/CO/sub 2/ into 3-methylcrotonyl-CoA (MC-CoA) by liver mitochondria and required the presence of ATP and biotin. Subsequently, the authors determined the activity (nmol /sup 14/CO/sub 2/ incorporated/mg protein/min, mean +/- SE, 6 rats) of MCase under normal (2.80 +/- 0.09) and altered conditions. The activity of MCase was not affected either by starvation (2.88 +/- 0.13), diabetes (2.95 +/- 0.11), low-protein (2.35 +/- 0.05), or high-protein diet (3.06 +/- 0.06). To further substantiate these results, they measured accumulation of MC-CoA by incubating liver mitochondria with ..cap alpha..-ketoisocaproate (KIC). Of the KIC fraction that underwent flux through BCKDH (2.15 +/- 0.14 nmol/mg protein/min), only 0.26% accumulated as MC-CoA. Even if diabetes, when the flux through BCKDH was significantly increased (3.62 +/- 0.36), the accumulation of MC-CoA was negligible (0.33%). The authors conclude that a) MCase activity does not change in response to dietary and hormonal alterations, and b) MCase is not a rate-limiting reaction in pathways of leucine oxidation.

Effect of level of dietary protein on the distribution of 14C-activity from exogenous 14C-inosine in chicks

International Nuclear Information System (INIS)

Masushige, Shoichi; Tadokoro, Tadahiro; Suzuki, Takao; Nakajima, Hisao.

1983-01-01

The effect of dietary protein level on the metabolic fate of intraperitoneally administered (exogeneous) 8- 14 C-inosine in chicks was studied. Three different protein level diets (low, standard and high) were prepared. Chicks were fed on these diets for 10 days, respectively and the following results were found: (1) RNA content of liver, small intestine and muscle in chicks fed on a low protein diet was decreased as compared to other diet groups, but no difference was observed in kidney or pancreas. (2) 14 C uptake by organs from exogeneous 8- 14 C-inosine was determined. The uptake of 14 C in kidney, pancreas and small intestine was higher than that in liver and muscle. Moreover, the uptake by tissues in the low protein groups was significantly higher than that in either the standard or high protein groups, but no difference was observed between these latter two groups. (3) The rate of incorporation of 14 C labelled purine by acid soluble materials and RNA was higher in kidney, pancreas and small intestine than in liver and muscle, and also higher in chicks fed on a low protein diet than in either the standard or high protein groups. (4) It was revealed that the 14 C-labelled purine rings from 8- 14 C-inosine were incorporated into AMP and GMP as constituents of RNA. (author)

Breast Regression Protein-39/Chitinase 3-Like 1 Promotes Renal Fibrosis after Kidney Injury via Activation of Myofibroblasts.

Science.gov (United States)

Montgomery, Tinika A; Xu, Leyuan; Mason, Sherene; Chinnadurai, Amirtha; Lee, Chun Geun; Elias, Jack A; Cantley, Lloyd G

2017-11-01

The normal response to kidney injury includes a robust inflammatory infiltrate of PMNs and macrophages. We previously showed that the small secreted protein breast regression protein-39 (BRP-39), also known as chitinase 3-like 1 (CHI3L1) and encoded by the Chi3l1 gene, is expressed at high levels by macrophages during the early stages of kidney repair and promotes tubular cell survival via IL-13 receptor α 2 (IL13R α 2)-mediated signaling. Here, we investigated the role of BRP-39 in profibrotic responses after AKI. In wild-type mice, failure to resolve tubular injury after unilateral ischemia-reperfusion injury (U-IRI) led to sustained low-level Chi3l1 mRNA expression by renal cells and promoted macrophage persistence and severe interstitial fibrosis. Analysis of macrophages isolated from wild-type kidneys 14 days after U-IRI revealed high-level expression of the profibrotic BRP-39 receptor Ptgdr2 / Crth2 and expression of the profibrotic markers Lgals3 , Pdgfb , Egf , and Tgfb In comparison, injured kidneys from mice lacking BRP-39 had significantly fewer macrophages, reduced expression of profibrotic growth factors, and decreased accumulation of extracellular matrix. BRP-39 depletion did not affect myofibroblast accumulation but did attenuate myofibroblast expression of Col1a1 , Col3a1 , and Fn1 Together, these results identify BRP-39 as an important activator of macrophage-myofibroblast crosstalk and profibrotic signaling in the setting of maladaptive kidney repair. Copyright © 2017 by the American Society of Nephrology.

CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein-Coupled Receptor CXCR2.

Science.gov (United States)

Boro, Monoranjan; Balaji, Kithiganahalli Narayanaswamy

2017-09-01

Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein-coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis -induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase C μ-dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C μ either by small interfering RNA-mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1β. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed therapeutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1β. Copyright © 2017 by The American Association of Immunologists, Inc.

The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence

DEFF Research Database (Denmark)

Agger, Karl; Cloos, Paul A C; Rudkjaer, Lise

2009-01-01

The tumor suppressor proteins p16INK4A and p14ARF, encoded by the INK4A-ARF locus, are key regulators of cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing cells, but becomes activated in response to oncogenic stress. Here, we show that e...... in mouse embryonic fibroblasts results in suppression of p16Ink4a and p19Arf expression and in their immortalization....

PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b.

Directory of Open Access Journals (Sweden)

Chao Luo

2016-09-01

Full Text Available Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3 as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Science.gov (United States)

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

Directory of Open Access Journals (Sweden)

Josef eBrandauer

2015-03-01

Full Text Available The mitochondrial protein deacetylase sirtuin (SIRT 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS handling. We determined the requirement of AMP-activated protein kinase (AMPK for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p<0.01 and superoxide dismutase 2 (MnSOD; p<0.05 protein abundance in quadriceps muscle of wild-type (WT; n=13-15, but not AMPK α2 kinase dead (KD; n=12-13 mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p=0.051; n=6. To further elucidate a role for AMPK in mediating these effects, we treated WT (n=7-8 and AMPK α2 KD (n=7-9 mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR. Four weeks of daily AICAR injections (500 mg/kg resulted in AMPK-dependent increases in SIRT3 (p<0.05 and MnSOD (p<0.01 in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n=9-10. Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p<0.01 and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p<0.05. Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122 or oligomycin-sensitivity conferring protein (OSCP; K139 was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training.

G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

Science.gov (United States)

Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

2016-12-30

G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulated production and anti-HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G.

Science.gov (United States)

Rose, Kristine M; Marin, Mariana; Kozak, Susan L; Kabat, David

2005-07-01

APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.

Microstructure and tensile properties of oxide dispersion strengthened Fe–14Cr–0.3Y2O3 and Fe–14Cr–2W–0.3Ti–0.3Y2O3

International Nuclear Information System (INIS)

Auger, M.A.; Castro, V. de; Leguey, T.; Monge, M.A.; Muñoz, A.; Pareja, R.

