MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

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Mirlekar, Bhalchandra; Patil, Sachin; Bopanna, Ramanamurthy; Chattopadhyay, Samit

2015-01-01

T reg cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T reg cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T reg phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T reg cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T reg cells in SMAR1 −/− mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T reg cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T reg physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T reg cells. • SMAR1 −/− T reg cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1 −/− mice. • Both Foxp3 and SMAR1 maintain T reg phenotype that controls colitis

An AML1-ETO/miR-29b-1 regulatory circuit modulates phenotypic properties of acute myeloid leukemia cells.

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Zaidi, Sayyed K; Perez, Andrew W; White, Elizabeth S; Lian, Jane B; Stein, Janet L; Stein, Gary S

2017-06-20

Acute myeloid leukemia (AML) is characterized by an aggressive clinical course and frequent cytogenetic abnormalities that include specific chromosomal translocations. The 8;21 chromosomal rearrangement disrupts the key hematopoietic RUNX1 transcription factor, and contributes to leukemia through recruitment of co-repressor complexes to RUNX1 target genes, altered subnuclear localization, and deregulation of the myeloid gene regulatory program. However, a role of non-coding microRNAs (miRs) in t(8;21)-mediated leukemogenesis is minimally understood. We present evidence of an interplay between the tumor suppressor miR-29b-1 and the AML1-ETO (also designated RUNX1-RUNX1T1) oncogene that is encoded by the t(8;21). We find that AML1-ETO and corepressor NCoR co-occupy the miR-29a/b-1 locus and downregulate its expression in leukemia cells. Conversely, re-introduction of miR-29b-1 in leukemia cells expressing AML1-ETO causes significant downregulation at the protein level through direct targeting of the 3' untranslated region of the chimeric transcript. Restoration of miR-29b-1 expression in leukemia cells results in decreased cell growth and increased apoptosis. The AML1-ETO-dependent differentiation block and transcriptional program are partially reversed by miR-29b-1. Our findings establish a novel regulatory circuit between the tumor-suppressive miR-29b-1 and the oncogenic AML1-ETO that controls the leukemic phenotype in t(8;21)-carrying acute myeloid leukemia.

Trans-acting translational regulatory RNA binding proteins.

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Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

2018-05-01

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

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Mirlekar, Bhalchandra; Patil, Sachin [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Bopanna, Ramanamurthy [Experimental Animal Facility, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Chattopadhyay, Samit, E-mail: samit@nccs.res.in [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India)

2015-08-21

T{sub reg} cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T{sub reg} cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T{sub reg} phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T{sub reg} cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T{sub reg} cells in SMAR1{sup −/−} mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T{sub reg} cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T{sub reg} physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T{sub reg} cells. • SMAR1{sup −/−} T{sub reg} cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1{sup −/−} mice. • Both Foxp3 and SMAR1 maintain T{sub reg} phenotype that controls colitis.

Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

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Volkman, Brian Finley [Univ. of California, Berkeley, CA (United States)

1995-02-01

Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

The steroidogenic response and corpus luteum expression of the steroidogenic acute regulatory protein after human chorionic gonadotropin administration at different times in the human luteal phase.

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Kohen, Paulina; Castro, Olga; Palomino, Alberto; Muñoz, Alex; Christenson, Lane K; Sierralta, Walter; Carvallo, Pilar; Strauss, Jerome F; Devoto, Luigi

2003-07-01

This study was designed 1) to assess corpus luteum (CL) steroidogenesis in response to exogenous human chorionic gonadotropin (hCG) at different times during the luteal phase, 2) to examine the effect of hCG on steroidogenic acute regulatory protein (StAR) expression within the CL, 3) to correlate StAR expression and luteal steroidogenic responses to hCG, and 4) to determine whether endogenous LH regulates ovarian steroidogenesis in the early luteal phase. Blood was collected before and after hCG treatment for steroid and hCGbeta determinations. CL were obtained at the time of surgery to assess StAR gene and protein expression. During the early luteal phase various women received the GnRH antagonist for 24-48 h; some of them also received hCG 24 h after the GnRH antagonist. A slight steroidogenic response to hCG was observed in early luteal phase; 17alpha-hydroxyprogesterone, but not progesterone (P4), levels were significantly increased 8 h post-hCG, indicating a differential response by the granulosa and theca-lutein cells. The 1.6- and 4.4-kb StAR transcripts and the 37-kDa preprotein and 30-kDa mature StAR protein did not change post-hCG administration in early luteal phase CL. In contrast, the StAR 4.4- and 1.6-kb transcripts diminished significantly (P < 0.05) after the antagonist treatment. Immunohistochemical staining for StAR protein was weak, particularly in granulosa-lutein cells. Treatment with hCG restored StAR mRNA and protein and plasma P4 levels within 24 h in antagonist-treated women. hCG stimulated the highest plasma concentrations of P4 and estradiol in the midluteal phase, indicating its greatest steroidogenic capacity. Midluteal tissue StAR gene and protein expression increased by 1.6- and 1.4-fold after 24 h of hCG treatment, respectively. Administration of hCG resulted in the greatest increment in plasma P4 (4-fold) and 17alpha-hydroxyprogesterone (3-fold) levels over baseline in the late luteal phase. This was associated with an increase in

Immunomodulatory effects of high-protein diet with resveratrol supplementation on radiation-induced acute-phase inflammation in rats.

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Kim, Kyoung-Ok; Park, HyunJin; Chun, Mison; Kim, Hyun-Sook

2014-09-01

We hypothesized that a high-protein diet and/or resveratrol supplementation will improve acute inflammatory responses in rats after receiving experimental abdominal radiation treatment (ART). Based on our previous study, the period of 10 days after ART was used as an acute inflammation model. Rats were exposed to a radiation dose of 17.5 Gy and were supplied with a control (C), 30% high-protein diet (HP), resveratrol supplementation (RES), or HP with RES diet ([HP+RES]). At day 10 after ART, we measured profiles of lipids, proteins, and immune cells in blood. The levels of clusters of differentiating 4(+) (CD4(+)) cells and regulatory T cells, serum proinflammatory cytokines, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine were also measured. ART caused significant disturbances of lipid profiles by increasing triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), and decreasing high-density lipoprotein cholesterol. The proinflammatroy cytokine levels were also increased by ART. All the experimental diets (HP, RES, and [HP+RES]) significantly decreased levels of TG, monocytes, proinflammatory cytokines, and 8-OHdG, whereas the platelet counts were increased. In addition, the HP and [HP+RES] diets decreased the concentrations of plasma LDL-C and total cholesterol. Also, the HP and RES diets decreased regulatory T cells compared with those of the control diet in ART group. Further, the HP diet led to a significant recovery of white blood cell counts, as well as increased percentages of lymphocyte and decreased percentages of neutrophils. In summary, RES appeared to be significantly effective in minimizing radiation-induced damage to lipid metabolism and immune responses. Our study also demonstrated the importance of dietary protein intake in recovering from acute inflammation by radiation.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

Science.gov (United States)

Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

Science.gov (United States)

Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

2006-01-01

Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

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Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

PROGRESSIVE ALTERATION OF SERUM PROTEINS IN RATS SEVERAL MONTHS AFTER AN ACUTE OR PROTRACTED IRRADIATION

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Ghys, R.; Reuter, A.

1963-06-15

Delayed changes of the serum proteins in male Sprague Dawley rats that survived the acute radiation syndrome were investigated. Doses of Co/sup 60/ gamma ranging from LD/sub O/ to LD/sub 50/ were given to rats six to eight weeks of age. Paper electrophoreses and microdosage of proteins by the buiret method were performed on plasma proteins for 206 rats: 29 with acute irradiation; 73 chronic irradiation; 44 acute irradiation following cold acclimatization; and 80 normal animals. No significant variations in the total serum proteins were found in andy one group. Alpha globulins were found to be slightly above normal in some irradiated rats, but there was no significant variation in the BETA globulin fraction. Gamma globulins showed a marked and consistent increase following irradiation. Thus for observed protein chandges in irradiated rats have not proven to be dose dependent. It is suggested that the changes may provide a link between early irradiation syndrome and late effects. (H.M.G.)

Acute fluoride poisoning alters myocardial cytoskeletal and AMPK signaling proteins in rats.

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Panneerselvam, Lakshmikanthan; Raghunath, Azhwar; Perumal, Ekambaram

2017-02-15

Our previous findings revealed that increased oxidative stress, apoptosis and necrosis were implicated in acute fluoride (F - ) induced cardiac dysfunction apart from hypocalcemia and hyperkalemia. Cardiac intermediate filaments (desmin and vimentin) and cytoskeleton linker molecule vinculin plays an imperative role in maintaining the architecture of cardiac cytoskeleton. In addition, AMPK is a stress activated kinase that regulates the energy homeostasis during stressed state. The present study was aimed to examine the role of cytoskeletal proteins and AMPK signaling molecules in acute F - induced cardiotoxicity in rats. In order to study this, male Wistar rats were treated with single oral doses of 45 and 90mg/kgF - for 24h. Acute F - intoxicated rats showed declined cytoskeletal protein expression of desmin, vimentin and vinculin in a dose dependent manner compared to control. A significant increase in phosphorylation of AMPKα (Thr172), AMPKß1 (Ser108) and Acetyl-coA carboxylase (ACC) (Ser79) in the myocardium and associated ATP deprivation were found in acute F - intoxicated rats. Further, ultra-structural studies confirmed myofibril lysis with interruption of Z lines, dilated sarcoplasmic reticulum and damaged mitochondrion were observed in both the groups of F - intoxicated rats. Taken together, these findings reveal that acute F - exposure causes sudden heart failure by altering the expression of cytoskeletal proteins and AMPK signaling molecules. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Acute phase protein response during acute ruminal acidosis in cattle

DEFF Research Database (Denmark)

Danscher, A. M.; Thoefner, M. B.; Heegaard, Peter M. H.

2011-01-01

The aim of the study was to describe the acute phase protein and leukocyte responses in dairy heifers during acute, oligofructose-induced ruminal acidosis. The study included 2 trials involving oral oligofructose overload (17g/kg BW) to nonpregnant Danish Holstein heifers. Trial 1 included 12...... performed.Heifers receiving oligofructose developed a profound ruminal and systemic acidosis (in Trial 1 and 2 lowest ruminal pH was 4.3±0.2 and 3.8±0.02, respectively, and minimum SBE was −9.3±4.1 and −8.9±2.8, respectively). In Trial 1, SAA concentrations were higher than baseline concentrations on all...... than control heifers at 18 and 24h after overload (max. 13.7±4.3 billions/L). Feeding had no effect on plasma fibrinogen concentrations or WBC in Trial 1.Acute ruminal and systemic acidosis caused by oligofructose overload resulted in distinct acute phase protein and leukocyte responses in dairy...

Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride.

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Panneerselvam, Lakshmikanthan; Raghunath, Azhwar; Perumal, Ekambaram

2017-09-01

Acute fluoride (F - ) toxicity is known to cause severe cardiac complications and leads to sudden heart failure. Previously, we reported that increased myocardial oxidative damage, apoptosis, altered cytoskeleton and AMPK signaling proteins associated with energy deprivation in acute F - induced cardiac dysfunction. The present study was aimed to decipher the status of myocardial heat shock proteins (Hsps-Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, Hsp90) and heat shock transcription factor 1 (Hsf1) in acute F - -intoxicated rats. In order to study the expression of myocardial Hsps, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F - for 24 h. The expression levels of myocardial Hsps were determined using RT-PCR, western blotting, and immunohistochemical studies. Acute F - -intoxicated rats showed elevated levels of both the transcripts and protein expression of Hsf1, Hsp27, Hsp32, Hsp60, and Hsp70 when compared to control. In addition, the expression levels of Hsp40 and Hsp90 were significantly declined in a dose-dependent fashion in F - -treated animals. Our result suggests that differential expression of Hsps in the rat myocardium could serve as a balance between pro-survival and death signal during acute F - -induced heart failure.

The porcine acute phase response to infection with Actinobacillus pleuropneumoniae. Haptoglobin, C-reactive protein, major acute phase protein and serum amyloid a protein are sensitive indicators of infection

DEFF Research Database (Denmark)

Heegaard, Peter M. H.; Klausen, Joan; Nielsen, J.P.

1998-01-01

response peaking at around 2 days after infection. Haptoglobin, C-reactive protein (CRP), and major acute phase protein (MAP) responded with large increases in serum levels, preceding the development of specific antibodies by 4-5 days. Serum amyloid A protein (SAA) was also strongly induced. The increase......, kinetics of induction and normalization were different between these proteins. It is concluded that experimental Ap-infection by the aerosol route induces a typical acute phase reaction in the pig, and that pig Hp, CRP, MAP, and SAA are major acute phase reactants. These findings indicate the possibility...

The Severe Acute Respiratory Syndrome (SARS-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein

Directory of Open Access Journals (Sweden)

Tan Yee-Joo

2005-02-01

Full Text Available Abstract Background A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV, resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro. Presentation of the hypothesis It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression. Testing the hypothesis The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology. Implication of the hypothesis If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe

Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing.

Science.gov (United States)

Hamm, Jon; Sullivan, Kristie; Clippinger, Amy J; Strickland, Judy; Bell, Shannon; Bhhatarai, Barun; Blaauboer, Bas; Casey, Warren; Dorman, David; Forsby, Anna; Garcia-Reyero, Natàlia; Gehen, Sean; Graepel, Rabea; Hotchkiss, Jon; Lowit, Anna; Matheson, Joanna; Reaves, Elissa; Scarano, Louis; Sprankle, Catherine; Tunkel, Jay; Wilson, Dan; Xia, Menghang; Zhu, Hao; Allen, David

2017-06-01

Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

Science.gov (United States)

Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

2016-11-25

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A conserved regulatory mechanism in bifunctional biotin protein ligases.

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Wang, Jingheng; Beckett, Dorothy

2017-08-01

Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

Impaired circulating CD4+ LAP+ regulatory T cells in patients with acute coronary syndrome and its mechanistic study.

Directory of Open Access Journals (Sweden)

Zheng-Feng Zhu

Full Text Available OBJECTIVE: CD4(+ latency-associated peptide (LAP(+ regulatory T cells (Tregs are a newly discovered T cell subset in humans and the role of these cells in patients with acute coronary syndrome (ACS has not been explored. We designed to investigate whether circulating frequency and function of CD4(+LAP(+ Tregs are defective in ACS. METHODS: One hundred eleven ACS patients (acute myocardial infarction and unstable angina and 117 control patients were enrolled in the study. The control patients consisted of chronic stable angina (CSA and chest pain syndrome (CPS. The frequencies of circulating CD4(+LAP(+ Tregs and the expression of the transmembrane protein glycoprotein-A repetitions predominant (GARP on CD4(+ T cells were determined by flow cytometry. The function of CD4(+LAP(+ Tregs was detected using thymidine uptake. Serum interleukin-10 (IL-10 and transforming growth factor-β protein (TGF-β levels were detected using ELISA and expression of GARP mRNA in peripheral blood mononuclear cells (PBMCs was measured by real time-polymerase chain reaction. RESULTS: We found ACS patients had a significantly lower frequency of circulating CD4(+LAP(+ Tregs, and the function of these cells was reduced compared to controls. The expression of GARP in CD4(+ T cells and the serum levels of TGF-β in ACS patients were lower than those of control patients. The serum levels of IL-10 were similar between the two cohorts. CONCLUSIONS: A novel regulatory T cell subset, defined as CD4(+LAP(+ T cells is defective in ACS patients.

Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells.

Science.gov (United States)

Curti, Antonio; Trabanelli, Sara; Onofri, Chiara; Aluigi, Michela; Salvestrini, Valentina; Ocadlikova, Darina; Evangelisti, Cecilia; Rutella, Sergio; De Cristofaro, Raimondo; Ottaviani, Emanuela; Baccarani, Michele; Lemoli, Roberto M

2010-12-01

The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia. Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels. We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4(+)CD25(+) Foxp3(+) T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4(+)CD25(+) T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms' tumor protein. These data identify

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

International Nuclear Information System (INIS)

Hua, Fang; Wang, Jun; Sayeed, Iqbal; Ishrat, Tauheed; Atif, Fahim; Stein, Donald G.

2009-01-01

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.

Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

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Daria eMolodtsova

2014-12-01

Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

Science.gov (United States)

Kesby, James P; Markou, Athina; Semenova, Svetlana

2016-10-01

Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.

Science.gov (United States)

Shi, Feng-Tao; Cheung, Anthony P; Klausen, Christian; Huang, He-Feng; Leung, Peter C K

2010-10-01

We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (β(A)β(A))-induced inhibin B (αβ(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P progesterone production were attenuated (P progesterone secretion than those observed with activin A treatment alone. GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Serum level of soluble fibrinogen-like protein 2 in renal allograft recipients with acute rejection: a preliminary study.

Science.gov (United States)

Zhao, Z; Yang, C; Tang, Q; Zhao, T; Jia, Y; Ma, Z; Rong, R; Xu, M; Zhu, T

2012-12-01

Soluble fibrinogen-like protein 2 (sfgl2), which is mainly secreted by T cells, is a novel effector of regulatory T cells with immunosuppressive functions. The aim of this study was to investigate serum levels of sfgl2 among renal allograft recipients. From November 2010 to August 2011 we retrospectively divided 47 renal allograft recipients into an acute rejection (n = 19) versus a stable group (n = 28) according to allograft biopsy results, using the Banff 2007 classification. The acute rejection group was subdivided into grade I (n = 8) versus grade II T-cell-mediated (n = 6) or antibody-mediated rejection episodes (n = 5). Peripheral blood samples were collected at the time of biopsy. Fourteen healthy volunteers were included as normal group controls. Serum levels of sfgl2 were analyzed by enzyme-linked immunosorbent assay. Serum levels of sfgl2 were increased among renal allograft recipients suffering from biopsy-proven acute rejection episodes (61.91 ± 45.68 ng/mL), versus those with stable allografts (38.59 ± 19.92 ng/mL, P rejection episodes (41.71 ± 16.44 ng/mL, P rejection (34.10 ± 9.26 ng/mL, P rejection episodes to an extent dependent upon the pathological type and severity of the response. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Increased regulatory T cells in acute lymphoblastic leukemia patients.

Science.gov (United States)

Idris, Siti-Zuleha; Hassan, Norfarazieda; Lee, Le-Jie; Md Noor, Sabariah; Osman, Raudhawati; Abdul-Jalil, Marsitah; Nordin, Abdul-Jalil; Abdullah, Maha

2015-10-01

Regulation in adaptive immune response balances a fine line that prevents instigation of self-damage or fall into unresponsiveness permitting abnormal cell growth. Mechanisms that keep this balance in check include regulatory T cells (Tregs). Tregs consist of a small but heterogeneous population which may be identified by the phenotype, CD3+CD4+CD25+CD127-. Role of Tregs in pathogenesis of cancers is thus far supported by evidence of increased Tregs in various cancers and may contribute to poorer prognosis. Tregs may also be important in acute leukemias. A review of the literature on Tregs in acute leukemias was conducted and Tregs were determined in B-cell acute lymphoblastic leukemias (ALLs). Studies on Tregs in B-cell ALL are few and controversial. We observed a significantly increased percentage of Tregs (mean ± SD, 9.72 ± 3.79% vs. 7.05 ± 1.74%; P = 0.047) in the bone marrow/peripheral blood of ALL (n = 17) compared to peripheral blood of normal controls (n = 35). A positive trend between Tregs and age (R = 0.474, P = 0.055, n = 17) implicates this factor of poor prognosis in B-cell ALL. Tregs in cancer are particularly significant in immunotherapy. The manipulation of the immune system to treat cancer has for a long time ignored regulatory mechanisms inducible or in place. In lymphoma studies tumor-specific mechanisms that are unlike conventional methods in the induction of Tregs have been hypothesized. In addition, tumor-infiltrating Tregs may present different profiles from peripheral blood pictures. Tregs will continue to be dissected to reveal their mysteries and their impact on clinical significance.

The level of neuron-specific enolase and S-100 protein in the cerebrospinal fluid of patients with acute bacterial meningitis

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A. V. Sokhan

2016-08-01

Full Text Available Aim. To evaluate the diagnostic and prognostic role of neuron-specific enolase (NSE and S-100 protein levels in cerebrospinal fluid (CSF of patients with acute bacterial meningitis in the course of the disease. Materials and Methods. 54 cases of acute bacterial meningitis were analyzed, among them – 26 with pneumococcal and 28 with meningococcal etiology. Patients were divided into groups depending on the severity and etiology of disease. In addition to routine laboratory methods, we analyzed the CSF levels of S-100 protein and NSE at admission and after 10 – 12 days of treatment. 12 patients with acute respiratory infections and meningism were examined as a comparison group. Results. In all patients with acute bacterial meningitis CSF NSE and protein S-100 levels were significantly higher than in the control group (P <0,05. CSF neuro specific proteins level was in direct dependence on severity of the disease, and in patients with severe disease was significantly higher than in patients with moderate severity and in the control group (P <0,01. After 10 – 12 days of treatment, the level of the NSE and S-100 protein decreased, but in severe cases was still higher than in the control group (P <0,05. Conclusions. Increased cerebrospinal fluid NSE and S – 100 protein levels shows the presence and value of neurons and glial cells damage in patients with acute bacterial meningitis. CSF S-100 protein and neuron-specific enolase levels help to determine the severity of neurons destruction and glial cells in patients with acute bacterial meningitis. Level of neurospecific protein is in direct proportion to the severity of the disease and is the highest in patients with severe cases (P<0,05. It confirms the diagnostic and prognostic value of CSF neurospecific protein determination in patients with bacterial meningitis.

Density-dependence as a size-independent regulatory mechanism

NARCIS (Netherlands)

De Vladar, H.P.

2006-01-01

The growth function of populations is central in biomathematics. The main dogma is the existence of density-dependence mechanisms, which can be modelled with distinct functional forms that depend on the size of the Population. One important class of regulatory functions is the theta-logistic, which

Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells.

Science.gov (United States)

Haushalter, Kristofer J; Casteel, Darren E; Raffeiner, Andrea; Stefan, Eduard; Patel, Hemal H; Taylor, Susan S

2018-03-23

cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

Science.gov (United States)

Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

2017-11-15

A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

International Nuclear Information System (INIS)

Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

2013-01-01

Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

Energy Technology Data Exchange (ETDEWEB)

Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

2013-07-12

Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

Energy Technology Data Exchange (ETDEWEB)

Hua, Fang, E-mail: fhua2@emory.edu [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States); Wang, Jun; Sayeed, Iqbal; Ishrat, Tauheed; Atif, Fahim; Stein, Donald G. [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States)

2009-12-18

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-{kappa}B). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-{kappa}B and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-{kappa}B activity and phosphorylation of the inhibitor of kappa B (I{kappa}B{alpha}) increased in ischemic brains, but IRF3, inhibitor of {kappa}B kinase complex-{epsilon} (IKK{epsilon}), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-{kappa}B activity or p-I{kappa}B{alpha} induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-{kappa}B signaling and brain injury after acute cerebral I/R.

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

Science.gov (United States)

Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

2016-05-17

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Science.gov (United States)

Zhang, Jin-You; Wu, Yi; Zhao, Shuan; Liu, Zhen-Xing; Zeng, Shen-Ming; Zhang, Gui-Xue

2015-09-15

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL. Copyright © 2015 Elsevier Inc. All rights reserved.

GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells

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Koichi R. Katsumura

2016-08-01

Full Text Available The master regulatory transcription factor GATA-2 triggers hematopoietic stem and progenitor cell generation. GATA2 haploinsufficiency is implicated in myelodysplastic syndrome (MDS and acute myeloid leukemia (AML, and GATA2 overexpression portends a poor prognosis for AML. However, the constituents of the GATA-2-dependent genetic network mediating pathogenesis are unknown. We described a p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation. We demonstrate that this mechanism establishes a growth-promoting chemokine/cytokine circuit in AML cells. p38/ERK-dependent GATA-2 phosphorylation facilitated positive autoregulation of GATA2 transcription and expression of target genes, including IL1B and CXCL2. IL-1β and CXCL2 enhanced GATA-2 phosphorylation, which increased GATA-2-mediated transcriptional activation. p38/ERK-GATA-2 stimulated AML cell proliferation via CXCL2 induction. As GATA2 mRNA correlated with IL1B and CXCL2 mRNAs in AML-M5 and high expression of these genes predicted poor prognosis of cytogenetically normal AML, we propose that the circuit is functionally important in specific AML contexts.

Regulatory network of GATA3 in pediatric acute lymphoblastic leukemia

OpenAIRE

Hou, Qianqian; Liao, Fei; Zhang, Shouyue; Zhang, Duyu; Zhang, Yan; Zhou, Xueyan; Xia, Xuyang; Ye, Yuanxin; Yang, Hanshuo; Li, Zhaozhi; Wang, Leiming; Wang, Xi; Ma, Zhigui; Zhu, Yiping; Ouyang, Liang

2017-01-01

GATA3 polymorphisms were reported to be significantly associated with susceptibility of pediatric B-lineage acute lymphoblastic leukemia (ALL), by impacting on GATA3 expression. We noticed that ALL-related GATA3 polymorphism located around in the tissue-specific enhancer, and significantly associated with GATA3 expression. Although the regulatory network of GATA3 has been well reported in T cells, the functional status of GATA3 is poorly understood in B-ALL. We thus conducted genome-wide gene...

Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements.

Science.gov (United States)

Kyrchanova, Olga; Chetverina, Darya; Maksimenko, Oksana; Kullyev, Andrey; Georgiev, Pavel

2008-12-01

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs-scs, scs'-scs', 1A2-1A2 and Wari-Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5-Su(Hw), dCTCF-Su(Hw), or dCTCF-Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

Activated Protein C Drives the Hyperfibrinolysis of Acute Traumatic Coagulopathy.

Science.gov (United States)

Davenport, Ross A; Guerreiro, Maria; Frith, Daniel; Rourke, Claire; Platton, Sean; Cohen, Mitchell; Pearse, Rupert; Thiemermann, Chris; Brohi, Karim

2017-01-01

Major trauma is a leading cause of morbidity and mortality worldwide with hemorrhage accounting for 40% of deaths. Acute traumatic coagulopathy exacerbates bleeding, but controversy remains over the degree to which inhibition of procoagulant pathways (anticoagulation), fibrinogen loss, and fibrinolysis drive the pathologic process. Through a combination of experimental study in a murine model of trauma hemorrhage and human observation, the authors' objective was to determine the predominant pathophysiology of acute traumatic coagulopathy. First, a prospective cohort study of 300 trauma patients admitted to a single level 1 trauma center with blood samples collected on arrival was performed. Second, a murine model of acute traumatic coagulopathy with suppressed protein C activation via genetic mutation of thrombomodulin was used. In both studies, analysis for coagulation screen, activated protein C levels, and rotational thromboelastometry (ROTEM) was performed. In patients with acute traumatic coagulopathy, the authors have demonstrated elevated activated protein C levels with profound fibrinolytic activity and early depletion of fibrinogen. Procoagulant pathways were only minimally inhibited with preservation of capacity to generate thrombin. Compared to factors V and VIII, proteases that do not undergo activated protein C-mediated cleavage were reduced but maintained within normal levels. In transgenic mice with reduced capacity to activate protein C, both fibrinolysis and fibrinogen depletion were significantly attenuated. Other recognized drivers of coagulopathy were associated with less significant perturbations of coagulation. Activated protein C-associated fibrinolysis and fibrinogenolysis, rather than inhibition of procoagulant pathways, predominate in acute traumatic coagulopathy. In combination, these findings suggest a central role for the protein C pathway in acute traumatic coagulopathy and provide new translational opportunities for management of

Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

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Allen C T Teng

Full Text Available Interferon regulatory factor 2 binding protein 2 (IRF2BP2 is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known.Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS to an evolutionarily conserved sequence (354ARKRKPSP(361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360. Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2C(12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2C(12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2.Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status

The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

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Grassmann Ralph

2005-09-01

Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

Robust and global delay-dependent stability for genetic regulatory networks with parameter uncertainties.

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Tian, Li-Ping; Wang, Jianxin; Wu, Fang-Xiang

2012-09-01

The study of stability is essential for designing or controlling genetic regulatory networks, which can be described by nonlinear differential equations with time delays. Much attention has been paid to the study of delay-independent stability of genetic regulatory networks and as a result, many sufficient conditions have been derived for delay-independent stability. Although it might be more interesting in practice, delay-dependent stability of genetic regulatory networks has been studied insufficiently. Based on the linear matrix inequality (LMI) approach, in this study we will present some delay-dependent stability conditions for genetic regulatory networks. Then we extend these results to genetic regulatory networks with parameter uncertainties. To illustrate the effectiveness of our theoretical results, gene repressilatory networks are analyzed .

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

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Child, Matthew A.; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A.; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A.; Boothroyd, John C.; Reese, Michael L.

2017-01-01

ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. PMID:28246362

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

Science.gov (United States)

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

International Nuclear Information System (INIS)

Mao, Grace; Brody, James P.

2007-01-01

Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s -1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase

Identification and quantitation of signal molecule-dependent protein phosphorylation

KAUST Repository

Groen, Arnoud J.

2013-09-03

Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

OpenAIRE

Jiang, M; Pandey, S; Tran, V T; Fong, H K

1991-01-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

Acute Phase Proteins and Variables of Protein Metabolism in Dairy Cows during the Pre- and Postpartal Period

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Cs. Tóthová

2008-01-01

Full Text Available The objective of the present study was to compare the concentrations of acute phase proteins and selected variables of protein metabolism in dairy cows of the Slovak Spotted breed from 4 weeks before parturition to 10 weeks after parturition. Acute phase proteins - haptoglobin (Hp and serum amyloid A (SAA - and variables of protein metabolism - total proteins, albumin, urea, creatinine, total immunoglobulins - were evaluated in blood serum. Significant differences were found in average values of the Hp and SAA concentrations in several groups during the monitored period (P P P P P P P P P < 0.001. The above mentioned results indicate that in the time around parturition there are significant changes in concentrations of acute phase proteins, as well as in the whole protein metabolism of dairy cows. These facts suggest that the postparturient period is a critical biological phase, throughout which there is the highest incidence of metabolic disorders.

Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.