2013-01-01

Two ODS ferritic steels with nominal compositions (wt.%): Fe–14Cr–0.3Y 2 O 3 and Fe–14Cr–2W–0.3Ti–0.3Y 2 O 3 have been produced by mechanical alloying and consolidation by hot isostatic pressing. The microstructure and tensile properties of these materials after being forged and heat-treated at 1123 K have been investigated to clarify the interrelation between composition, microstructure and mechanical properties. The second-phase precipitates in these alloys have been analyzed by high-angle annular dark-field imaging in scanning TEM mode and electron diffraction. Fe–14Cr–2W–0.3Ti–0.3Y 2 O 3 exhibits a duplex microstructure consisting of large recrystallized grains, as large as 1.5 μm, and unrecovered regions containing submicron equiaxed grains. In addition, three types of secondary phase particles have been found: large M 23 C 6 particles containing W and Cr, (Cr + Ti) rich spherical particles with diameters between 50 and 500 nm, and fine (Y + Ti) oxide particles with sizes below 30 nm. In contrast, Fe14CrY shows a uniform structure of equiaxed grains, with sizes in the range 0.5–3 μm, containing a fine dispersion of Y oxide particles ( 2 O 3 at temperatures up to 773 K, but the opposite appears to occur beyond this temperature

14 CFR 99.3 - Definitions.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Definitions. 99.3 Section 99.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES SECURITY CONTROL OF AIR TRAFFIC General § 99.3 Definitions. Aeronautical facility...

14 CFR 77.3 - Standards.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Standards. 77.3 Section 77.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRSPACE OBJECTS... section 16 of the Federal Airport Act; (3) Developing technical standards and guidance in the design and...

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

Science.gov (United States)

Li, Congmin; Lim, Sunghyuk; Braunewell, Karl H; Ames, James B

2016-01-01

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

Synthesis of methyl ((chloro-2 ethyl)-3 nitroso-3 Ureido)-3 Didesoxy-2,3. alpha. -D-Arabino-hexopyrannoside labelled with carbon-14 or carbon-13 (CY 233 - SR 90008). Synthese du methyl ((chloro-2 ethyl)-3 nitroso-3 Ureido)-3 Didesoxy-2,3. alpha. -D-Arabino-hexopyrannoside marque au carbone-14 ou carbone-13 (CY 233 - SR 90008)

Energy Technology Data Exchange (ETDEWEB)

Sion, R.; Schumer, A.; Durme, E. van (Sanofi Recherche, Brussels (Belgium)); Gouyette, A. (Centre de Lutte Contre le Cancer Gustave-Roussy, 94 - Villejuif (France)); Geslin, M.; Fournier, J.P.; Roger, P. (Sanofi Recherche, Montrouge (France). Inst. Choay); Berger, Y. (Sanofi Recherche, Montpellier (France))

1990-06-01

CY 233 (Ecomustine or SR 90098) is a new antitumour nitrosourea: it is characterized by a 2-chloroethylnitrosourea substituent on a dideoxycarbohydrate. It has been labelled with {sup 14}C on (a) the carbonyl group of the urea in four stages starting with {sup 14}COCl{sub 2}, (b) the second carbon of the chloroethyl group in four stages starting with ({sup 14}C) ethanolamine, and (c) on the methyl group on the anomeric centre of the carbohydrate in three stages starting with {sup 14}CH{sub 3}OH. The final position was also labelled with {sup 13}C starting with {sup 13}CH{sub 3}OH. These differently labelled compounds are suitable for mechanistic studies of antitumour activity. (author).

32 CFR 14.3 - Policies and procedures.

Science.gov (United States)

2010-07-01

... 32 National Defense 1 2010-07-01 2010-07-01 false Policies and procedures. 14.3 Section 14.3... QUALIFICATION OF CIVILIAN DEFENSE COUNSEL § 14.3 Policies and procedures. (a) Application procedures. (1... the subject of any sanction or disciplinary action by any court, bar, or other competent governmental...

VEGFR-3 signaling is regulated by a G-protein activator, activator of G-protein signaling 8, in lymphatic endothelial cells.

Science.gov (United States)

Sakima, Miho; Hayashi, Hisaki; Mamun, Abdullah Al; Sato, Motohiko

2018-07-01

Vascular endothelial growth factor C (VEGFC) and its cognate receptor VEGFR-3 play a key role in lymphangiogenesis. We previously reported that an ischemia-inducible Gβγ signal regulator, activator of G-protein signaling 8 (AGS8), regulated the subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR-2) and influenced VEGFA-induced signaling in vascular endothelial cells. Here, we report that AGS8 regulates VEGFR-3, which is another subtype of the VEGF receptor family, and mediates VEGFC signaling in human dermal lymphatic endothelial cells (HDLECs). VEGFC stimulated the proliferation of HDLECs and tube formation by HDLECs, which were inhibited by knocking down AGS8 by small interfering RNA (siRNA). AGS8 siRNA inhibited VEGFC-mediated phosphorylation of VEGFR-3 and its downstream molecules, including ERK1/2 and AKT. Analysis of fluorescence-activated cell sorting and immunofluorescence staining demonstrated that AGS8 knockdown was associated with a reduction of VEGFR-3 at the cell surface. Endocytosis inhibitors did not rescue the decrease of cell-surface VEGFR-3, suggesting that AGS8 regulated the trafficking of VEGFR-3 to the plasma membrane. An immunoprecipitation assay indicated that VEGFR-3 formed a complex including AGS8 and Gβγ in cells. These data suggest the novel regulation of VEGFC-VEGFR-3 by AGS8 in HDLECs and a potential role for AGS8 in lymphangiogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Fluorography--limitations on its use for the quantitative detection of 3H- and 14-C-labeled proteins in polyacrylamide gels

International Nuclear Information System (INIS)

Harding, C.R.; Scott, I.R.

1983-01-01

The suitability of fluorography for the detection of 3 H- and 14 C-labeled proteins on polyacrylamide gradient gels has been investigated. If was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration

Insulin receptor substrate-3, interacting with Bcl-3, enhances p50 NF-{kappa}B activity

Energy Technology Data Exchange (ETDEWEB)

Kabuta, Tomohiro [Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657 (Japan); Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502 (Japan); Hakuno, Fumihiko; Cho, Yoshitake; Yamanaka, Daisuke; Chida, Kazuhiro [Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657 (Japan); Asano, Tomoichiro [Graduate School of Biomedical Science, Hiroshima University, Hiroshima 734-8551 (Japan); Wada, Keiji [Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502 (Japan); Takahashi, Shin-Ichiro, E-mail: atkshin@mail.ecc.u-tokyo.ac.jp [Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657 (Japan)

2010-04-09

The insulin receptor substrate (IRS) proteins are major substrates of both insulin receptor and insulin-like growth factor (IGF)-I receptor tyrosine kinases. Previously, we reported that IRS-3 is localized to both cytosol and nucleus, and possesses transcriptional activity. In the present study, we identified Bcl-3 as a novel binding protein to IRS-3. Bcl-3 is a nuclear protein, which forms a complex with the homodimer of p50 NF-{kappa}B, leading to enhancement of transcription through p50 NF-{kappa}B. We found that Bcl-3 interacts with the pleckstrin homology domain and the phosphotyrosine binding domain of IRS-3, and that IRS-3 interacts with the ankyrin repeat domain of Bcl-3. In addition, IRS-3 augmented the binding activity of p50 to the NF-{kappa}B DNA binding site, as well as the tumor necrosis factor (TNF)-{alpha}-induced transcriptional activity of NF-{kappa}B. Lastly, IRS-3 enhanced NF-{kappa}B-dependent anti-apoptotic gene induction and consequently inhibited TNF-{alpha}-induced cell death. This series of results proposes a novel function for IRS-3 as a transcriptional regulator in TNF-{alpha} signaling, distinct from its function as a substrate of insulin/IGF receptor kinases.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

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Raheem Ullah

Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

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Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Synthesis and characterization of n-alkylamino derivatives of vitamin K3: Molecular structure of 2-propylamino-3-methyl-1,4-naphthoquinone and antibacterial activities

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Chadar, Dattatray; Camilles, Maria; Patil, Rishikesh; Khan, Ayesha; Weyhermüller, Thomas; Salunke-Gawali, Sunita

2015-04-01

We would like to introduce eight analogues of n-alkylamino derivatives of vitamin K3 (2-methyl-1,4-naphthoquinone) viz, 2-(n-alkylamino)-3-methyl-1,4-naphthoquinone (where n-alkyl is methyl; LM-1, ethyl; LM-2, propyl; LM-3, butyl; LM-4, pentyl; LM-5, hexyl; LM-6, heptyl; LM-7, octyl; LM-8). All the above analogues have been successfully synthesized from vitamin K3 and characterized using different analytical techniques. Furthermore, in order to understand the mechanistic aspects of formation of LM-1 to LM-8 compounds, we could propose the mechanism. The FT-IR analysis of LM-1 to LM-8 indicate the presence of characteristic band of Nsbnd H group ∼3287-3364 cm-1, the variation was attributed to extensive intramolecular hydrogen bonding interaction. The molecular structure of LM-3 compound has been confirmed by single crystal X-ray diffraction analysis. LM-3 compound crystallises in triclinic space group P1. There were four independent molecules in asymmetric unit cell and their molecular interactions observed via Nsbnd H⋯O, Csbnd H⋯O and π-π stacking of quinonoid rings. Pharmacological potential of all compounds has been evaluated in terms of their antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus. All the compounds were active against both the strains while LM-2 was found to be more effective with a minimum inhibition concentration of 0.3125 μg/mL and 0.156 μg/mL respectively.

14 CFR 95.3 - Symbols.

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2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Symbols. 95.3 Section 95.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES IFR ALTITUDES General § 95.3 Symbols. For the purposes of this part— (a) COP means...

14 CFR 298.3 - Classification.

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2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Classification. 298.3 Section 298.3... REGULATIONS EXEMPTIONS FOR AIR TAXI AND COMMUTER AIR CARRIER OPERATIONS General § 298.3 Classification. (a) There is hereby established a classification of air carriers, designated as “air taxi operators,” which...

Agmatine Reverses Sub-chronic Stress induced Nod-like Receptor Protein 3 (NLRP3) Activation and Cytokine Response in Rats.

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Sahin, Ceren; Albayrak, Ozgur; Akdeniz, Tuğba F; Akbulut, Zeynep; Yanikkaya Demirel, Gulderen; Aricioglu, Feyza

2016-10-01

The activation of Nod-like receptor protein 3 (NLRP3) has lately been implicated in stress and depression as an initiator mechanism required for the production of interleukin (IL)-1β and IL-18. Agmatine, an endogenous polyamine widely distributed in mammalian brain, is a novel neurotransmitter/neuromodulator, with antistress, anxiolytic and antidepressant-like effects. In this study, we examined the effect of exogenously administered agmatine on NLRP3 inflammasome pathway/cytokine responses in rats exposed to restraint stress for 7 days. The rats were divided into three groups: stress, stress+agmatine (40 mg/kg; i.p.) and control groups. Agmatine significantly down-regulated the gene expressions of all stress-induced NLRP3 inflammasome components (NLRP3, NF-κB, PYCARD, caspase-1, IL-1β and IL-18) in the hippocampus and prefrontal cortex (PFC) and reduced pro-inflammatory cytokine levels not only in both brain regions, but also in serum. Stress-reduced levels of IL-4 and IL-10, two major anti-inflammatory cytokines, were restored back to normal by agmatine treatment in the PFC. The findings of the present study suggest that stress-activated NLRP3 inflammasome and cytokine responses are reversed by an acute administration of agmatine. Whether antidepressant-like effect of agmatine can somehow, at least partially, be mediated by the inhibition of NLRP3 inflammasome cascade and relevant inflammatory responses requires further studies in animal models of depression. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

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An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

2008-09-05

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism.

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McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

2016-02-12

Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

Characterization of the third component of complement (C3) after activation by cigarette smoke

International Nuclear Information System (INIS)

Kew, R.R.; Ghebrehiwet, B.; Janoff, A.

1987-01-01

Activation of lung complement by tobacco smoke may be an important pathogenetic factor in the development of pulmonary emphysema in smokers. We previously showed that cigarette smoke can modify C3 and activate the alternative pathway of complement in vitro. However, the mechanism of C3 activation was not fully delineated in these earlier studies. In the present report, we show that smoke-treated C3 induces cleavage of the alternative pathway protein, Factor B, when added to serum containing Mg-EGTA. This effect of cigarette smoke is specific for C3 since smoke-treated C4, when added to Mg-EGTA-treated serum, fails to activate the alternative pathway and fails to induce Factor B cleavage. Smoke-modified C3 no longer binds significant amounts of [ 14 C]methylamine (as does native C3), and relatively little [ 14 C]methylamine is incorporated into its alpha-chain. Thus, prior internal thiolester bond cleavage appears to have occurred in C3 activated by cigarette smoke. Cigarette smoke components also induce formation of noncovalently associated, soluble C3 multimers, with a Mr ranging from 1 to 10 million. However, prior cleavage of the thiolester bond in C3 with methylamine prevents the subsequent formation of these smoke-induced aggregates. These data indicate that cigarette smoke activates the alternative pathway of complement by specifically modifying C3 and that these modifications include cleavage of the thiolester bond in C3 and formation of noncovalently linked C3 multimers

Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

International Nuclear Information System (INIS)

Weber, N.; Mangold, H.K.

1985-01-01

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'- 14 C]hexadecyl-sn-glycerol or rac-1-O-[1'- 14 C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'- 14 C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity

BAG3: a multifaceted protein that regulates major cell pathways

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Rosati, A; Graziano, V; De Laurenzi, V; Pascale, M; Turco, M C

2011-01-01

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. PMID:21472004

Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism.

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Ogi, Kazuhiro; Nakashima, Kenji; Chihara, Kazuyasu; Takeuchi, Kenji; Horiguchi, Tomoko; Fujieda, Shigeharu; Sada, Kiyonao

2011-09-01

Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

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Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

Calcium-Induced Activation of a Mutant G-Protein-Coupled Receptor Causes In Vitro Transformation of NIH/3T3 Cells

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Ana O. Hoff

1999-12-01

Full Text Available The calcium-sensing receptor (CaR is a G-proteincoupled receptor that is widely expressed, has tissuespecific functions, regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151 Met in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

Incineration of radioactive wastes containing only C-14 and H-3

International Nuclear Information System (INIS)

Garcia, Corazon M.