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Bruni, R; Roizman, B

1998-11-01

The herpes simplex virus 1 infected cell protein 22 (ICP22), the product of the alpha22 gene, is a nucleotidylylated and phosphorylated nuclear protein with properties of a transcriptional factor required for the expression of a subset of viral genes. Here, we report the following. (i) ICP22 interacts with a previously unknown cellular factor designated p78 in the yeast two-hybrid system. The p78 cDNA encodes a polypeptide with a distribution of leucines reminiscent of a leucine zipper. (ii) In uninfected and infected cells, antibody to p78 reacts with two major bands with an apparent Mr of 78,000 and two minor bands with apparent Mrs of 62, 000 and 55,000. (ii) p78 also interacts with ICP22 in vitro. (iii) In uninfected cells, p78 was dispersed largely in the nucleoplasm in HeLa cells and in the nucleoplasm and cytoplasm in HEp-2 cells. After infection, p78 formed large dense bodies which did not colocalize with the viral regulatory protein ICP0. (iv) Accumulation of p78 was cell cycle dependent, being highest very early in S phase. (v) The accumulation of ICP22 in synchronized cells was highest in early S phase, in contrast to the accumulation of another protein, ICP27, which was relatively independent of the cell cycle. (vi) In the course of the cell cycle, ICP22 was transiently modified in an aberrant fashion, and this modification coincided with expression of p78. The results suggest that ICP22 interacts with and may be stabilized by cell cycle-dependent proteins.

Photoaffinity labeling of cAMP-dependent protein kinase by 4-azido-2-nitrophenyladenylyl pyrophosphate

International Nuclear Information System (INIS)

Johnson, D.R.; Ho, H.T.; Wong, S.S.

1986-01-01

A photoaffinity analogue of ATP, 4-azido-2-nitrophenyl-adenylyl pyrophosphate (ANAP) has been synthesized to investigate the topographical interaction between the catalytic and the regulatory subunits of the bovine heart type II cAMP-dependent protein kinase. The synthesis involves coupling of 4-azido-2-nitrophenyl phosphate with adenosine 5'-monophosphomorpholidate. ANAP has an absorption maximum at 260 nm (molar absorptivity = 35.4 x 10 3 M -1 cm -1 ) and a shoulder at 320 nm. Kinetically, ANAP inhibits the enzyme competitively against ATP with a Ki of 0.37 mM. The catalytic subunit is inactivated by ANAP upon photolysis in the presence of magnesium ion. ATP protects the enzyme from photoinactivation but the regulatory subunit does not. Gel electrophoretic analysis of the enzyme labeled by [ 14 C]ANAP shows that the photoincorporated ANAP is associated mainly with the catalytic subunit, even when the regulator dimer is in twelve fold excess. Little or no ANAP is found incorporated into the regulator subunit. The data suggest that the photoreactive portion of ANAP does not lie within reach of the regulatory protein when the analogue is bound to the catalytic subunit

Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke

International Nuclear Information System (INIS)

Lee, Heung M.; Reed, Jason; Greeley, George H.; Englander, Ella W.

2009-01-01

Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase α subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

OpenAIRE

Sanda, Takaomi; Lawton, Lee N.; Barrasa, M. Inmaculada; Fan, Zi Peng; Kohlhammer, Holger; Gutierrez, Alejandro; Ma, Wenxue; Tatarek, Jessica; Ahn, Yebin; Kelliher, Michelle A.; Jamieson, Catriona H.M.; Staudt, Louis M.; Young, Richard A.; Look, A. Thomas

2012-01-01

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3 and RUNX1. We show that TAL1 forms a positive interconnected auto-regulatory loop with GATA3 and RUNX1, and that the TAL1 complex directly activates the MY...

Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

Science.gov (United States)

Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

2014-09-01

Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

Probes of the Mitochondrial cAMP-dependent Protein Kinase

Science.gov (United States)

Shell, Jennifer R.; Lawrence, David S.

2013-01-01

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca2+, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R2C2) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). PMID:23410952

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

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Mohan Babu

Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

Reishi Protein LZ-8 Induces FOXP3+ Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice

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Hsien-Yeh Hsu

2013-01-01

Full Text Available LZ-8, an immunomodulatory protein isolated from Ganoderma lucidum (also known as Ling-Zhi or Reishi, has been shown to promote cell proliferation and IL-2 production in T cells. In this study, we show that LZ-8 induces the expansion of both murine and human CD4+ T cells into FOXP3+ regulatory T (Treg cells. LZ-8 treatment was found to stimulate a 4-fold and a 10-fold expansion in the Treg populations of murine and human primary CD4+ T cells, respectively. In addition, the expression of CTLA-4 and IL-10 was induced in LZ-8-treated CD4+ T cells. Using neutralizing antibodies and gene-deficient T-cell lines, we also found that LZ-8 promotes Treg expansion through a CD45-mediated signaling pathway and that the CD18-dependent induction of IL-2 was involved in Treg formation and IL-10 production. The suppressive activity of LZ-8 was confirmed using a murine model of DSS-induced colitis; the disease was alleviated by the adoptive transfer of LZ-8-treated CD4+ T cells. In conclusion, a new regulatory function for LZ-8 was identified, and the molecular mechanisms underlying this function were elucidated.

Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

International Nuclear Information System (INIS)

Yang, Weng-Lang; Ravatn, Roald; Kudoh, Kazuya; Alabanza, Leah; Chin, Khew-Voon

2010-01-01

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R 2 C 2 . The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIα, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIα, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIα subunit of PKA may have functions independent of the kinase. We show here that the RIα subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIα results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIα and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIα modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIα with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

Foxo4- and Stat3-dependent IL-10 production by progranulin in regulatory T cells restrains inflammatory arthritis

Science.gov (United States)

Fu, Wenyu; Hu, Wenhuo; Shi, Lei; Mundra, Jyoti Joshi; Xiao, GuoZhi; Dustin, Michael L.; Liu, Chuan-ju

2017-01-01

Progranulin (PGRN) restrains inflammation and is therapeutic against inflammatory arthritis; however, the underlying immunological mechanism remains unknown. In this study, we demonstrated that anti-inflammatory cytokine IL-10 was a critical mediator for PGRN-mediated anti-inflammation in collagen-induced arthritis by using PGRN and IL-10 genetically modified mouse models. IL-10 green fluorescent protein reporter mice revealed that regulatory T (Treg) cells were the predominant source of IL-10 in response to PGRN. In addition, PGRN-mediated expansion and activation of Treg cells, as well as IL-10 production, depends on JNK signaling, but not on known PGRN-activated ERK and PI3K pathways. Furthermore, microarray and chromatin immunoprecipitation sequencing screens led to the discovery of forkhead box protein O4 and signal transducer and activator of transcription 3 as the transcription factors required for PGRN induction of IL-10 in Treg cells. These findings define a previously unrecognized signaling pathway that underlies IL-10 production by PGRN in Treg cells and present new insights into the mechanisms by which PGRN resolves inflammation in inflammatory conditions and autoimmune diseases, particularly inflammatory arthritis.—Fu, W., Hu, W., Shi, L., Mundra, J. J. Xiao, G., Dustin, M. L., Liu, C. Foxo4- and Stat3-dependent IL-10 production by progranulin in regulatory T cells restrains inflammatory arthritis. PMID:28011648

Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome.

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Olivia Hoffman

Full Text Available A hallmark of acute respiratory distress syndrome (ARDS is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.

The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis

DEFF Research Database (Denmark)

Sørensen, Nanna Skall; Tegtmeier, C.; Andresen, Lars Ole

2006-01-01

The pig acute phase protein (APP) response to experimental Streptococcus suis (S. suis) infection was mapped by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pig-MAP) and the negative APPs albumin...... and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic, infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p...... the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes...

Sleep and protein synthesis-dependent synaptic plasticity: impacts of sleep loss and stress

Science.gov (United States)

Grønli, Janne; Soulé, Jonathan; Bramham, Clive R.

2014-01-01

Sleep has been ascribed a critical role in cognitive functioning. Several lines of evidence implicate sleep in the consolidation of synaptic plasticity and long-term memory. Stress disrupts sleep while impairing synaptic plasticity and cognitive performance. Here, we discuss evidence linking sleep to mechanisms of protein synthesis-dependent synaptic plasticity and synaptic scaling. We then consider how disruption of sleep by acute and chronic stress may impair these mechanisms and degrade sleep function. PMID:24478645

Cold shock protein YB-1 is involved in hypoxia-dependent gene transcription

International Nuclear Information System (INIS)

Rauen, Thomas; Frye, Bjoern C.; Wang, Jialin; Raffetseder, Ute; Alidousty, Christina; En-Nia, Abdelaziz; Floege, Jürgen; Mertens, Peter R.

2016-01-01

Hypoxia-dependent gene regulation is largely orchestrated by hypoxia-inducible factors (HIFs), which associate with defined nucleotide sequences of hypoxia-responsive elements (HREs). Comparison of the regulatory HRE within the 3′ enhancer of the human erythropoietin (EPO) gene with known binding motifs for cold shock protein Y-box (YB) protein-1 yielded strong similarities within the Y-box element and 3′ adjacent sequences. DNA binding assays confirmed YB-1 binding to both, single- and double-stranded HRE templates. Under hypoxia, we observed nuclear shuttling of YB-1 and co-immunoprecipitation assays demonstrated that YB-1 and HIF-1α physically interact with each other. Cellular YB-1 depletion using siRNA significantly induced hypoxia-dependent EPO production at both, promoter and mRNA level. Vice versa, overexpressed YB-1 significantly reduced EPO-HRE-dependent gene transcription, whereas this effect was minor under normoxia. HIF-1α overexpression induced hypoxia-dependent gene transcription through the same element and accordingly, co-expression with YB-1 reduced HIF-1α-mediated EPO induction under hypoxic conditions. Taken together, we identified YB-1 as a novel binding factor for HREs that participates in fine-tuning of the hypoxia transcriptome. - Highlights: • Hypoxia drives nuclear translocation of cold shock protein YB-1. • YB-1 physically interacts with hypoxia-inducible factor (HIF)-1α. • YB-1 binds to the hypoxia-responsive element (HRE) within the erythropoietin (EPO) 3′ enhancer. • YB-1 trans-regulates transcription of hypoxia-dependent genes such as EPO and VEGF.

CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

Science.gov (United States)

IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

International Nuclear Information System (INIS)

Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

2005-01-01

Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

Latent Growth Modeling of nursing care dependency of acute neurological inpatients.

Science.gov (United States)

Piredda, M; Ghezzi, V; De Marinis, M G; Palese, A

2015-01-01

Longitudinal three-time point study, addressing how neurological adult patient care dependency varies from the admission time to the 3rd day of acute hospitalization. Nursing care dependency was measured with the Care Dependency Scale (CDS) and a Latent Growth Modeling approach was used to analyse the CDS trend in 124 neurosurgical and stroke inpatients. Care dependence followed a decreasing linear trend. Results can help nurse-managers planning an appropriate amount of nursing care for acute neurological patients during their initial stage of hospitalization. Further studies are needed aimed at investigating the determinants of nursing care dependence during the entire in-hospital stay.

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

International Nuclear Information System (INIS)

Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

1988-01-01

The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

Energy Technology Data Exchange (ETDEWEB)

Yang, Weng-Lang [Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030 (United States); Ravatn, Roald [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Kudoh, Kazuya [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Department of Obstetrics and Gynecology, National Defense Medical College, Tokorozawa, Saitama (Japan); Alabanza, Leah [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Chin, Khew-Voon, E-mail: khew-voon.chin@utoledo.edu [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States)

2010-01-15

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R{sub 2}C{sub 2}. The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RI{alpha}, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RI{alpha}, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RI{alpha} subunit of PKA may have functions independent of the kinase. We show here that the RI{alpha} subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RI{alpha} results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RI{alpha} and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RI{alpha} modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RI{alpha} with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population

DEFF Research Database (Denmark)

Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine

2014-01-01

Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum...... proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type....... Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations...

Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

DEFF Research Database (Denmark)

Skovgaard, Kerstin; Mortensen, Shila; Boye, Mette

2009-01-01

The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other...... parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we studied gene expression changes in peripheral lymphoid tissues as compared to hepatic expression changes, 14-18 h after lung...... with measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14-18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase...

Regulatory T Cells Contribute to the Inhibition of Radiation-Induced Acute Lung Inflammation via Bee Venom Phospholipase A₂ in Mice.

Science.gov (United States)

Shin, Dasom; Lee, Gihyun; Sohn, Sung-Hwa; Park, Soojin; Jung, Kyung-Hwa; Lee, Ji Min; Yang, Jieun; Cho, Jaeho; Bae, Hyunsu

2016-04-30

Bee venom has long been used to treat various inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Previously, we reported that bee venom phospholipase A₂ (bvPLA₂) has an anti-inflammatory effect through the induction of regulatory T cells. Radiotherapy is a common anti-cancer method, but often causes adverse effects, such as inflammation. This study was conducted to evaluate the protective effects of bvPLA₂ in radiation-induced acute lung inflammation. Mice were focally irradiated with 75 Gy of X-rays in the lung and administered bvPLA₂ six times after radiation. To evaluate the level of inflammation, the number of immune cells, mRNA level of inflammatory cytokine, and histological changes in the lung were measured. BvPLA₂ treatment reduced the accumulation of immune cells, such as macrophages, neutrophils, lymphocytes, and eosinophils. In addition, bvPLA₂ treatment decreased inflammasome-, chemokine-, cytokine- and fibrosis-related genes' mRNA expression. The histological results also demonstrated the attenuating effect of bvPLA₂ on radiation-induced lung inflammation. Furthermore, regulatory T cell depletion abolished the therapeutic effects of bvPLA₂ in radiation-induced pneumonitis, implicating the anti-inflammatory effects of bvPLA₂ are dependent upon regulatory T cells. These results support the therapeutic potential of bvPLA₂ in radiation pneumonitis and fibrosis treatments.

High-throughput assessment of context-dependent effects of chromatin proteins

NARCIS (Netherlands)

Brueckner, L. (Laura); Van Arensbergen, J. (Joris); Akhtar, W. (Waseem); L. Pagie (Ludo); B. van Steensel (Bas)

2016-01-01

textabstractBackground: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy

5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

Science.gov (United States)

Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

2004-01-01

The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

Low-Density Lipoprotein Receptor–Related Protein-1 Is a Therapeutic Target in Acute Myocardial Infarction

Directory of Open Access Journals (Sweden)

Stefano Toldo, PhD

2017-10-01

Full Text Available Low-density lipoprotein receptor–related protein-1 (LRP1 is a ubiquitous membrane receptor functioning as a scavenger and regulatory receptor, inducing anti-inflammatory and prosurvival signals. Based on the known structure–activity of the LRP1 receptor binding site, the authors synthesized a small peptide (SP16. SP16 induced a >50% reduction in infarct size (p < 0.001 and preservation of left ventricular systolic function (p < 0.001, and treatment with an LRP1 blocking antibody eliminated the protective effects of SP16. In conclusion, LRP1 activation with SP16 given within 30 min of reperfusion during experimental acute myocardial infarction leads to a cardioprotective signal reducing infarct size and preservation of cardiac systolic function.

Genome-wide discovery of drug-dependent human liver regulatory elements.

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Robin P Smith

2014-10-01

Full Text Available Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR and three active regulatory marks (p300, H3K4me1, H3K27ac on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4% that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.

Novel adenosine 3',5'-cyclic monophosphate dependent protein kinases in a marine diatom

International Nuclear Information System (INIS)

Lin, P.P.C.; Volcani, B.E.

1989-01-01

Two novel adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg 2+ and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser( 32 P)-Ser-Asn-Ala-Arg and have an apparent M r of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M r of about 78,000 is photolabeled with 8-azido[ 32 P]cAMP and is also phosphorylated with [γ- 32 P]ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids

Type 2 diabetes mellitus with early phase acute inflammatory protein on serum protein electrophoresis

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ET Tuladhar

2012-03-01

Full Text Available Background: The onset of Type 2 diabetes has been associated with low grade systemic inflammation. The inflammatory status has been studied by measuring acute phase reactant proteins like hsCRP, α1- antitrypsin, α1-acid glycoprotein, ceruloplasmin, fibrinogen. Most of these acute phase reactants form α1 and α2 bands on electropherogram of serum proteins. The aim of this study was to evaluate inflammatory status in controlled and uncontrolled type 2 diabetes using cellulose acetate electrophoresis and to find the impact of glycemic status as indicated by HbA1c on inflammation process. Materials and Methods: Serum protein electrophoresis was done on serum samples of 60 cases of Diabetes [controlled and uncontrolled] using cellulose acetate paper technique. The electropherogram obtained was stained with Ponseu S and then quantitated using densitometer. Glycemic status was studied by HbA1c analysis. The density of α1and α2 bands in electropherogram were correlated with HbA1c level. Result: A significant increase in the percentage of α1 and α2 band proteins (0.765 and 0.716, p<0.001 were found with the increasing level of HbA1c. With cutoff of HbA1c 7% (American Diabetic Association recommended, the α1 and α2 serum proteins concentration are significantly higher (p<0.001 in uncontrolled diabetes mellitus compared to controlled diabetes mellitus Conclusion: Cellulose acetate electrophoresis of serum proteins show early phase acute inflammatory status in uncontrolled type 2 diabetes mellitus. The process of systemic inflammation worsens with uncontrolled glycemia as indicated by HbA1c. Inflammatory status should be studied adjunct to glycemic status. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6024 JPN 2012; 2(3: 211-214

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

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Victoria A. Meliopoulos

2016-11-01

Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

A protein-dependent side-chain rotamer library.

KAUST Repository

Bhuyan, M.S.; Gao, Xin

2011-01-01

Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

A protein-dependent side-chain rotamer library.

KAUST Repository

Bhuyan, M.S.

2011-12-14

Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

Steroidogenic acute regulatory protein (StAR) gene expression construct: Development, nanodelivery and effect on reproduction in air-breathing catfish, Clarias batrachus.

Science.gov (United States)

Rathor, Pravesh Kumar; Bhat, Irfan Ahmad; Rather, Mohd Ashraf; Gireesh-Babu, Pathakota; Kumar, Kundan; Purayil, Suresh Babu Padinhate; Sharma, Rupam

2017-11-01

Steroidogenic acute regulatory protein (StAR) is responsible for the relocation of cholesterol across mitochondrial membrane in vertebrates and is, therefore, a key factor in regulating the rate and timing of steroidogenesis. In the present study, we developed chitosan nanoparticle (CNP) conjugated StAR gene construct (CNP-pcDNA4-StAR) in a eukaryotic expression vector, pcDNA4/HisMax A. CNPs of 135.4nm diameter, 26.7mV zeta potential and 0.381 polydispersity index were used for conjugation. The loading efficiency (LE) of pcDNA4-StAR construct with CNPs was found to be 86%. After the 24h of intramuscular injection, the CNP-pcDNA4-StAR plasmid could be detected from testis, brain, kidney and muscle tissues of Clarias batrachus. The transcript levels of important reproductive genes viz. cyp11a1, cyp17a1, 3β-hsd, 17β-hsd and cyp19a1 in CNP-pcDNA4-StAR treated group were initially low up to 24h, but significantly increased subsequently up to 120h. In naked pcDNA4-StAR treated group, the mRNA level of 3β-hsd, 17β-hsd and cyp19a1 increased initially up to 24h, while cyp11a1 and cyp17a1 increased up to 48h and then started declining. Similar results were obtained for 11-Ketotestosterone and 17β-estradiol. The results indicate relatively long lasting effects of nano-conjugated construct compared to the construct alone. Furthermore, the histopathology of gonads and liver authenticates its possible role in the gonadal development in fish without any adverse effect. Copyright © 2017 Elsevier B.V. All rights reserved.

C-reactive protein: an aid for diagnosis of acute appendicitis

International Nuclear Information System (INIS)

Ahmed, N.

2017-01-01

Delayed or wrong diagnosis of acute appendicitis in patients results in complications like perforation, gangrene, etc. which carries a significant amount of morbidity and mortality to the patients. Thus, timely diagnosis of acute appendicitis is crucial to prevent these complications. Recently, it was found that serum C-reactive protein (CRP) individually can be a useful marker, thus in resource limited settings (i.e., access to ultrasonography) simple laboratory investigation can be of extreme utility for the diagnosis of acute appendicitis. Current study aimed to ascertain and determine the role of C Reactive Protein (CRP) as a complementary test to decrease the rate of negative appendectomies in tertiary care hospitals of Pakistan. Methods: Using non-probability consecutive sampling, 112 patients with the initial diagnosis of acute appendicitis on history and clinical examination were enrolled. A blood sample was taken for serum level of CRP. Results: Mean age was 20.8+-8.6 years and 51 (45.5 %) patients were males. Pathologic review revealed 100 cases (89.3%) of acute appendicitis, 4 patients (3.6%) had perforated appendix while 8 patients (7.1%) had normal appendix. Sensitivity, specificity, positive and negative predictive value and diagnostic accuracy of C reactive protein >24 mg/lit taking histology as gold standard came out 25.9%, 100%, 100%, 9.4% and 31.25% respectively. Conclusion: It was concluded that CRP >48 mg/lit is an indication of perforated appendix and when the surgeon is in fix whether to go conservatively or apply some intervention, CRP can be a good diagnostic aid. (author)

Nasopharyngeal Protein Biomarkers of Acute Respiratory Virus Infection

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Thomas W. Burke

2017-03-01

Full Text Available Infection of respiratory mucosa with viral pathogens triggers complex immunologic events in the affected host. We sought to characterize this response through proteomic analysis of nasopharyngeal lavage in human subjects experimentally challenged with influenza A/H3N2 or human rhinovirus, and to develop targeted assays measuring peptides involved in this host response allowing classification of acute respiratory virus infection. Unbiased proteomic discovery analysis identified 3285 peptides corresponding to 438 unique proteins, and revealed that infection with H3N2 induces significant alterations in protein expression. These include proteins involved in acute inflammatory response, innate immune response, and the complement cascade. These data provide insights into the nature of the biological response to viral infection of the upper respiratory tract, and the proteins that are dysregulated by viral infection form the basis of signature that accurately classifies the infected state. Verification of this signature using targeted mass spectrometry in independent cohorts of subjects challenged with influenza or rhinovirus demonstrates that it performs with high accuracy (0.8623 AUROC, 75% TPR, 97.46% TNR. With further development as a clinical diagnostic, this signature may have utility in rapid screening for emerging infections, avoidance of inappropriate antibacterial therapy, and more rapid implementation of appropriate therapeutic and public health strategies.

Optimal combinations of acute phase proteins for detecting infectious disease in pigs

DEFF Research Database (Denmark)

Heegaard, Peter M. H.; Stockmarr, Anders; Piñeiro, Matilde

2011-01-01

The acute phase protein (APP) response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect...... gondii) and one viral (porcine respiratory and reproductive syndrome virus) infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP), haptoglobin (Hp), pig major acute phase protein (pigMAP) and serum amyloid A (SAA)) and three negative...

Serum acute phase protein concentrations in female dogs with mammary tumors.

Science.gov (United States)

Tecles, Fernando; Caldín, Marco; Zanella, Anna; Membiela, Francisco; Tvarijonaviciute, Asta; Subiela, Silvia Martínez; Cerón, José Joaquín

2009-03-01

Acute phase proteins (APPs) are proteins whose concentrations in serum change after any inflammatory stimulus or tissue damage. The aim of the current study was to evaluate 3 positive APPs (C-reactive protein, serum amyloid A, and haptoglobin) and 1 negative APP (albumin) in female dogs with mammary neoplasia. Acute phase proteins were studied in 70 female dogs aged 8-12 years in the following groups: healthy (n = 10); mammary tumors in stages I (n = 19), II (n = 5), III (n = 6), IV (n = 5), and V (n = 7); and with mammary neoplasia plus a concomitant disease (n = 18). In animals with mammary neoplasia, significant increases of positive APPs were only detected in those that had metastasis or a neoplasm with a diameter greater than 5 cm and ulceration. Dogs with mammary neoplasia and a concomitant disease also had high C-reactive protein concentrations. Albumin concentration was decreased in animals with metastasis and with a concomitant disease. The results of the present study indicate that the acute phase response could be stimulated in female dogs with mammary gland tumors because of different factors, such as metastasis, large size of the primary mass, and ulceration or secondary inflammation of the neoplasm.

EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

DEFF Research Database (Denmark)

Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel

2015-01-01

at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.......Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins...

Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

Science.gov (United States)

De Silva, Matharage S I; Dayton, Adam W; Rhoten, Lance R; Mallett, John W; Reese, Jared C; Squires, Mathieu D; Dalley, Andrew P; Porter, James P; Judd, Allan M

2018-06-01

In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1. Copyright © 2018 Elsevier Inc. All rights reserved.

Activation of counter-regulatory mechanisms in a rat renal acute rejection model

Directory of Open Access Journals (Sweden)

Salomon Daniel R

2008-02-01

Full Text Available Abstract Background Microarray analysis provides a powerful approach to identify gene expression alterations following transplantation. In patients the heterogeneity of graft specimens, co-morbidity, co-medications and the challenges in sample collection and preparation complicate conclusions regarding the underlying mechanisms of graft injury, rejection and immune regulation. Results We used a rat kidney transplantation model with strict transplant and sample preparation procedures to analyze genome wide changes in gene expression four days after syngeneic and allogeneic transplantation. Both interventions were associated with substantial changes in gene expression. After allogeneic transplantation, genes and pathways related to transport and metabolism were predominantly down-regulated consistent with rejection-mediated graft injury and dysfunction. Up-regulated genes were primarily related to the acute immune response including antigen presentation, T-cell receptor signaling, apoptosis, interferon signaling and complement cascades. We observed a cytokine and chemokine expression profile consistent with activation of a Th1-cell response. A novel finding was up-regulation of several regulatory and protective genes after allogeneic transplantation, specifically IL10, Bcl2a1, C4bpa, Ctla4, HO-1 and the SOCS family. Conclusion Our data indicate that in parallel with the predicted activation of immune response and tissue injury pathways, there is simultaneous activation of pathways for counter regulatory and protective mechanisms that would balance and limit the ongoing inflammatory/immune responses. The pathophysiological mechanisms behind and the clinical consequences of alterations in expression of these gene classes in acute rejection, injury and dysfunction vs. protection and immunoregulation, prompt further analyses and open new aspects for therapeutic approaches.

Acute differential effects of dietary protein quality on postprandial lipemia in obese non-diabetic subjects

DEFF Research Database (Denmark)

Holmer-Jensen, Jens; Mortensen, Lene Sundahl; Astrup, Arne

2013-01-01

Non-fasting triglyceridemia is much closer associated to cardiovascular risk compared to fasting triglyceridemia. We hypothesized that there would be acute differential effects of four common dietary proteins (cod protein, whey isolate, gluten, and casein) on postprandial lipemia in obese non......-diabetic subjects. To test the hypothesis we conducted a randomized, acute clinical intervention study with crossover design. We supplemented a fat rich mixed meal with one of four dietary proteins i.e. cod protein, whey protein, gluten or casein. Eleven obese non-diabetic subjects (age: 40-68, body mass index: 30...... concentration in the chylomicron rich fraction (P = .0293). Thus, we have demonstrated acute differential effects on postprandial metabolism of four dietary proteins supplemented to a fat rich mixed meal in obese non-diabetic subjects. Supplementation with whey protein caused lower postprandial lipemia compared...

Enzymatic Mercury Detoxification: The Regulatory Protein MerR

CERN Multimedia

Ctortecka, B; Walsh, C T; Comess, K M

2002-01-01

Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

Regulatory T cells-derived IL-35 promotes the growth of adult acute myeloid leukemia blasts.

Science.gov (United States)

Tao, Qianshan; Pan, Ying; Wang, Yiping; Wang, Huiping; Xiong, Shudao; Li, Qing; Wang, Jia; Tao, Lili; Wang, Zhitao; Wu, Fan; Zhang, Rui; Zhai, Zhimin

2015-11-15

Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL-35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL-35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL-35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs-derived from adult AML patients produced IL-35 in a stimulation-dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML. © 2015 UICC.

Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

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Tsubasa Munakata

2007-09-01

Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

Contrast media induced acute renal failure in diabetics

International Nuclear Information System (INIS)

Rambausek, M.

1985-01-01

Dehydration, preexisting renal insufficiency, multiple myeloma and insulin-dependent diabetes mellitus are known risk factors for a radiocontrast medium induced acute renal failure. In 90% of patients with insulin-dependent diabetes mellitus, renal insufficiency and proteinuria, a further detoriation of renal function can be expected after i.v. administration of radiocontrast medium. Recent concepts on the genesis of acute renal failure after radiocontrast medium in multiple myeloma emphasize the role of tubular blocade (tubular precipitation of myeloma protein with contrast medium). In insulin-dependent diabetic patients we found altered carbohydrate composition of urinary Tamm Horsfall Protein (THP), with increased glucose and diminished N-acetyl-neuraminicacid content. This was paralleled by a difference in an in-vitro system of coprecipitation where THP of diabetes triggered more pronounced calcium dependent coprecipitation of contrast medium and albumin. These in-vitro findings might be important for the explanation of the genesis of radiocontrast medium-induced acute renal failure in insulin-dependent diabetes mellitus. (orig.) [de

Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

Science.gov (United States)

Iyengar, Bharat Ravi; Pillai, Beena; Venkatesh, K V; Gadgil, Chetan J

2017-05-30

We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

Targeted proteomics as a tool for porcine acute phase proteins measurements

DEFF Research Database (Denmark)

Marco-Ramell, Anna; Bassols, Anna; Bislev, Stine Lønnerup

2013-01-01

. Selected reaction monitoring (SRM), a targeted quantitative proteomic technique, may be used as an alternative to commercial kits for the measurement and validation of target proteins. Acute phase proteins (APPs) are widely recognized inflammation and infection biomarkers (Eckersall, 2010...

Interaction of dependent and non-dependent regions of the acutely injured lung during a stepwise recruitment manoeuvre

International Nuclear Information System (INIS)

Gómez-Laberge, Camille; Rettig, Jordan S; Arnold, John H; Wolf, Gerhard K; Smallwood, Craig D; Boyd, Theonia K

2013-01-01

The benefit of treating acute lung injury with recruitment manoeuvres is controversial. An impediment to settling this debate is the difficulty in visualizing how distinct lung regions respond to the manoeuvre. Here, regional lung mechanics were studied by electrical impedance tomography (EIT) during a stepwise recruitment manoeuvre in a porcine model with acute lung injury. The following interaction between dependent and non-dependent regions consistently occurred: atelectasis in the most dependent region was reversed only after the non-dependent region became overdistended. EIT estimates of overdistension and atelectasis were validated by histological examination of lung tissue, confirming that the dependent region was primarily atelectatic and the non-dependent region was primarily overdistended. The pulmonary pressure–volume equation, originally designed for modelling measurements at the airway opening, was adapted for EIT-based regional estimates of overdistension and atelectasis. The adaptation accurately modelled the regional EIT data from dependent and non-dependent regions (R 2 > 0.93, P < 0.0001) and predicted their interaction during recruitment. In conclusion, EIT imaging of regional lung mechanics reveals that overdistension in the non-dependent region precedes atelectasis reversal in the dependent region during a stepwise recruitment manoeuvre. (paper)

A role for barley calcium-dependent protein kinase CPK2a in the response to drought

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Agata Cieśla

2016-10-01

Full Text Available Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L., one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index, an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger.