1992-01-01

C-14 and H-3 arc popularly used in chemical and biological research institutions in the Philippines. Most of the solid radioactive wastes generated by these institutions consist of combustible materials such as paper and accumulated environmental samples. Liquid wastes usually contain organic substances. The method proposed for managing C-14 and H-3 wastes is incineration which is expected to provide an acceptable means of disposal for C-14 and H-3 and their hazardous organic constituent. In the incineration process) the radioactively contaminated waste will be mixed with non-radioactive combustible wastes to lower the activity concentration and to improve the efficiency of combustion which will be carried out in a locally fabricated drum incinerator. The calculations presented determines the concentration limit for the incinerable wastes and the restriction on specific activity of the particles of the incinerable wastes containing C-14 or H-3 on the basis of the accepted air concentration and on the annual dose limit for an average radiation worker in the country. In the calculations for C-14, considerations were taken on the exposure received from the deposition of radioactive particles in the lungs containing unoxidized carbon. Calculations for H-3, however, is based on the assumption that the concentration of the radionuclide in the body water is the same as that in the environment. (author)

HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3 Transcriptional Activity.

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Gary T ZeRuth

Full Text Available The transcription factor Gli-similar 3 (Glis3 plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells

International Nuclear Information System (INIS)

Grossman, A.; Richardson, S.B.; Altszuler, N.; Lane, B.

1985-01-01

Extracts of rat pancreas contain significant amounts of an [ 3 H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [ 3 H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [ 3 H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [ 3 H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [ 3 H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [ 3 H]estradiol-binding activity

Synthesis of methyl [(chloro-2 ethyl)-3 nitroso-3 Ureido]-3 Didesoxy-2,3 α-D-Arabino-hexopyrannoside labelled with carbon-14 or carbon-13 (CY 233 - SR 90008)

International Nuclear Information System (INIS)

Sion, R.; Schumer, A.; Durme, E. van; Gouyette, A.; Geslin, M.; Fournier, J.P.; Roger, P.

1990-01-01

CY 233 (Ecomustine or SR 90098) is a new antitumour nitrosourea: it is characterized by a 2-chloroethylnitrosourea substituent on a dideoxycarbohydrate. It has been labelled with 14 C on a) the carbonyl group of the urea in four stages starting with 14 COCl 2 , b) the second carbon of the chloroethyl group in four stages starting with [ 14 C] ethanolamine, and c) on the methyl group on the anomeric centre of the carbohydrate in three stages starting with 14 CH 3 OH. The final position was also labelled with 13 C starting with 13 CH 3 OH. These differently labelled compounds are suitable for mechanistic studies of antitumour activity. (author)

Synthesis and Biological Activity of 3-Chloro-1-(4-perimidine methylcarbonylamino-4-phenyl-azetidin-2-one

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Dinesh R. Panchasara

2009-01-01

Full Text Available Perimidine-1-acetic acid hydrazide (1 undergoes facile condensation with aromatic aldehydes to afford the corresponding azomethine (i.e. Schiff base derivatives (2a-h in good yield. Cyclocondensation of compounds (2a-h with chloro acetyl chloride affords 3-chloro-1-(4-perimidine methylcarbonylamino-4-phenyl-azetidin-2-ones (3a-h. The structures of these compounds were established on the basis of analytical and spectral data. The newly synthesized compounds were evaluated for their antibacterial and antifungal activities.

ANTI-ALLERGIC EFFECTS OF 1,5-BIS(4’-HYDROXY-3’-METHOXYPHENYL-1,4-PENTADIENE-3-ONE ON MAST CELL-MEDIATED ALLERGY MODEL

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AGUNG ENDRO NUGROHO

2009-01-01

Full Text Available 1,5-bis(4’-hydroxy-3’-methoxyphenyl-1,4-pentadiene-3-one is a 1,5-diphenyl-1,4-pentadiene-3-one analogue of curcumin that is produced by modifying the middle site of curcumin leading to 1,4-pentadiene-3-ones to maintain the hydroxy moiety at the aromatic rings that are responsible for its biological activities. Curcumin has been reported to have anti-allergic effects and can inhibit the release of histamine from mast cells. In the present study, we evaluated the anti-allergic effects of 1,5-bis(4’-hydroxy-3’-methoxyphenyl-1,4-pentadiene-3-one in a mast cell-mediated allergy mode in order to provide information about a newly synthesised-compound for an alternative allergy drug. The study was performed using (1 a rat basophilic leukaemia (RBL-2H3 cell line, which is a tumour analogue of mast cells, with DNP24-BSA, thapsigargin and ionomycin as inducers for secretory markers from mast cells, and (2 an active cutaneous anaphylaxis (ACA reaction, with ovalbumin as an inductor of mast cell degranulation. Treatment with 1,5-bis(4’-hydroxy-3’-methoxyphenyl-1,4-pentadiene-3-one strongly inhibited the DNP24-BSA, thapsigargin and ionomycin-mediated release of histamine and β-hexosaminidase from the RBL-2H3 cell line. The results indicated that this compound influenced the activation processes of FcεRI by antigen and intracellular Ca2+ signalling events in mast cells. In type 1 allergy model, this compound also inhibited the active cutaneous anaphylactic reaction on rat dorsal skins generated by ovalbumin. We conclude that the compound 1,5-bis(4’-hydroxy-3’-methoxyphenyl-1,4-pentadiene-3-one showed anti-allergic activities mediated by mechanisms related to intracellular signalling events in mast cells.

Multiple roles for nuclear localization signal (NLS, aa 442-472) of receptor interacting protein 3 (RIP3)

International Nuclear Information System (INIS)

Li Mei; Feng Shanshan; Wu Mian

2008-01-01

RIP3, a Ser/Thr kinase of RIP (Receptor Interacting Protein) family, is recruited to the TNFR1 signaling complex through RIP and has been shown to mediate apoptosis induction and NF-κB activation. RIP3 is a nucleocytoplasmic shuttling protein and its unconventional nuclear localization signal (NLS, 442-472 aa) is sufficient to trigger apoptosis in the nucleus. In this study, we demonstrate that this NLS exhibits several other roles besides apoptotic function. Firstly, this NLS was found to be required for both RIP3-induced apoptosis and RIP3-mediated NF-κB activation. Next, similar to RHIM motif (RIP homotypic interaction motif), NLS of RIP3 was found to be involved in RIP3-RIP interaction. Furthermore, this NLS was found to be both sufficient and necessary for RIP3 self-association. Our primary data also showed that RIP3 might form a homodimer within cells, and its apoptotic activity may not be required for this dimerization, rather the intactness of NLS determines RIP3-induced apoptosis, since a point mutation at amino acid residue 452 (Ile to Ala) within NLS greatly reduced its apoptotic ability, despite that RIP3 point mutant RIP3/I452A is able to dimerize with wild type RIP3 or itself

Hypoxic activation of the unfolded protein response (UPR) induces expression of the metastasis-associated gene LAMP3

International Nuclear Information System (INIS)

Mujcic, Hilda; Rzymski, Tomasz; Rouschop, Kasper M.A.; Koritzinsky, Marianne; Milani, Manuela; Harris, Adrian L.; Wouters, Bradly G.