Science.gov (United States)

Jernejc, Katarina; Bencina, Mojca

2003-08-29

Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

Relationship between Acute Phase Proteins and Serum Fatty Acid Composition in Morbidly Obese Patients

Science.gov (United States)

Fernandes, Ricardo; Beserra, Bruna Teles Soares; Cunha, Raphael Salles Granato; Hillesheim, Elaine; Camargo, Carolina de Quadros; Pequito, Danielle Cristina Tonello; de Castro, Isabela Coelho; Fernandes, Luiz Cláudio; Nunes, Everson Araújo; Trindade, Erasmo Benício Santos de Moraes

2013-01-01

Background. Obesity is considered a low-grade inflammatory state and has been associated with increased acute phase proteins as well as changes in serum fatty acids. Few studies have assessed associations between acute phase proteins and serum fatty acids in morbidly obese patients. Objective. To investigate the relationship between acute phase proteins (C-Reactive Protein, Orosomucoid, and Albumin) and serum fatty acids in morbidly obese patients. Methods. Twenty-two morbidly obese patients were enrolled in this study. Biochemical and clinical data were obtained before bariatric surgery, and fatty acids measured in preoperative serum. Results. Orosomucoid was negatively correlated with lauric acid (P = 0.027) and eicosapentaenoic acid (EPA) (P = 0.037) and positively with arachidonic acid (AA) (P = 0.035), AA/EPA ratio (P = 0.005), and n-6/n-3 polyunsaturated fatty acids ratio (P = 0.035). C-Reactive Protein (CRP) was negatively correlated with lauric acid (P = 0.048), and both CRP and CRP/Albumin ratio were negatively correlated with margaric acid (P = 0.010, P = 0.008, resp.). Albumin was positively correlated with EPA (P = 0.027) and margaric acid (P = 0.008). Other correlations were not statistically significant. Conclusion. Our findings suggest that serum fatty acids are linked to acute phase proteins in morbidly obese patients. PMID:24167354

Pig-MAP, porcine acute phase proteins and standardisation of assays in Europe

DEFF Research Database (Denmark)

Alava, M.A.; Gonzalez-Ramon, N.; Heegaard, Peter M. H.

1997-01-01

The pattern of plasma proteins changes greatly following infection, inflammation or tissue injury. The concentration of some proteins referred to as acute phase proteins (APPs) significantly increases within hours or days after the onset of these processes. In contrast, the concentration of other...... during the inflammation. In addition to CRP and Hp, a serum alpha(2)-globulin was observed to be the major acute phase (MAP) protein in pigs. Pig-MAP is a new mammalian plasma protein, which is the pig counterpart of a recently cloned human serum protein denominated PK-120 or MRP. Pig-MAP shows promise...... for the presence of infectious, inflammatory and pathological lesions in animals. The ability to monitor the APP concentration in serum of pigs will improve the quality and safety of the meat produced as well as provide important diagnostic information for animal health and welfare. The serum concentration of APP...

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

Science.gov (United States)

Chen, Jiahuan; Ganguly, Anutosh; Mucsi, Ashley D; Meng, Junchen; Yan, Jiacong; Detampel, Pascal; Munro, Fay; Zhang, Zongde; Wu, Mei; Hari, Aswin; Stenner, Melanie D; Zheng, Wencheng; Kubes, Paul; Xia, Tie; Amrein, Matthias W; Qi, Hai; Shi, Yan

2017-02-01

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner. © 2017 Chen et al.

Bcl-2 Protein Expression in Egyptian Acute Myeloid Leukemia

International Nuclear Information System (INIS)

El-Shakankiry, N.; El-Sayed, Gh.M.M.; El-Maghraby, Sh.; Moneer, M.M.

2009-01-01

Objective: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. Patients and methods: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. Results: The mean bcl-2 protein expression was significantly higher in patients (0.68610.592) compared to controls (0.313±0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups 0»=0.86). Conclusion: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy.

Effects of sterol regulatory element-binding protein (SREBP in chickens

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Alipour Fahimeh

2012-02-01

Full Text Available Abstract Sterol regulatory element binding protein- 1 and -2 (SREBP-1 and -2 are key transcription factors involved in the biosynthesis of cholesterol and fatty acids. The SREBP have mostly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, though, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. Since a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we review Size and Tissue expression Pattern of SREBP and role of this protein in chickens.

Variation in the Sodium-Dependent Vitamin C Transporter 2 Gene Is Associated with Risk of Acute Coronary Syndrome among Women

DEFF Research Database (Denmark)

Dalgård, Christine; Christiansen, Lene; Vogel, Ulla

2013-01-01

Vitamin C is associated with a lower risk of coronary heart disease possibly due to its anti-oxidative effects, beneficial effects on endothelial function and importance in collagen synthesis. The sodium-dependent vitamin C transporter 2 is responsible for the transport of vitamin C into various...... cells and malfunction of this protein leads to reduced vitamin C in tissue, including the arterial wall. We tested the hypothesis that candidate variations rs6139591 and rs1776964 in the gene coding for sodium-dependent vitamin C transporter 2 are associated with development of acute coronary syndrome....

Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

KAUST Repository

Marondedze, Claudius

2013-01-05

Background: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. Findings. Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. Conclusions: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. 2013 Marondedze et al.; licensee BioMed Central Ltd.

Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle.

Science.gov (United States)

Kanzaki, Keita; Watanabe, Daiki; Kuratani, Mai; Yamada, Takashi; Matsunaga, Satoshi; Wada, Masanobu

2017-02-01

The aim of this study was to examine the in vivo effects of eccentric contraction (ECC) on calpain-dependent proteolysis of Ca 2+ -regulatory proteins and force production in fast-twitch skeletal muscles. Rat extensor digitorum longus muscles were exposed to 200 repeated ECC in situ and excised immediately [recovery 0 (REC0)] or 3 days [recovery 3 (REC3)] after cessation of ECC. Calpain inhibitor (CI)-treated rats were intraperitoneally injected with MDL-28170 before ECC and during REC3. Tetanic force was markedly reduced at REC0 and remained reduced at REC3. CI treatment ameliorated the ECC-induced force decline but only at REC3. No evidence was found for proteolysis of dihydropyridine receptor (DHPR), junctophilin (JP)1, JP2, ryanodine receptor (RyR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)1a, or junctional face protein-45 at REC0. At REC3, ECC resulted in decreases in DHPR, JP1, JP2, RyR, and SERCA1a. CI treatment prevented the decreases in DHPR, JP1, and JP2, whereas it had little effect on RyR and SERCA1a. These findings suggest that DHPR, JP1, and JP2, but not RyR and SERCA1a, undergo calpain-dependent proteolysis in in vivo muscles subjected to ECC and that impaired function of DHPR and/or JP might cause prolonged force deficits with ECC. NEW & NOTEWORTHY Calpain-dependent proteolysis is one of the contributing factors to muscle damage that occurs with eccentric contraction (ECC). It is unclear, however, whether calpains account for proteolysis of Ca 2+ -regulatory proteins in in vivo muscles subjected to ECC. Here, we provide evidence that dihydropyridine receptor and junctophilin, but not ryanodine receptor and sarcoplasmic reticulum Ca 2+ -ATPase, undergo calpain-dependent proteolysis. Copyright © 2017 the American Physiological Society.

Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression

DEFF Research Database (Denmark)

Sullivan, B.E.; Carroll, C.C.; Jemiolo, B.

2009-01-01

Sullivan BE, Carroll CC, Jemiolo B, Trappe SW, Magnusson SP, Dossing S, Kjaer M, Trappe TA. Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression. J Appl Physiol 106: 468-475, 2009. First published November 20, 2008; doi: 10.1152/japplphysiol.......91341.2008.-Tendon is mainly composed of collagen and an aqueous matrix of proteoglycans that are regulated by enzymes called matrix metalloproteinases ( MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Although it is known that resistance exercise (RE) and sex influence tendon metabolism...... and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP...

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

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Elisa E. Figueroa-Angulo

2015-11-01

Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

Science.gov (United States)

Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

2015-11-26

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

Effects of montelukast sodium combined with pidotimod on acute phase protein and immune function in children with acute bronchitis

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Jing Wang

2017-08-01

Full Text Available Objective: To observe the effects of montelukast sodium combined with pidotimod on acute phase protein (APP and indexes of immunologic function in pediatric acute bronchitis treatment. Methods: A total of 180 cases children with acute bronchitis acted as research objects were randomly divided into control group (n=65 and observation group (n=63. On the basis of conventional therapy, control group was treated by plus pidotimod. On this base, observation group was treated with montelukast sodium. The changes of acute phase proteins (CRP, HP, a1-AAG and CER and immune function (CD3+ , CD4+ , CD8+ and CD4+ /CD8+ levels before and after treatment were observed after 2 months. Results: Before treatment, CRP, HP, a1-AAG, CER, CD3+ , CD4+ , CD8+ and CD4+ /CD8+ levels of two groups had no statistically significant difference; CRP, HP, a1-AAG, CER, and CD8+ levels of control and observation groups decreased significantly after treatment, the decreases of observation group were more obvious than that of control group, and the levels after treatment were significantly lower than that of control groups. The levels of CD3+ , CD4+ and CD4+ /CD8+ in two groups after treatment were significantly higher than those before treatment. For observation group, the levels of CD3+ , CD4+ and CD4+ /CD8+ increased more significantly after treatment, which were significantly higher than that of the control group. Conclusion: Using Montelukast sodium combined with pidotimod can effectively reduce the children's acute phase protein levels, improve immune function, which has clinical value for the treatment of children with acute bronchitis.

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

Science.gov (United States)

Sun, Xingmin; Mierke, Dale F; Biswas, Tapan; Lee, Sang Yeol; Landy, Arthur; Radman-Livaja, Marta

2006-11-17

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.

Science.gov (United States)

Zoncu, Roberto; Perera, Rushika M; Sebastian, Rafael; Nakatsu, Fubito; Chen, Hong; Balla, Tamas; Ayala, Guillermo; Toomre, Derek; De Camilli, Pietro V

2007-03-06

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P(2) breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P(2) breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P(2) depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P(2) in clathrin coat dynamics and Arp2/3-dependent actin regulation.

Polycomb domain formation depends on short and long distance regulatory cues.

Directory of Open Access Journals (Sweden)

Bernd Schuettengruber

Full Text Available BACKGROUND: Polycomb group (PcG proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In Drosophila, cis-regulatory regions termed PcG response elements (PREs act as nucleation sites for PcG proteins to create large repressive PcG domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3. In addition to an action in cis, PREs can interact over long distances, thereby enhancing PcG dependent silencing. How PcG domains are established, which factors limit their propagation in cis, and how long range interactions of PREs in trans affect the chromatin structure is largely unknown. PRINCIPAL FINDINGS: We demonstrate that the insertion of a PRE-containing transgene in the Drosophila genome generates an artificial PcG domain and we analyze its organization by quantitative ChIP and ChIP-on-chip experiments. Intriguingly, a boundary element and known insulator proteins do not necessarily interfere with spreading of H3K27me3. Instead, domain borders correlate with the presence of promoter regions bound by RNA Polymerase II and active chromatin marks. In contrast, genes that are silent during early fly development get included within the PcG domain and this incorporation interferes with gene activation at later developmental stages. Moreover, trans-interaction of the transgenic PRE with its homologous endogenous PRE results in increased PcG binding, correlating with reinforced silencing of genes within the domain borders. CONCLUSIONS: Our results suggest that higher-order organization of PcG-bound chromatin can stabilize gene silencing within PcG domains. Further we propose that multi-protein complexes associated with active promoters are able to define the limits of PcG domains. Future work aimed to pinpoint the factors providing this barrier function will be required to understand the precise molecular mechanism by which active promoter regions can act as boundaries to stop

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

DEFF Research Database (Denmark)

Issinger, O G; Beier, H; Speichermann, N

1980-01-01

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

International Nuclear Information System (INIS)

Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

1987-01-01

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

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Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

2015-02-01

sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

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Luo, Yonglun; Blechingberg, Jenny; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders D; Bolund, Lars; Nielsen, Anders Lade

2015-11-14

FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

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Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

2014-01-31

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

dependent/calmodulin- stimulated protein kinase from moss

Indian Academy of Sciences (India)

Unknown

stimulated protein kinase; CDPK, calmodulin domain-like protein kinase; KM14, 14 amino acid synthetic peptide; .... used were obtained from Sigma Chemical Company, USA, ..... Plant chimeric Ca2+/Calmodulin-dependent protein kinase.

StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

Science.gov (United States)

Clark, Barbara J; Hudson, Elizabeth A

2015-03-04

The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

Acute Heat Stress Changes Protein Expression in the Testes of a Broiler-Type Strain of Taiwan Country Chickens.

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Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chan, Hong-Lin; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2018-03-19

Heat stress leads to decreased fertility in roosters. This study investigated the global protein expression in response to acute heat stress in the testes of a broiler-type strain of Taiwan country chickens (TCCs). Twelve 45-week-old roosters were randomly allocated to the control group maintained at 25°C, and three groups subjected to acute heat stress at 38°C for 4 h, with 0, 2, and 6 h of recovery, respectively. Testis samples were collected for hematoxylin and eosin staining, apoptosis assay, and protein analysis. The results revealed 101 protein spots that differed significantly from the control following exposure to acute heat stress. The proteins that were differentially expressed participated mainly in protein metabolism and other metabolic processes, responses to stimuli, apoptosis, cellular organization, and spermatogenesis. Proteins that negatively regulate apoptosis were downregulated and proteins involved in autophagy and major heat shock proteins (HSP90α, HSPA5, and HSPA8) were upregulated in the testes of heat-stressed chickens. In conclusion, acute heat stress causes a change in protein expression in the testes of broiler-type B strain TCCs and may thus impair cell morphology, spermatogenesis, and apoptosis. The expression of heat shock proteins increased to attenuate the testicular injury induced by acute heat stress.

Anti-thrombin III, Protein C, and Protein S deficiency in acute coronary syndrome

Directory of Open Access Journals (Sweden)

Dasnan Ismail

2002-06-01

Full Text Available The final most common pathway for the majority of coronary artery disease is occlusion of a coronary vessel. Under normal conditions, antithrombin III (AT III, protein C, and protein S as an active protein C cofactor, are natural anticoagulants (hemostatic control that balances procoagulant activity (thrombin antithrombin complex balance to prevent thrombosis. If the condition becomes unbalanced, natural anticoagulants and the procoagulants can lead to thrombosis. Thirty subjects with acute coronary syndrome (ACS were studied for the incidence of antithrombin III (AT III, protein C, and protein S deficiencies, and the result were compare to the control group. Among patients with ACS, the frequency of distribution of AT-III with activity < 75% were 23,3% (7 of 30, and only 6,7% ( 2 of 30 in control subject. No one of the 30 control subject have protein C activity deficient, in ACS with activity < 70% were 13,3% (4 of 30. Fifteen out of the 30 (50% control subjects had protein S activity deficiency, while protein S deficiency activity < 70% was found 73.3.% (22 out of 30. On linear regression, the deterministic coefficient of AT-III activity deficiency to the development ACS was 13,25 %, and the deterministic coefficient of protein C activity deficient to the development of ACS was 9,06 %. The cut-off point for AT-III without protein S deficiency expected to contribute to the development of vessel disease was 45%. On discriminant analysis, protein C activity deficiency posed a risk for ACS of 4,5 greater than non deficient subjects, and AT-III activity deficiency posed a risk for ACS of 3,5 times greater than non deficient subjects. On binary logistic regression, protein S activity acted only as a reinforcing factor of AT-III activity deficiency in the development of ACS. Protein C and AT III deficiency can trigger ACS, with determinant coefficients of 9,06% and 13,25% respectively. Low levels of protein C posed a greater risk of

Core transcriptional regulatory circuit controlled by the TAL1 complex in human T cell acute lymphoblastic leukemia.