2009-01-01

Background and purpose: Tumour hypoxia contributes to failure of cancer treatment through its ability to protect against therapy and adversely influence tumour biology. In particular, several studies suggest that hypoxia promotes metastasis. Hypoxia-induced cellular changes are mediated by oxygen-sensitive signaling pathways that activate downstream transcription factors. We have investigated the induction and transcriptional regulation of a novel metastasis-associated gene, LAMP3 during hypoxia. Materials and methods: Microarray, quantitative PCR, Western blot analysis and immunohistochemistry were used to investigate hypoxic regulation of LAMP3. The mechanism for LAMP3 induction was investigated using transient RNAi and stable shRNA targeting components of the hypoxic response. Endoplasmic reticulum stress inducing agents, including proteasome inhibitors were assessed for their ability to regulate LAMP3. Results: LAMP3 is strongly induced by hypoxia at both the mRNA and protein levels in a large panel of human tumour cell lines. Induction of LAMP3 occurs as a consequence of the activation of the PERK/eIF2α/ATF4 arm of the unfolded protein response (UPR) and is independent of HIF-1α. LAMP3 is expressed heterogeneously within the microenvironment of tumours, overexpressed in breast cancer, and increases in tumours treated with avastin. Conclusions: These data identify LAMP3 as a novel hypoxia-inducible gene regulated by the UPR. LAMP3 is a new candidate biomarker of UPR activation by hypoxia in tumours and is a potential mediator of hypoxia-induced metastasis.

Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

Science.gov (United States)

Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

2016-03-21

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

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Zhigang Yi

2011-01-01

Full Text Available Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14 that when overexpressed was able to mediate protection from yellow fever virus (YFV-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV, a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV, all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work

Synthesis of 7-[α-(2-amino-[2-14C]thiazol-4-yl)-α-(Z)-methoxyimin oacetamido]-3-(1-methylpyrrolidinio)methyl-3-cephem-4-carboxylate hydrochloride ([14C]cefepime hydrochloride)

International Nuclear Information System (INIS)

Standridge, R.T.; Swigor, J.E.

1993-01-01

The title compound ([ 14 C]cefepime hydrochloride) was prepared as follows:- [ 14 C]Thiourea was condensed with ethyl 4-bromo-3-oxo-2-methoxyimino-acetate providing ethyl 2-(2-amino-4-[2- 14 C] thiazolyl)-2-methoxyi-minoacetate as the pure Z-isomer. Saponification gave the amino acid this was reacted with 1-hydroxybenzotriazole to give the activated ester. Condensation in situ with 7-amino-3-(1-methylpyrrolidinio) methyl-3-cephem-4-carboxylate yielded the product as the pure sulfate salt. Treatment of the sulfate salt with base provided the zwitterion isolated as the stable N-methyl-2-pyrrolidinone adduct. An aqueous solution of the adduct was converted to the crystalline title compound, [ 14 C]Cefepime hydrochloride hydrate, with hydrochloric acid/acetone. Radiochemical purity was 99.0% and specific activity, 34.2 μCi/mg. Overall yield from [ 14 C]thiourea was 18%. (Author)

Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

International Nuclear Information System (INIS)

Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.

1986-01-01

The authors have determined the ontogeny of vitamin D 3 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM 3 H-vitamin D 3 in the presence of an NADPH generating system, oxygen, KCl, and MgCl 2 . Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D 3 (250HD 3 ) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD 3 in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D 3 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D 3 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D 3 25-hydroxylase

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

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Hoyt, Laura R.; Ather, Jennifer L.; Randall, Matthew J.; DePuccio, Daniel P.; Landry, Christopher C.; Wewers, Mark D.; Gavrilin, Mikhail A.; Poynter, Matthew E.

2016-01-01

Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished ASC speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of GABAA receptor activation or NMDA receptor inhibition, but was associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC, were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols. PMID:27421477

Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

Science.gov (United States)

Tran, Si C.; Pham, Tu M.; Nguyen, Lam N.; Park, Eun-Mee; Lim, Yun-Sook

2016-01-01

ABSTRACT Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCE TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of

Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

International Nuclear Information System (INIS)

Zhang, Yi; Gonzalez, Rachel M; Zangar, Richard C

2011-01-01

Protein secretion by mammary cells results in autocrine and paracrine signaling that defines cell growth, migration and the extracellular environment. Even so, we have a limited understanding of the cellular processes that regulate protein secretion. In this study, we utilize human epithelial mammary cell (HMEC) lines that were engineered to express different levels of HER1, HER2 and HER3. Using an ELISA microarray platform, we evaluate the effects of epidermal growth factor family receptor (HER) expression on protein secretion in the HMEC lines upon initiation of HER1 receptor activation. The secreted proteins include three HER1 ligands, interleukins 1α and 18, RANTES, vascular-endothelial and platelet-derived growth factors, matrix metalloproteases 1, 2 and 9, and the extracellular portion of the HER1 and HER2 proteins. In addition, we investigate whether MAPK/Erk and PI3K/Akt signaling regulate protein secretion in these cell lines and if so, whether the involvement of HER2 or HER3 receptor alters their response to MAPK/Erk and PI3K/Akt signal pathway inhibition in terms of protein secretion. Differential expression of HER2 and HER3 receptors alters the secretion of a variety of growth factors, cytokines, and proteases. Some alterations in protein secretion are still observed when MAPK/Erk or PI3K/Akt signaling is inhibited. This study suggests that HER overexpression orchestrates broad changes in the tumor microenvironment by altering the secretion of a diverse variety of biologically active proteins

14 CFR 155.3 - Applicable law.

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2010-01-01

... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Applicable law. 155.3 Section 155.3... RELEASE OF AIRPORT PROPERTY FROM SURPLUS PROPERTY DISPOSAL RESTRICTIONS § 155.3 Applicable law. (a... transfer to the requirements of applicable law. Based on the laws cited in this paragraph, the...

The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data

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Lee, Woonghee, E-mail: whlee@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison and Biochemistry Department (United States); Petit, Chad M. [University of Alabama at Birmingham, Department of Biochemistry and Molecular Genetics (United States); Cornilescu, Gabriel; Stark, Jaime L.; Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison and Biochemistry Department (United States)

2016-06-15

We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27–98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data

International Nuclear Information System (INIS)

Lee, Woonghee; Petit, Chad M.; Cornilescu, Gabriel; Stark, Jaime L.; Markley, John L.

2016-01-01

We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27–98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

Tunable paramagnetic relaxation enhancements by [Gd(DPA)3]3- for protein structure analysis

International Nuclear Information System (INIS)

Yagi, Hiromasa; Loscha, Karin V.; Su, Xun-Cheng; Stanton-Cook, Mitchell; Huber, Thomas; Otting, Gottfried

2010-01-01

Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein-ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA) 3 ] 3- , can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA) 3 ] 3- to protein, allowing quantitative distance measurements for nuclear spins within about 15 A of the Gd 3+ ion. Such data accurately define the metal position relative to the protein, greatly enhancing the interpretation of pseudocontact shifts induced by [Ln(DPA) 3 ] 3- complexes of paramagnetic lanthanide (Ln 3+ ) ions other than gadolinium. As an example we studied the quaternary structure of the homodimeric GCN4 leucine zipper.