Science.gov (United States)

Sanda, Takaomi; Lawton, Lee N; Barrasa, M Inmaculada; Fan, Zi Peng; Kohlhammer, Holger; Gutierrez, Alejandro; Ma, Wenxue; Tatarek, Jessica; Ahn, Yebin; Kelliher, Michelle A; Jamieson, Catriona H M; Staudt, Louis M; Young, Richard A; Look, A Thomas

2012-08-14

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Alterations of proteins in MDCK cells during acute potassium deficiency.

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Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

2016-06-01

Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury. Copyright © 2016 Elsevier B.V. All rights reserved.

Maternal deprivation affects the neuromuscular protein profile of the rat colon in response to an acute stressor later in life.

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Lopes, Luísa V; Marvin-Guy, Laure F; Fuerholz, Andreas; Affolter, Michael; Ramadan, Ziad; Kussmann, Martin; Fay, Laurent B; Bergonzelli, Gabriela E

2008-04-30

Early life stress as neonatal maternal deprivation (MD) predisposes rats to alter gut functions in response to acute psychological stressors in adulthood, mimicking features of irritable bowel syndrome (IBS). We applied proteomics to investigate whether MD permanently changes the protein profile of the external colonic neuromuscular layer that may condition the molecular response to an acute stressor later in life. Male rat pups were separated 3 h/day from their mothers during the perinatal period and further submitted to water avoidance (WA) stress during adulthood. Proteins were extracted from the myenteric plexus-longitudinal muscle of control (C), WA and MD+WA rat colon, separated on 2D gels, and identified by mass spectrometry. MD amplified the WA-induced protein changes involved in muscle contractile function, suggesting that stress accumulation along life imbalances the muscle tone towards hypercontractility. Our results also propose a stress dependent regulation of gluconeogenesis. Secretogranin II - the secretoneurin precursor - was induced by MD. The presence of secretoneurin in myenteric ganglia may partially explain the stress-mediated modulation of gastrointestinal motility and/or mucosal inflammation previously described in MD rats. In conclusion, our findings suggest that neonatal stress alters the responses to acute stress in adulthood in intestinal smooth muscle and enteric neurons.

BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

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Sanja Aveic

Full Text Available BCL2 associated Athano-Gene 1 (BAG1 is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

International Nuclear Information System (INIS)

Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

2014-01-01

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

Energy Technology Data Exchange (ETDEWEB)

Carvalho-Costa, P.G. [Programa de Graduação em Psicobiologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Branco, L.G.S. [Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Leite-Panissi, C.R.A. [Programa de Graduação em Psicobiologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

2014-09-19

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells.

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Liana Verinaud

Full Text Available Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola, a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 μg of LPS and were treated with viola (3.5mg/kg via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+. We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation and stimulation of regulatory T cells (in chronic inflammation. New studies must be

Estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract of female rats upon colon-bladder cross-sensitization.

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Xiao-Qing Pan

Full Text Available Co-morbidity of bladder, bowel, and non-specific pelvic pain symptoms is highly prevalent in women. Little evidence is present on modulation of pelvic pain syndromes by sex hormones, therefore, the objective of this study was to clarify the effects of hormonal fluctuations within the estrous cycle on regulatory neuropeptides in female rats using a model of neurogenic bladder dysfunction. The estrous cycle in female rats (Sprague-Dawley, 230-250 g was assessed by vaginal smears and weight of uterine horns. Neurogenic bladder dysfunction was induced by a single inflammatory insult to the distal colon. Protein expression of calcitonin gene related peptide (CGRP, substance P (SP, nerve growth factor (NGF, and brain derived neurotrophic factor (BDNF in the pelvic organs, sensory ganglia and lumbosacral spinal cord was compared in rats in proestrus (high estrogen vs diestrus (low estrogen. Under normal physiological conditions, concentration of SP and CGRP was similar in the distal colon and urinary bladder during all phases of the estrous cycle, however, acute colitis induced a significant up-regulation of CGRP content in the colon (by 63% and urinary bladder (by 54%, p≤0.05 to control of rats in proestrus. These changes were accompanied by a significant diminution of CGRP content in L6-S2 DRG after colonic treatment, likely associated with its release in the periphery. In rats with high estrogen at the time of testing (proestrus, experimental colitis caused a significant up-regulation of BDNF colonic content from 26.1±8.5 pg/ml to 83.4±32.5 pg/ml (N = 7, p≤0.05 to control and also induced similar effects on BDNF in the urinary bladder which was also up-regulated by 5-fold in rats in proestrus (p≤0.05 to respective control. Our results demonstrate estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract upon colon-bladder cross-sensitization, which may contribute to pain fluctuations in female patients

Severe acute intermittent hypoxia elicits phrenic long-term facilitation by a novel adenosine-dependent mechanism

Science.gov (United States)

Nichols, Nicole L.; Dale, Erica A.

2012-01-01

Acute intermittent hypoxia [AIH; 3, 5-min episodes; 35–45 mmHg arterial Po2 (PaO2)] elicits serotonin-dependent phrenic long-term facilitation (pLTF), a form of phrenic motor facilitation (pMF) initiated by Gq protein-coupled metabotropic 5-HT2 receptors. An alternate pathway to pMF is induced by Gs protein-coupled metabotropic receptors, including adenosine A2A receptors. AIH-induced pLTF is dominated by the serotonin-dependent pathway and is actually restrained via inhibition from the adenosine-dependent pathway. Here, we hypothesized that severe AIH shifts pLTF from a serotonin-dependent to an adenosine-dependent form of pMF. pLTF induced by severe (25–30 mmHg PaO2) and moderate (45–55 mmHg PaO2) AIH were compared in anesthetized rats, with and without intrathecal (C4) spinal A2A (MSX-3, 130 ng/kg, 12 μl) or 5-HT receptor antagonist (methysergide, 300 μg/kg, 15 μl) injections. During severe, but not moderate AIH, progressive augmentation of the phrenic response during hypoxic episodes was observed. Severe AIH (78% ± 8% 90 min post-AIH, n = 6) elicited greater pLTF vs. moderate AIH (41% ± 12%, n = 8; P MSX-3 (28% ± 6%; n = 6; P 0.05). Thus severe AIH shifts pLTF from a serotonin-dependent to an adenosine-dependent mechanism; the adenosinergic pathway inhibits the serotonergic pathway following moderate AIH. Here we demonstrate a novel adenosine-dependent pathway to pLTF following severe AIH. Shifts in the mechanisms of respiratory plasticity provide the ventilatory control system greater flexibility as challenges that differ in severity are confronted. PMID:22403346

Depression of Complement Regulatory Factors in Rat and Human Renal Grafts Is Associated with the Progress of Acute T-Cell Mediated Rejection.

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Kazuaki Yamanaka

Full Text Available The association of complement with the progression of acute T cell mediated rejection (ATCMR is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs in acute T-cell mediated rejection using rat and human renal allografts.We prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP by immunohistochemical staining in human renal grafts and their clinical course.qRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry, was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate.We conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.

Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

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Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

2017-09-08

Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

Science.gov (United States)

Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

2017-05-01

Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

Acute guttate psoriasis patients have positive streptococcus hemolyticus throat cultures and elevated antistreptococcal M6 protein titers.

Science.gov (United States)

Zhao, Guang; Feng, Xiaoling; Na, Aihua; Yongqiang, Jiang; Cai, Qing; Kong, Jian; Ma, Huijun

2005-02-01

To further study the role of Streptococci hemolyticus infection and streptococcal M6 protein in the pathogenesis of acute guttate psoriasis, streptococcal cultures were taken from the throats of 68 patients with acute guttate psoriasis. PCR technique was applied to detect M6 protein encoding DNA from those cultured streptococci. Pure M6 protein was obtained by Sephacry/S-200HR and Mono-Q chromatography from proliferated Streptococcus hemolyticus. Antistreptococcal M6 protein titers were measured in the serum of patients with acute guttate psoriasis, plaque psoriasis and healthy controls by ELISA. A high incidence of Streptococcus hemolyticus culture was observed in the guttate psoriatic group compared with the plaque psoriasis and control groups. Fourteen strains of Streptococcus hemolyticus were cultured from the throats of 68 acute guttate psoriasis patients. Of these, 5 strains contain DNA encoding the M6 protein gene as confirmed by PCR technique. More than 85% purification of M6 protein was obtained from Streptococcus pyogenes. Applying our pure M6 protein with the ELISA methods, we found that the titer of antistreptococcal M6 protein was significantly higher in the serum of guttate psoriasis patients than in the control or plaque psoriasis groups (P M6 protein in their sera.

HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse.

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Martelli, Massimo F; Di Ianni, Mauro; Ruggeri, Loredana; Falzetti, Franca; Carotti, Alessandra; Terenzi, Adelmo; Pierini, Antonio; Massei, Maria Speranza; Amico, Lucia; Urbani, Elena; Del Papa, Beatrice; Zei, Tiziana; Iacucci Ostini, Roberta; Cecchini, Debora; Tognellini, Rita; Reisner, Yair; Aversa, Franco; Falini, Brunangelo; Velardi, Andrea

2014-07-24

Posttransplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation, we reported that donor-derived T regulatory cells (Tregs), coinfused with conventional T cells (Tcons), protected recipients against graft-versus-host disease (GVHD). The present phase 2 study investigated whether Treg-Tcon adoptive immunotherapy prevents posttransplant leukemia relapse. Forty-three adults with high-risk acute leukemia (acute myeloid leukemia 33; acute lymphoblastic leukemia 10) were conditioned with a total body irradiation-based regimen. Grafts included CD34(+) cells (mean 9.7 × 10(6)/kg), Tregs (mean 2.5 × 10(6)/kg), and Tcons (mean 1.1 × 10(6)/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed ≥grade 2 acute GVHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GVHD without loss of the benefits of graft-versus-leukemia (GVL) activity. Humanized murine models provided insights into the mechanisms underlying separation of GVL from GVHD, suggesting the GVL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow. © 2014 by The American Society of Hematology.

Current Knowledge on the Acute Regulation of Steroidogenesis.

Science.gov (United States)

Selvaraj, Vimal; Stocco, Douglas M; Clark, Barbara J

2018-04-27

How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years, and facts surrounding their rise or decline. Only two have persisted-Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). We present a detailed summary of the work that has been published for each of these two proteins, the specific data that has appeared in support of their role in cholesterol transport and steroidogenesis, and the ensuing observations that have arisen in recent years that have refuted the role of TSPO in this process. We believe that the only viable candidate that has been shown to be indispensable is the StAR protein. Lastly, we provide our view on what may be the most important questions concerning the acute regulation of steroidogenesis that need to be asked in future.

Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia.

Science.gov (United States)

Le Sommer, Samantha; Morrice, Nicola; Pesaresi, Martina; Thompson, Dawn; Vickers, Mark A; Murray, Graeme I; Mody, Nimesh; Neel, Benjamin G; Bence, Kendra K; Wilson, Heather M; Delibegović, Mirela

2018-01-01

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B -/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75-87. ©2017 AACR . ©2017 American Association for Cancer Research.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

Science.gov (United States)

Ying, Sheng; Hill, Allyson T; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

2017-06-01

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds ( Ricinus communis ) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca 2+ -dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca 2+ -dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: ( i ) a pair of Ca 2+ binding sites with identical dissociation constants of 5.03 μM, ( ii ) a Ca 2+ -dependent electrophoretic mobility shift, and ( iii ) a marked Ca 2+ -independent hydrophobicity. Pull-down experiments established the Ca 2+ -dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca 2+ -dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis ( Arabidopsis thaliana ) CPK4 and soybean ( Glycine max ) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca 2+ signaling and the posttranslational control of respiratory CO 2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. © 2017 American Society of Plant

An antisense oligodeoxynucleotide targeted against the type IIβ regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

International Nuclear Information System (INIS)

Tortora, G.; Clair, T.; Cho-Chung, Y.S.

1990-01-01

The type II β regulatory subunit of cAMP-dependent protein kinase (RII β ) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII β antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII β antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII β protein. Exposure to RII β sense, RI α and RII α antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII β regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells

No relationship between most polymorphisms of steroidogenic acute regulatory (StAR gene with polycystic ovarian syndrome

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Azadeh-Sadat Nazouri

2015-12-01

Full Text Available Background: Polycystic ovary syndrome (PCOS is one of the most common endocrine women’s disorders in reproductive age. Hyperandrogenism has a critical role in the etiology of PCOS and it can cause fault in Steroidogenesis process. During steroidogenesis, steroidogenic acute regulatory protein (StAR seems to increase the delivery of cholesterol through mitochondrial membrane. Therefore, polymorphisms of StAR might effect on this protein and play a role in the etiology of PCOS. Objective: The aim of this study was to investigate the association between StAR SNPs with PCOS. Thus, seven polymorphisms in this gene: rs104894086, rs104894089, rs104894090, rs137852689, rs10489487, rs104894085 were detected. Materials and Methods: In this case control study, 45 PCOS women, 40 male factor/unexplained infertile women, and 40 fertile women as two control groups were participated from 2008-2012. Polymorphisms were detected using restriction fragment length polymorphism (PCR-RFLP method. Results: Heterozygote genotyping for rs137852689 SNP (amino acid 218 C > T was only seen in seven PCOS patients, one in normal ovulatory women, and five in male factor/unexplained infertile women (15.5%, 2.5%, 12.5%, respectively (p= 0.12. While, it has shown no association between other SNPS with PCOs. Conclusion: The RFLP results for seven chosen SNPs, which located in exon 5 and 7 showed normal status in three groups, it means no heterozygous or homozygous forms of selected SNPs were observed. So, it seems evaluation of the active amino acid sites should be investigated and also the study population should be increased.

Time-dependent negative reinforcement of ethanol intake by alleviation of acute withdrawal.

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Cunningham, Christopher L; Fidler, Tara L; Murphy, Kevin V; Mulgrew, Jennifer A; Smitasin, Phoebe J

2013-02-01

Drinking to alleviate the symptoms of acute withdrawal is included in diagnostic criteria for alcoholism, but the contribution of acute withdrawal relief to high alcohol intake has been difficult to model in animals. Ethanol dependence was induced by passive intragastric ethanol infusions in C57BL/6J (B6) and DBA/2J (D2) mice; nondependent control animals received water infusions. Mice were then allowed to self-administer ethanol or water intragastrically. The time course of acute withdrawal was similar to that produced by chronic ethanol vapor exposure in mice, reaching a peak at 7 to 9 hours and returning to baseline within 24 hours; withdrawal severity was greater in D2 than in B6 mice (experiment 1). Postwithdrawal delays in initial ethanol access (1, 3, or 5 days) reduced the enhancement in later ethanol intake normally seen in D2 (but not B6) mice allowed to self-infuse ethanol during acute withdrawal (experiment 2). The postwithdrawal enhancement of ethanol intake persisted over a 5-day abstinence period in D2 mice (experiment 3). D2 mice allowed to drink ethanol during acute withdrawal drank more ethanol and self-infused more ethanol than nondependent mice (experiment 4). Alcohol access during acute withdrawal increased later alcohol intake in a time-dependent manner, an effect that may be related to a genetic difference in sensitivity to acute withdrawal. This promising model of negative reinforcement encourages additional research on the mechanisms underlying acute withdrawal relief and its role in determining risk for alcoholism. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Usefulness of acute phase proteins for monitoring development of ...

African Journals Online (AJOL)

Background: Serum levels of acute phase proteins (APP) have been used to diagnose and follow up treatment of liver diseases. This study was carried out to determine the usefulness of APP to predict development of hepatocellular carcinoma (HCC) among Hepatitis B virus (HBV) carriers. Study design: In a prospective ...

Density-dependence as a size-independent regulatory mechanism.

Science.gov (United States)

de Vladar, Harold P

2006-01-21

The growth function of populations is central in biomathematics. The main dogma is the existence of density-dependence mechanisms, which can be modelled with distinct functional forms that depend on the size of the population. One important class of regulatory functions is the theta-logistic, which generalizes the logistic equation. Using this model as a motivation, this paper introduces a simple dynamical reformulation that generalizes many growth functions. The reformulation consists of two equations, one for population size, and one for the growth rate. Furthermore, the model shows that although population is density-dependent, the dynamics of the growth rate does not depend either on population size, nor on the carrying capacity. Actually, the growth equation is uncoupled from the population size equation, and the model has only two parameters, a Malthusian parameter rho and a competition coefficient theta. Distinct sign combinations of these parameters reproduce not only the family of theta-logistics, but also the van Bertalanffy, Gompertz and Potential Growth equations, among other possibilities. It is also shown that, except for two critical points, there is a general size-scaling relation that includes those appearing in the most important allometric theories, including the recently proposed Metabolic Theory of Ecology. With this model, several issues of general interest are discussed such as the growth of animal population, extinctions, cell growth and allometry, and the effect of environment over a population.

Inference of hierarchical regulatory network of estrogen-dependent breast cancer through ChIP-based data

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Parvin Jeffrey

2010-12-01

Full Text Available Abstract Background Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks. Results In this study, we applied computational approaches to analyze three public available ChIP-based datasets: ChIP-seq, ChIP-PET and ChIP-chip, and to investigate the hierarchical regulatory network for ERα and ERα partner TFs regulation in estrogen-dependent breast cancer MCF7 cells. 16 common TFs and two common new TF partners (RORA and PITX2 were found among ChIP-seq, ChIP-chip and ChIP-PET datasets. The regulatory networks were constructed by scanning the ChIP-peak region with TF specific position weight matrix (PWM. A permutation test was performed to test the reliability of each connection of the network. We then used DREM software to perform gene ontology function analysis on the common genes. We found that FOS, PITX2, RORA and FOXA1 were involved in the up-regulated genes. We also conducted the ERα and Pol-II ChIP-seq experiments in tamoxifen resistance MCF7 cells (denoted as MCF7-T in this study and compared the difference between MCF7 and MCF7-T cells. The result showed very little overlap between these two cells in terms of targeted genes (21.2% of common genes and targeted TFs (25% of common TFs. The significant dissimilarity may indicate totally different transcriptional regulatory mechanisms between these two cancer cells. Conclusions Our study uncovers new estrogen-mediated regulatory networks by mining three ChIP-based data in MCF7 cells and ChIP-seq data in MCF7-T cells. We compared the different ChIP-based technologies as well as different breast cancer cells. Our computational analytical approach may guide biologists to

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

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Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

Science.gov (United States)

Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

2014-01-01

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

Science.gov (United States)

Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

2002-01-01

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

[Change of CD4(+) CD25(+) regulatory T cells and NK Cells in peripheral blood of children with acute leukemia and its possible significance in tumor immunity].

Science.gov (United States)

Wu, Ze-Lin; Hu, Guan-Yu; Chen, Fu-Xiong; Lu, Hui-Min; Wu, Zi-Liang; Li, Hua-Mei; Wei, Feng-Gui; Guan, Jing-Ming; Wu, Li-Ping

2010-06-01

This study was purposed to investigate the changes of CD4(+) CD25(+) regulatory T cells and NK cells in peripheral blood of acute leukemia children at different stages, the function of immune system and the possible roles of the CD4(+) CD25(+) regulatory T cells as well as NK cells in leukemia immunity. The number and proportion of CD4(+) CD25(+) regulatory T cells and NK cells were detected by flow cytometry in the peripheral blood of 53 acute leukemia children, including 25 patients in new diagnosis and 28 patients in continuous complete remission (CCR), and were compared with that of 20 normal children. The results indicated that the mean proportion of CD4(+) CD25(+) CD127(+) in CD4(+) T cells of peripheral blood in newly diagnosed patients, patients with CCR and normal children were (9.55 +/- 2.41)%, (8.54 +/- 2.51)% and (6.25 +/- 0.85)% respectively, the mean proportions of CD4(+)CD25(+)CD127(+) in newly diagnosed patients and patients with CCR were higher than that in normal children, the mean proportion of CD4(+)CD25(+)CD127(+) in newly diagnosed patients were higher than that in patients with CCR (p cell count in patients with acute leukaemia decreased as compared with normal control, while after achieving CCR, the NK cell count in patients were also less than that in normal control (4.11 +/- 3.87% and 10.41 +/- 7.20% vs 14.06 +/- 5.95%, p regulatory T cells is a simple, reproductive and accurate method, and the CD4(+) CD25(+) CD127(+) T cells can better reflect the proportion of CD4(+)CD25(+) regulatory T cells. The increase of regulatory T cells and decrease of NK cells in pediatric patients with acute leukemia indicate that the function of NK cells may be depressed. Treg T cells play a role in occurrence and development of leukemia, and are involved in down-regulating NK cell function.

Predictive value of c-reactive protein for thrombolytic therapy in acute myocardial infarction

International Nuclear Information System (INIS)

Majeed, N.; Bashir, F.

2014-01-01

The serum levels of C-reactive protein on admission may predict the efficacy of reperfusion in patients with acute myocardial infarction. Objectives: This study was conducted to know the predictive value of CRP for success of thrombolysis and to know the prognostic value of C-reactive protein in patients having acute myocardial infarction. Study Design: It was single center, open labeled cross sectional study. Materials and Methods: Sixty patients of acute myocardial infarction diagnosed on clinical and ECG criteria, who received thrombolytic therapy with strepto- kinase, were included in the study. The diagnosis of acute rnyocardial infarction was made on clinical para meters and ECG criteria. The ECG changes were noted before starting thrombolysis. The baseline sample for C-reactive protein (CRP,) was taken before starting thrombolysis. The time duration between onset of symptoms and start of thrombolysis was also noted. The thrombolysis was done with streptokinase infusion, 1.5 million units diluted in 100ml normal saline, intravenously over one hour. The ECG was repeated after six hours of completion of thrombolysis and, changes were noted and compared with ECG changes before thrombolysis. Now second sample for C-reactive protein (CRP2) was taken after six hours of completion of thrombolysis. CRP was measured by a high sensitivity assay which can accurately measure basal levels of CRP throughout the currently accepted cardiovascular risk assessment range (0.20 - 10.0 mg/L). According to ECG findings after thrombolysis, all patients were divided into two groups. Group A was considered as successful group to thrombolysis, in whom ECG changes were settled. Group B was considered as unsuccessful group to thrombolysis, in whom ECG changes remained same as before thrombolysis. Both values of C-reactive protein, CRP, and CRP2 were compared in both groups group A and group B. Results: Plasma CRP values before and after thrombolysis had strong predictive value for

Application of acute phase protein measurements in veterinary clinical chemistry

DEFF Research Database (Denmark)

Petersen, Henning; Nielsen, J. P.; Heegaard, Peter M. H.

2004-01-01

The body's early defence in response to trauma, inflammation or infection, the acute phase response, is a complex set of systemic reactions seen shortly after exposure to a triggering event. One of the many components is an acute phase protein response in which increased hepatic synthesis leads t...... A and their possible use as non-specific indicators of health in large animal veterinary medicine such as in the health status surveillance of pigs at the herd level, for the detection of mastitis in dairy cattle and for the prognosis of respiratory diseases in horses....

Effect of microinjections of subunits of cAMP-dependent protein kinase on development, proliferation, and RNA synthesis in early embryos of the loach Misgurnus fossilis L

International Nuclear Information System (INIS)

Glukhov, A.I.; Benyumov, A.O.; Nesterova, M.V.; Severin, E.S.; Gazaryan, K.G.

1986-01-01

The effect of the catalytic and regulatory subunits of cAMP-dependent protein kinase type II on development, proliferation, and RNA synthesis was studied in loach embryos. It was found that injection of the catalytic subunit in a physiological concentration leads to a disturbance in the course of development and inhibits proliferation and RNA synthesis in the embryos. An increase in the concentration of this protein above the physiological level leads to death of the embryos in the first hours of development. Injection of the regulatory subunit stimulated the incorporation of labeled uridine into the acid-insoluble fraction of the embryos, beginning with the gastrula stage. The cell nuclei of loach embryos injected with subunits of protein kinase type II were transplanted into activated loach egg cells: subunits of protein kinase type I had no effect on the ability of nuclei of undetermined loach embryo cells to provide de novo development and their effect was reversible

Early lymphocyte recovery after intensive timed sequential chemotherapy for acute myelogenous leukemia: peripheral oligoclonal expansion of regulatory T cells.

Science.gov (United States)

Kanakry, Christopher G; Hess, Allan D; Gocke, Christopher D; Thoburn, Christopher; Kos, Ferdynand; Meyer, Christian; Briel, Janet; Luznik, Leo; Smith, B Douglas; Levitsky, Hyam; Karp, Judith E

2011-01-13

Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4(+) and included a greatly expanded population of CD3(+)CD4(+)CD25(+)Foxp3(+) T cells. Recovering CD3(+)CD4(+)CD25(+)Foxp3(+) T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.

A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ

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Roepcke Stefan

2011-12-01

Full Text Available Abstract Background Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM. In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms. Results Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site. Conclusions Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.

Regulatory function of a novel population of mouse autoantigen-specific Foxp3 regulatory T cells depends on IFN-gamma, NO, and contact with target cells.

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Cyndi Chen

Full Text Available BACKGROUND: Both naturally arising Foxp3(+ and antigen-induced Foxp3(- regulatory T cells (Treg play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel class II MHC tetramer, we have isolated a population of CD4(+ Foxp3(- T cells specific for the autoantigen glutamic acid decarboxylase p286-300 peptide (NR286 T cells from diabetes-resistant non-obese resistant (NOR mice. These Foxp3(- NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-gamma was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg. CONCLUSIONS/SIGNIFICANCE: These studies identified a unique population of autoantigen-specific Foxp3(- Treg that can exert their regulatory function dependent on not only IFN-gamma and NO but also cell contact with target cells.

Calcium-dependent but calmodulin-independent protein kinase from soybean

International Nuclear Information System (INIS)

Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

1987-01-01

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≥ 2 micromolar). The protein kinase activity was stimulated 100-fold by ≥ 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤ 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45 Ca 2+ in the presence of KCl and MgCl 2 , which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity

Reconstructing the regulatory circuit of cell fate determination in yeast mating response.

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Shao, Bin; Yuan, Haiyu; Zhang, Rongfei; Wang, Xuan; Zhang, Shuwen; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

2017-07-01

Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our

Meperidine (pethidine versus morphine in acute pain management of opioid-dependent patients

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Solhi H

2016-08-01

Full Text Available Hassan Solhi,1 Hossein Sanaei-Zadeh,2 Sadra Solhi,1 Mohammad Ali Azizi Nadian,1 Morteza Gharibi,3 Bahman Sadeghi Sedeh4 1Department of Internal Medicine, Arak University of Medical Sciences, Arak, 2Emergency Room, Division of Medical Toxicology, Hazrat Ali-Asghar (p Hospital, Shiraz University of Medical Sciences, Shiraz, 3Department of Emergency Medicine, Arak University of Medical Sciences, 4Department of Social Medicine, Arak University of Medical Sciences, Arak, Iran Abstract: The present study aimed to evaluate the effectiveness of morphine and meperidine (pethidine as pain relief in opioid-dependent patients with acute pain. A total of 122 opioid-dependent patients with acute pain were included in the study. Their pain severity was assessed, using visual analog scale (VAS scores ranging from 0 to 10. The patients randomly received intravenous morphine (up to 0.15 mg/kg or meperidine (up to 1.5 mg/kg for pain control by patient control analgesia (PCA pump. The clinical opioid withdrawal scale (COWS was employed for the assessment of withdrawal symptoms. The pain relief and the emergence of withdrawal symptoms were measured at 15, 30, and 60 minutes after drug administration. The patients who received morphine reported a better pain control compared to those who received meperidine (mean ± standard deviation [SD] VAS scores 4.11±1.90 vs 5.85±2.08 at the end of the study; P<0.001. On the other hand, the patients who received meperidine indicated prominent withdrawal symptoms (mean ± SD COWS scores 4.80±2.18 vs. 1.98±0.82 at the end of the study; P<0.001. Our findings revealed that morphine can be recommended in acute pain management of opioid-dependent patients. In addition, emergency physicians should ask their patients about any drug dependence before selecting the appropriate drug for their acute pain management. Keywords: pain management, meperidine, morphine, opioid dependency, withdrawal symptoms

Expression of Th17 and CD4+ CD25+ T regulatory cells in peripheral blood of acute leukemia patients and their prognostic significance.

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Xiang, Mingli; Guo, Li; Ma, Yan; Li, Yi

2016-11-01

To discuss the expression of T helper cell 17 (Th17) cells and CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in peripheral blood (PB) of patients with acute leukemia (AL), and to explore the relationship between them and disease prognosis. 40 patients diagnosed with acute leukemia in The First Affiliated Hospital of Zhengzhou University from July 2012 to August 2014 were selected as the observation group. Meanwhile, 40 healthy people were taken as the control group. Flow Cytometry Method (FCM) was used to detect the level of Th17 cells and CD4 + CD25 + Foxp3 + cells in peripheral blood of the two groups, and enzyme-linked immuno sorbent assay (ELISA) method was used to test the level of IL17 and TGF-β in peripheral blood of two groups; reverse transcription-polymerase chain reaction (RT-PCR) was adopted to analyze the mRNA levels of RORγT and Foxp3 in peripheral blood. In addition, we examined the levels of Th17 and CD4 + CD25 + Foxp3 + cells and associated factor levels in patients with remission after AL chemotherapy. the Th17 cells (CD3 + CD4 + IL-17 + ) in acute leukemia patients accounted for (1.51±0.27)%, which was significantly higher than that of control group (0.36±0.23)%, with statistical significance (t=20.51, Pcells in AL patients was (3.37±0.48)%, which was significantly higher than that of control group of (1.26±0.27)%, with statistical significance (t=24.23, Pt=7.83, Pt=7.83, Pt=12.27, Pt=7.89, Pcells and CD4 + CD25 + Foxp3 + cells, and the serum levels of IL-17 and TGF-β in acute leukemia patients all decreased significantly after 6 months of chemotherapy, and the difference was statistically significant (Pcells, CD4+ CD25+ Foxp3 + cells and their secretory proteins IL-17, TGF-β and transcription factors were significantly increased in AL patients. Therefore, regular detection of peripheral blood Th17 and Treg cells, as well as their secretory proteins are useful for monitoring the immune status and prognosis of patients.

Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

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Petra Procházková

Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

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Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

2014-01-01

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

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Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

2016-09-01

Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

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Yoshikawa, Yuki; Nasuno, Ryo; Kawahara, Nobuhiro; Nishimura, Akira; Watanabe, Daisuke; Takagi, Hiroshi

2016-07-01

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

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Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells

Czech Academy of Sciences Publication Activity Database

Kanderová, V.; Kuzilkova, D.; Stuchlý, J.; Vašková, M.; Brdička, Tomáš; Fišer, K.; Hrušák, O.; Lund-Johansen, F.; Kalina, T.

2016-01-01

Roč. 15, č. 4 (2016), s. 1246-1261 ISSN 1535-9476 R&D Projects: GA MŠk 2B06064 Institutional support: RVO:68378050 Keywords : acute lymphoblastic-leukemia * acute promyelocytic leukemia * cytometric immunobead assay * caspase-dependent cleavage * acute myeloid-leukemia * gene-expression * fusion proteins * flow-cytometry * pcr data * b-cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.540, year: 2016

PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

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Osadnik, Hendrik; Schöpfel, Michael; Heidrich, Eyleen; Mehner, Denise; Lilie, Hauke; Parthier, Christoph; Risselada, H Jelger; Grubmüller, Helmut; Stubbs, Milton T; Brüser, Thomas

2015-11-01

Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains. © 2015 John Wiley & Sons Ltd.

Primary Screening for Proteins Differentially Expressed in the Myocardium of a Rat Model of Acute Methamphetamine Intoxication

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Guoqiang Qu

2016-01-01

Full Text Available The mechanism of myocardial injury induced by the cardiovascular toxicity of methamphetamine (MA has been shown to depend on alterations in myocardial proteins caused by MA. Primary screening of the expression of myocardial proteins in a rat model of MA intoxication was achieved by combining two-dimensional electrophoresis and mass spectrometry analyses, which revealed a total of 100 differentially expressed proteins. Of these, 13 displayed significantly altered expression. Moreover, Western blotting and real-time reverse transcription quantitative polymerase chain reaction analyses of several relative proteins demonstrated that acute MA intoxication lowers protein expression and mRNA transcription of aldehyde dehydrogenase-2 and NADH dehydrogenase (ubiquinone 1 alpha subcomplex subunit 10. In contrast, MA intoxication elevated the protein expression and mRNA transcription of heat shock protein family B (small member 1. By combining behavioral assessments of experimental rat models with the histological and pathological changes evident in cardiomyocytes, a mechanism accounting for MA myocardial toxicity was suggested. MA alters the regulation of gene transcription and the subsequent expression of certain proteins that participate in myocardial respiration and in responding to oxidative stress, resulting in myocardial dysfunction and structural changes that affect the functioning of the cardiovascular system.

Evidence for roles of the Escherichia coli Hda protein beyond regulatory inactivation of DnaA.

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Baxter, Jamie C; Sutton, Mark D

2012-08-01

The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication (oriC) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. Here, we used the mutant dnaN159 strain, which expresses the β159 clamp protein, to gain insight into how the actions of Hda are co-ordinated with replication. Elevated expression of Hda impeded growth of the dnaN159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of -1 frameshift mutations characteristic of Pol IV, while loss of hda conferred cold sensitivity. Using the dnaN159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Taken together, these findings suggest that although one or more Hda functions are essential for cell viability, RIDA may be dispensable. © 2012 Blackwell Publishing Ltd.

Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

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Michelle T. Barati

2016-01-01

Full Text Available The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG growth conditions as compared to isoosmotic/normal glucose control (NG⁎ conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1 proteins involved in cell survival/cell signaling and (2 proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB, supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase, suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

DECK: Distance and environment-dependent, coarse-grained, knowledge-based potentials for protein-protein docking

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Vakser Ilya A

2011-07-01

Full Text Available Abstract Background Computational approaches to protein-protein docking typically include scoring aimed at improving the rank of the near-native structure relative to the false-positive matches. Knowledge-based potentials improve modeling of protein complexes by taking advantage of the rapidly increasing amount of experimentally derived information on protein-protein association. An essential element of knowledge-based potentials is defining the reference state for an optimal description of the residue-residue (or atom-atom pairs in the non-interaction state. Results The study presents a new Distance- and Environment-dependent, Coarse-grained, Knowledge-based (DECK potential for scoring of protein-protein docking predictions. Training sets of protein-protein matches were generated based on bound and unbound forms of proteins taken from the DOCKGROUND resource. Each residue was represented by a pseudo-atom in the geometric center of the side chain. To capture the long-range and the multi-body interactions, residues in different secondary structure elements at protein-protein interfaces were considered as different residue types. Five reference states for the potentials were defined and tested. The optimal reference state was selected and the cutoff effect on the distance-dependent potentials investigated. The potentials were validated on the docking decoys sets, showing better performance than the existing potentials used in scoring of protein-protein docking results. Conclusions A novel residue-based statistical potential for protein-protein docking was developed and validated on docking decoy sets. The results show that the scoring function DECK can successfully identify near-native protein-protein matches and thus is useful in protein docking. In addition to the practical application of the potentials, the study provides insights into the relative utility of the reference states, the scope of the distance dependence, and the coarse-graining of

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

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Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2014-07-01

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

C-reactive protein as a marker of infection in children with severe acute malnutrition in Khartoum state, Sudan

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Abdelmoneim E.M. Kheir

2017-08-01

Full Text Available Severe acute malnutrition and acute systemic infection are often synergistic in children and lead to considerable mortality. The main aim of this research was to determine whether children with severe acute malnutrition can mount an acute phase reactant response measured by C-reactive protein. This was a descriptive, cross-sectional, hospital-based study that was carried out in the five main children hospitals in Khartoum state, from November 1st, 2012 to March 1st, 2013. 132 children with severe acute malnutrition were included in the study. Data collection included history, examination and C-reactive protein measurement. The data were analyzed using Statistical Package for Social Sciences (SPSS for descriptive and inferential statistics. The main results revealed that 93(70.5% children between 12-23 months of age and most of them had marasmus. Diarrhoea was the commonest presenting symptoms in 86.4%, followed by fever and vomiting. Most of the children (82.6% had positive C-reactive protein with variable levels. In conclusion malnourished children are able to synthesize C-reactive protein in response to an infectious process and the magnitude of this response is increased in those with severe infections.

Cytokine and acute phase protein gene expression in liver biopsies from dairy cows with a lipopolysaccharide - induced mastitis

DEFF Research Database (Denmark)

Vels, J; Røntved, Christine M.; Bjerring, Martin

2009-01-01

A minimally invasive liver biopsy technique was tested for its applicability to study the hepatic acute phase response (APR) in dairy cows with Escherichia coli lipopolysaccharide (LPS)-induced mastitis. The hepatic mRNA expression profiles of the inflammatory cytokines, tumor necrosis factor (TNF......, a minimally invasive liver biopsy technique can be used for studying the hepatic APR in diseased cattle. Lipopolysaccharide-induced mastitis resulted in a time-dependent production of inflammatory cytokines and SAA and Hp in the liver of dairy cows.......- ), IL-1β, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and 1-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200 µg of LPS (n = 8) or NaCl solution (n = 6...

Circulating microbial products and acute phase proteins as markers of pathogenesis in lymphatic filarial disease.

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R Anuradha

Full Text Available Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+ or without (CP Ag- active infection; with clinically asymptomatic infections (INF; and in those without infection (endemic normal [EN]. Comparisons between the two actively infected groups (CP Ag+ compared to INF and those without active infection (CP Ag- compared to EN were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein, acute phase proteins (haptoglobin and serum amyloid protein-A, and inflammatory cytokines (IL-1β, IL-12, and TNF-α are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.

Multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP and lung resistance protein (LRP gene expression in childhood acute lymphoblastic leukemia

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Elvis Terci Valera

Full Text Available CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP, and lung resistance protein (LRP may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR, and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005. The presence of the common acute lymphoblastic leukemia antigen (CALLA was correlated with increased LRP expression (p = 0.009 and increased risk of relapse or death (p = 0.05. The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05, as confirmed by multivariate analysis of the three genes studied (p = 0.035. DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

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Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

2008-02-22

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

The selective phosphorylation of a guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Carlson, K.E.

1989-01-01

Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds1[OPEN

Science.gov (United States)

Hill, Allyson T.; Anderson, Erin M.; She, Yi-Min

2017-01-01

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. PMID:28363991

The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

Science.gov (United States)

Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

2008-01-01

The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

HIV infection and drugs of abuse: role of acute phase proteins.

Science.gov (United States)

Samikkannu, Thangavel; Rao, Kurapati V K; Arias, Adriana Y; Kalaichezian, Aarthi; Sagar, Vidya; Yoo, Changwon; Nair, Madhavan P N

2013-09-17

HIV infection and drugs of abuse such as methamphetamine (METH), cocaine, and alcohol use have been identified as risk factors for triggering inflammation. Acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) are the biomarkers of inflammation. Hence, the interactive effect of drugs of abuse with acute phase proteins in HIV-positive subjects was investigated. Plasma samples were utilized from 75 subjects with METH use, cocaine use, alcohol use, and HIV-positive alone and HIV-positive METH, cocaine, and alcohol users, and age-matched control subjects. The plasma CRP and SAA levels were measured by ELISA and western blot respectively and the CD4 counts were also measured. Observed results indicated that the CRP and SAA levels in HIV-positive subjects who are METH, cocaine and alcohol users were significantly higher when compared with either drugs of abuse or HIV-positive alone. The CD4 counts were also dramatically reduced in HIV-positive with drugs of abuse subjects compared with only HIV-positive subjects. These results suggest that, in HIV-positive subjects, drugs of abuse increase the levels of CRP and SAA, which may impact on the HIV infection and disease progression.

Effect of acute maternal starvation on tyrosine metabolism and protein synthesis in fetal sheep

International Nuclear Information System (INIS)

Krishnamurti, C.R.; Schaefer, A.L.

1984-01-01

To determine the effects of acute maternal starvation on intrauterine growth, tyrosine concentration and specific activity values in plasma, intracellular free and protein bound pools were determined in catheterized ovine fetuses following an 8 h continuous infusion of L-[2,3,5,6 3 H] or L-[U- 14 C] tyrosine into the ewe and fetus respectively at 115-125 days of gestation. From the kinetic data the rates of whole body and tissue fractional protein synthesis were calculated. Although placental protein synthesis was not significantly changed as a result of acute maternal starvation, fetal whole body protein synthesis was reduced from 63 g/d/kg in the fed to 25 g/d/kg in the starved condition. There was also a 10 fold reduction in the net placental transfer of tyrosine to the fetus in the starved ewes. In addition, a three fold increase was observed in the quantity of tyrosine used for oxidation by the fetuses of starved ewes, changing from 5.2% of tyrosine net utilization in the fed to 13.7% in the starved condition. Significant reductions in tissue fractional protein synthesis rates were also seen in the liver, brain, lung kidney and GIT tissues from 78, 37, 65, 45 and 71%/d respectively in the fed to 12, 10, 23, 22 and 35%/d in the fetuses of starved ewes. The data indicate that during acute maternal starvation the sheep fetus utilizes more tyrosine for oxidation and less for anabolic purposes which is reflected in a decrease both in whole body and tissue fractional rates of protein synthesis

A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5 '-diphosphocholine coupled dendrimers

DEFF Research Database (Denmark)

Heegaard, Peter M. H.; Pedersen, H. G.; Jensen, A. L.

2009-01-01

C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosp......C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP...... was applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature...

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

Science.gov (United States)

Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

2006-08-01

Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

Regulatory proteins (inhibitors or activators) affect estimates of Msub(r) of enzymes and receptors by radiation inactivation

International Nuclear Information System (INIS)

Potier, M.; Giroux, S.

1985-01-01

The radiation-inactivation method allows the determination of the Msub(r) of enzymes and receptors by monitoring the decay of biological activity as a function of absorbed dose. The presence of regulatory or effector proteins (inhibitors or activators) associated with an enzyme or receptor, or released in the preparation after tissue homogenization, may affect the decay of biological activity. How the activity is affected, however, will depend on the type of inhibition (competitive or non-competitive), the inhibitor or activator concentration, the dissociation constant of the enzyme-effector system, and the effector Msub(r) relative to that of the enzyme. Since little is known on how effector proteins influence radiation inactivation of enzymes and receptors, we have considered a theoretical model in an effort to provide a framework for the interpretation of experimentally obtained data. Our model predicts that competitive and non-competitive inhibitors of enzymes could be distinguished by analysing irradiated samples with various substrate concentrations. Inhibitors will decrease whereas activators will increase the apparent target size of enzymes or receptors. (author)

Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

Directory of Open Access Journals (Sweden)

Shahram Golbabapour

2013-01-01

Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

Schiff base metal derivatives enhance the expression of HSP70 and suppress BAX proteins in prevention of acute gastric lesion.

Science.gov (United States)

Golbabapour, Shahram; Gwaram, Nura Suleiman; Al-Obaidi, Mazen M Jamil; Soleimani, A F; Ali, Hapipah Mohd; Abdul Majid, Nazia

2013-01-01

Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP) 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg), the positive control (Tween 20 5% v/v, 5 mL/kg), and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg). After 1 h, all of the groups received ethanol 95% (5 mL/kg) but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg). The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E), immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes

International Nuclear Information System (INIS)

Olsen, Jan Roger; Azeem, Waqas; Hellem, Margrete Reime; Marvyin, Kristo; Hua, Yaping; Qu, Yi; Li, Lisha; Lin, Biaoyang; Ke, XI- Song; Øyan, Anne Margrete; Kalland, Karl- Henning

2016-01-01

Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable. Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in

Phylogenetic conservation of the regulatory and functional properties of the Vav oncoprotein family

International Nuclear Information System (INIS)

Couceiro, Jose R.; Martin-Bermudo, Maria D.; Bustelo, Xose R.

2005-01-01

Vav proteins are phosphorylation-dependent GDP/GTP exchange factors for Rho/Rac GTPases. Despite intense characterization of mammalian Vav proteins both biochemically and genetically, there is little information regarding the conservation of their biological properties in lower organisms. To approach this issue, we have performed a characterization of the regulatory, catalytic, and functional properties of the single Vav family member of Drosophila melanogaster. These analyses have shown that the intramolecular mechanisms controlling the enzyme activity of mammalian Vav proteins are already present in Drosophila, suggesting that such properties have been set up before the divergence between protostomes and deuterostomes during evolution. We also show that Drosophila and mammalian Vav proteins have similar catalytic specificities. As a consequence, Drosophila Vav can trigger oncogenic transformation, morphological change, and enhanced cell motility in mammalian cells. Gain-of-function studies using transgenic flies support the implication of this protein in cytoskeletal-dependent processes such as embryonic dorsal closure, myoblast fusion, tracheal development, and the migration/guidance of different cell types. These results highlight the important roles of Vav proteins in the signal transduction pathways regulating cytoskeletal