Tomato leaf curl Kerala virus (ToLCKeV AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1

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Mukherjee Sunil K

2010-06-01

Full Text Available Abstract Background Geminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study. Results AC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro. Conclusions Highly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines.

Science.gov (United States)

Torres-Gómez, Héctor; Lehmkuhl, Kirstin; Frehland, Bastian; Daniliuc, Constantin; Schepmann, Dirk; Ehrhardt, Christina; Wünsch, Bernhard

2015-08-01

Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki=11 μM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn-3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

International Nuclear Information System (INIS)

Miziorko, H.M.; Behnke, C.E.

1985-01-01

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

Energy Technology Data Exchange (ETDEWEB)

Geng, Weijia; Li, Yang; Yu, Yongbo; Yu, Yang; Duan, Junchao; Jiang, Lizhen; Li, Qiuling; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn, E-mail: zwsun@hotmail.com [Capital Medical University, School of Public Health (China)

2016-04-15

Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.

Silica nanoparticles induce multinucleation through activation of PI3K/Akt/GSK-3β pathway and downregulation of chromosomal passenger proteins in L-02 cells

Science.gov (United States)

Geng, Weijia; Li, Yang; Yu, Yongbo; Yu, Yang; Duan, Junchao; Jiang, Lizhen; Li, Qiuling; Sun, Zhiwei

2016-04-01

Silica nanoparticles (SNPs) are applicable in various fields due to their unique physicochemical characteristics. However, concerns over their potential adverse effects have been raised. In our previous studies, we reported that SNPs could induce abnormal high incidence of multinucleation. The aim of this study is to further investigate the mechanisms of multinucleation induced by SNPs (68 nm) in human normal liver L-02 cells (L-02 cells). In order to determine the cytotoxicity of SNPs, MTT assay was performed, and the cell viability was decreased in a dose-dependent manner. The intracellular reactive oxygen species (ROS) detected by flow cytometry and multinucleation observed by Giemsa stain showed that ROS generation and rate of multinucleated cells increased after SNPs exposure. N-acetyl-cysteine (NAC), a glutathione precursor against SNP-induced toxicity, was used as a ROS inhibitor to elucidate the relationship between ROS and multinucleation. The presence of NAC resulted in inhibition of both ROS generation and rate of multinucleation. Moreover, Western blot analysis showed that the protein levels of Cdc20, Aurora B, and Survivin were down-regulated, and the PI3K/Akt/GSK-3β pathway was activated by SNPs. In conclusion, our findings strongly suggested that multinucleation induced by SNPs was related to PI3K/Akt/GSK-3β signal pathway activation and downregulation of G2/M phase-related protein and chromosomal passenger proteins.

Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

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Hirotaka Takahashi

Full Text Available Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3. Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1 targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

Some biological consequences of disintegration of 3H and 14C incorporated in an influenza virus

International Nuclear Information System (INIS)

Prokudina, E.N.; Semenova, N.P.; Yamnikova, S.S.; Zhdanov, V.M.

1987-01-01

An influenza virus labeled with 3 H-uridine losses its infectiousness when stored for a long time. It is suggested that disintegration of tritium incorporated into virus RNA causes lethal intramolecular modifications therein. At the same time, the antigenic activity of virus nucleoprotein decreases perhaps due to the direct effect of tritium. The comparison of the degree of inactivation of various antigenic sites of the nucleoprotein within a virus, labeled with 3 H-uridine, suggests that they are located at different distances from RNA. A long-term action of 3 H disintegration on RNA of a maturing virus decreased the yield probably due to the injury of the intracellular virus RNA during the infections process. Upon storage of the influenza virus labelle with 14 C-amino acids the antigenic properties are reduced by the nucleoprotein while the infectiousness remains unaffected. The long-term effect of 14 C disintegration on proteins of the maturing virus does not lead to fatal outcome

3DSwap: Curated knowledgebase of proteins involved in 3D domain swapping

KAUST Repository

Shameer, Khader

2011-09-29

Three-dimensional domain swapping is a unique protein structural phenomenon where two or more protein chains in a protein oligomer share a common structural segment between individual chains. This phenomenon is observed in an array of protein structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics analyses to develop an integrated knowledgebase of proteins involved in 3D domain swapping. The hallmark of 3D domain swapping is the presence of distinct structural segments such as the hinge and swapped regions. We have curated the literature to delineate the boundaries of these regions. In addition, we have defined several new concepts like \\'secondary major interface\\' to represent the interface properties arising as a result of 3D domain swapping, and a new quantitative measure for the \\'extent of swapping\\' in structures. The catalog of proteins reported in 3DSwap knowledgebase has been generated using an integrated structural bioinformatics workflow of database searches, literature curation, by structure visualization and sequence-structure-function analyses. The current version of the 3DSwap knowledgebase reports 293 protein structures, the analysis of such a compendium of protein structures will further the understanding molecular factors driving 3D domain swapping. The Author(s) 2011.

Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

International Nuclear Information System (INIS)

Lehto, Markku; Hynynen, Riikka; Karjalainen, Katja; Kuismanen, Esa; Hyvaerinen, Kati; Olkkonen, Vesa M.

2005-01-01

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P 2 and PI(3,4,5)P 3 . A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein

Turnover of whole body proteins and myofibrillar proteins in middle-aged active men

International Nuclear Information System (INIS)

Zackin, M.; Meredith, C.; Frontera, W.; Evans, W.

1986-01-01

Endurance-trained older men have a higher proportion of lean tissue and greater muscle cell oxidative capacity, reversing age-related trends and suggesting major changes in protein metabolism. In this study, protein turnover was determined in 6 middle-aged (52+/-1 yr) men who were well trained (VO 2 max 55.2+/-5.0 ml O 2 /kg.min) and lean (body fat 18.9+/-2.8%, muscle mass 36.6+/-0.6%). The maintained habitual exercise while consuming 0.6, 0.9 or 1.2 g protein/kg.day for 10-day periods. N flux was measured from 15 N in urea after oral 15 N-glycine administration. Myofibrillar protein breakdown was estimated from urinary 3-methyl-histidine. Dietary protein had no effect on turnover rates, even when N balance was negative. Whole body protein synthesis was 3.60+/-0.12 g/kg.day and breakdown was 3.40+/-0.14 g/kg.day for all N intakes. Whole body protein flux, synthesis and breakdown were similar to values reported for sedentary young (SY) or sedentary old (SO) men on comparable diets. 3-me-his (3.67+/-0.14 μmol/kg.day) was similar to values reported for SY but higher (p<0.01) than for SO. Myofibrillar protein breakdown per unit muscle mass (185+/-7 μmol 3-me-his/g creatinine) was higher (p<0.01) than for SY or SO. In active middle-aged men, myofibrillar proteins may account for a greater proportion of whole body protein turnover, despite an age-related reduction in muscle mass

Monocyte chemotactic protein-3: possible involvement in apical periodontitis chemotaxis.

Science.gov (United States)

Dezerega, A; Osorio, C; Mardones, J; Mundi, V; Dutzan, N; Franco, M; Gamonal, J; Oyarzún, A; Overall, C M; Hernández, M

2010-10-01

To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry.   MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis. © 2010 International Endodontic Journal.

Synthesis of 14C- and 2H-labeled 1,3 dihydro-3, 3-dimethyl-5-(1,4,5,6,- tetrahydro-6-oxo-3-pyridazinyl)-2H-indol-2-one (LY195115), an orally effective positive inotrope

International Nuclear Information System (INIS)

Robertson, D.W.; Krushinski, J.H.; Kau, D.

1986-01-01

The synthesis of 14 C- and 2 H-labeled 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-indol -2-one (LY195115), an extremely potent, orally-effective cardiotonic with inotropic and vasodilator activities is described. The 14 C-label was introduced in the antepenultimate step by reaction of a β-chloroketone precursor with Na 14 CN; acid-catalyzed hydrolysis and cyclization with hydrazine provided the tetrahydropyridazinone bearing the 14 C-label in the oxo-carbon. 1,3-Dihydro-3,3-di(methyl-d 3 ) -2H-indol-2-one was prepared by exhaustive methylation of 1-acetyl-1,3-dihydro-2H-indol-2-one with sodium hydride and iodomethane-d 3 , followed by removal of the nitrogen protecting group. This labeled material was converted in two steps to [ 2 H 6 ]-LY195115. (author)

3DProIN: Protein-Protein Interaction Networks and Structure Visualization.

Science.gov (United States)

Li, Hui; Liu, Chunmei

2014-06-14

3DProIN is a computational tool to visualize protein-protein interaction networks in both two dimensional (2D) and three dimensional (3D) view. It models protein-protein interactions in a graph and explores the biologically relevant features of the tertiary structures of each protein in the network. Properties such as color, shape and name of each node (protein) of the network can be edited in either 2D or 3D views. 3DProIN is implemented using 3D Java and C programming languages. The internet crawl technique is also used to parse dynamically grasped protein interactions from protein data bank (PDB). It is a java applet component that is embedded in the web page and it can be used on different platforms including Linux, Mac and Window using web browsers such as Firefox, Internet Explorer, Chrome and Safari. It also was converted into a mac app and submitted to the App store as a free app. Mac users can also download the app from our website. 3DProIN is available for academic research at http://bicompute.appspot.com.

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

International Nuclear Information System (INIS)

Hasan, Raghibul; Sharma, Rinu; Saraya, Anoop; Chattopadhyay, Tushar K; DattaGupta, Siddartha; Walfish, Paul G; Chauhan, Shyam S; Ralhan, Ranju

2014-01-01

Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (p trend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC

14 CFR 23.3 - Airplane categories.

Science.gov (United States)

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Airplane categories. 23.3 Section 23.3... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES General § 23.3 Airplane categories. (a) The normal category is limited to airplanes that have a seating configuration, excluding pilot...

Microstructure and tensile properties of oxide dispersion strengthened Fe–14Cr–0.3Y{sub 2}O{sub 3} and Fe–14Cr–2W–0.3Ti–0.3Y{sub 2}O{sub 3}

Energy Technology Data Exchange (ETDEWEB)

Auger, M.A., E-mail: mauger@fis.uc3m.es [Departamento de Física, Universidad Carlos III de Madrid, 28911 Leganés (Spain); Castro, V. de; Leguey, T.; Monge, M.A.; Muñoz, A.; Pareja, R. [Departamento de Física, Universidad Carlos III de Madrid, 28911 Leganés (Spain)

2013-11-15

Two ODS ferritic steels with nominal compositions (wt.%): Fe–14Cr–0.3Y{sub 2}O{sub 3} and Fe–14Cr–2W–0.3Ti–0.3Y{sub 2}O{sub 3} have been produced by mechanical alloying and consolidation by hot isostatic pressing. The microstructure and tensile properties of these materials after being forged and heat-treated at 1123 K have been investigated to clarify the interrelation between composition, microstructure and mechanical properties. The second-phase precipitates in these alloys have been analyzed by high-angle annular dark-field imaging in scanning TEM mode and electron diffraction. Fe–14Cr–2W–0.3Ti–0.3Y{sub 2}O{sub 3} exhibits a duplex microstructure consisting of large recrystallized grains, as large as 1.5 μm, and unrecovered regions containing submicron equiaxed grains. In addition, three types of secondary phase particles have been found: large M{sub 23}C{sub 6} particles containing W and Cr, (Cr + Ti) rich spherical particles with diameters between 50 and 500 nm, and fine (Y + Ti) oxide particles with sizes below 30 nm. In contrast, Fe14CrY shows a uniform structure of equiaxed grains, with sizes in the range 0.5–3 μm, containing a fine dispersion of Y oxide particles (<30 nm) homogeneously distributed inside the grains, as well as large carbide and oxide particles. Tensile tests performed over the temperature range 273–973 K have revealed that the alloy containing W and Ti has lower yield and tensile strengths than Fe–14Cr–0.3Y{sub 2}O{sub 3} at temperatures up to 773 K, but the opposite appears to occur beyond this temperature.

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

Directory of Open Access Journals (Sweden)

Leonie Unterholzner

2011-09-01

Full Text Available Recognition of viruses by pattern recognition receptors (PRRs causes interferon-β (IFN-β induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1 and IκB kinase-ε (IKKε, which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Energy Technology Data Exchange (ETDEWEB)

Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

International Nuclear Information System (INIS)

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

2010-01-01

Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Adenovirus Protein E4-ORF1 activation of PI3 kinase reveals differential regulation of downstream effector pathways in adipocytes

OpenAIRE

Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K.; McGraw, Timothy E.

2016-01-01

Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but...

CXCR3 expression and activation of eosinophils

DEFF Research Database (Denmark)

Jinquan, T; Jing, C; Jacobi, H H

2000-01-01

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce...... in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact...

3dRPC: a web server for 3D RNA-protein structure prediction.

Science.gov (United States)

Huang, Yangyu; Li, Haotian; Xiao, Yi

2018-04-01

RNA-protein interactions occur in many biological processes. To understand the mechanism of these interactions one needs to know three-dimensional (3D) structures of RNA-protein complexes. 3dRPC is an algorithm for prediction of 3D RNA-protein complex structures and consists of a docking algorithm RPDOCK and a scoring function 3dRPC-Score. RPDOCK is used to sample possible complex conformations of an RNA and a protein by calculating the geometric and electrostatic complementarities and stacking interactions at the RNA-protein interface according to the features of atom packing of the interface. 3dRPC-Score is a knowledge-based potential that uses the conformations of nucleotide-amino-acid pairs as statistical variables and that is used to choose the near-native complex-conformations obtained from the docking method above. Recently, we built a web server for 3dRPC. The users can easily use 3dRPC without installing it locally. RNA and protein structures in PDB (Protein Data Bank) format are the only needed input files. It can also incorporate the information of interface residues or residue-pairs obtained from experiments or theoretical predictions to improve the prediction. The address of 3dRPC web server is http://biophy.hust.edu.cn/3dRPC. yxiao@hust.edu.cn.

Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

Directory of Open Access Journals (Sweden)

Lu Frances Fangjia

2012-11-01

Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

In vitro and in vivo characterisation of [{sup 3}H]ANSTO-14 binding to the {sigma}{sub 1} binding sites

Energy Technology Data Exchange (ETDEWEB)

Nguyen, Vu H. E-mail: V.H.Nguyen@ansto.gov.au; Mardon, Karine; Kassiou, Michael; Christie, MacDonald J

1999-02-01

ABSTRACT. N-(4-phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0{sup 2,6}.0{sup 3,10}.0{sup 5,9}.0{sup 8}= {sup ,11}]dodecane (ANSTO-14) showed the highest activity for the {sigma}{sub 1} site ( K{sub i}=9.4 nM) and 19-fold {sigma}{sub 1}/{sigma}{sub 2} selectivity. The present study showed that [{sup 3}H]ANSTO-14 binds to a single high-affinity site in guinea pig brain membranes with an equilibrium K{sub d} of 8.0 {+-} 0.3 nM, in good agreement with the kinetic studies ( K{sub d}=13.3{+-}5.4 nM, n=4), and a B{sub max} of 3,199 {+-} 105 fmol/mg protein ( n=4). The in vivo biodistribution of [{sup 3}H]ANSTO-14 showed a high uptake in the diencephalon. Pretreatment of rats with {sigma} ligands including (+)-pentazocine ({sigma}{sub 1}), ANSTO-14 ({sigma}{sub 1}), and DTG ({sigma}{sub 1} and {sigma}{sub 2}) did not significantly reduce radiotracer uptake in the brain, but did in the spleen. A labelled metabolite was found in the liver and brain. Due to its insensitivity to {sigma} ligands, the accumulation of [{sup 3}H]ANSTO-14 in the brain indicates high nonspecific binding. Therefore, [{sup 3}H]ANSTO-14 is a suitable ligand for labelling {sigma}{sub 1} sites in vitro but is not suitable for brain imaging of {sigma} binding sites in vivo.

1,5-Diphenyl-1,4-pentadiene-3-ones and cyclic analogues as antioxidative agents. Synthesis and structure-activity relationship.

NARCIS (Netherlands)

Sardjiman, S.S.; Reksohadiprodjo, M.S.; Hakim, L.; van der Goot, H.; Timmerman, H.

1997-01-01

A series of 1,5-diphenyl-1,4-pentadiene-3-ones and cyclic analogues with OH-groups in the para position of the phenyl rings and various meta substituents were prepared and their antioxidant activity compared with that of curcumin. Most of them exhibited potent antioxidative activity, especially when

Labelling of pneumococcal penicillin-binding proteins with [3H]propionyl-ampicillin. A rapid method for monitoring penicillin-binding activity

International Nuclear Information System (INIS)

Hakenbeck, R.; Kohiyama, M.

1982-01-01

Penicillin-binding proteins (PBPs) are membrane components ubiquitous to all bacteria examined so far. Some of them are present in only a few copies per cell. The conventional method of visualizing these proteins consists in binding of radioactive penicillin to the fractions containing PBPs followed by SDS-PAGE and finally fluorography. Although this procedure is laborious, it is necessary for the determination of the identity as well as for the quantification of each PBP. On the other hand, when penicillin-binding conditions are to be examined or binding activity has to be followed through fractionation and purification of PBPs, no fast monitoring device for these proteins has been available. The authors developed a rapid and easy assay for penicillin-binding activity with a filter-binding technique using [ 3 H]propionyl ampicillin ( 3 H-PA) of high specific activity. As little 2μg of crude membranes obtained from the highly penicillin-sensitive, β-lactamase-negative organism Streptococcus pneumoniae, are sufficient to detect binding activity. In this paper they describe optimum conditions for the assay of PBPs and show that this binding activity correlates with the presence of native penicillin-binding proteins. (Auth.)

Self-association and domain rearrangements between complement C3 and C3u provide insight into the activation mechanism of C3.

Science.gov (United States)

Li, Keying; Gor, Jayesh; Perkins, Stephen J

2010-10-01

Component C3 is the central protein of the complement system. During complement activation, the thioester group in C3 is slowly hydrolysed to form C3u, then the presence of C3u enables the rapid conversion of C3 into functionally active C3b. C3u shows functional similarities to C3b. To clarify this mechanism, the self-association properties and solution structures of C3 and C3u were determined using analytical ultracentrifugation and X-ray scattering. Sedimentation coefficients identified two different dimerization events in both proteins. A fast dimerization was observed in 50 mM NaCl but not in 137 mM NaCl. Low amounts of a slow dimerization was observed for C3u and C3 in both buffers. The X-ray radius of gyration RG values were unchanged for both C3 and C3u in 137 mM NaCl, but depend on concentration in 50 mM NaCl. The C3 crystal structure gave good X-ray fits for C3 in 137 mM NaCl. By randomization of the TED (thioester-containing domain)/CUB (for complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains in the C3b crystal structure, X-ray fits showed that the TED/CUB domains in C3u are extended and differ from the more compact arrangement of C3b. This TED/CUB conformation is intermediate between those of C3 and C3b. The greater exposure of the TED domain in C3u (which possesses the hydrolysed reactive thioester) accounts for the greater self-association of C3u in low-salt conditions. This conformational variability of the TED/CUB domains would facilitate their interactions with a broad range of antigenic surfaces. The second dimerization of C3 and C3u may correspond to a dimer observed in one of the crystal structures of C3b.

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors.

Science.gov (United States)

Mototani, Yasumasa; Okamura, Tadashi; Goto, Motohito; Shimizu, Yukiko; Yanobu-Takanashi, Rieko; Ito, Aiko; Kawamura, Naoya; Yagisawa, Yuka; Umeki, Daisuke; Nariyama, Megumi; Suita, Kenji; Ohnuki, Yoshiki; Shiozawa, Kouichi; Sahara, Yoshinori; Kozasa, Tohru; Saeki, Yasutake; Okumura, Satoshi

2018-06-01

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

Protein: FEA3 [TP Atlas

Lifescience Database Archive (English)

Full Text Available FEA3 AREB pathway: Signaling proteins At4g11890/T26M18_100 At4g11890, Protein kinase family pr...otein, Putative uncharacterized protein At4g11890/T26M18_100 3702 Arabidopsis thaliana 826796 Q8GY82 22225700 ...

Early events elicited by Bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

International Nuclear Information System (INIS)

Zachary, I.; Sinnett-Smith, J.W.; Rozengurt, E.

1986-01-01

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an M/sub r/ 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent. The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125 I-labeled epidermal growth factor ( 125 I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca 2+ concentration does not mediate the bombesin inhibition of 125 I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125 I-EGF to its cellular receptor

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

International Nuclear Information System (INIS)

Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

2016-01-01

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0–15 μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. - Highlights: • Andrographolide is a diterpenoid phytochemical. �

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation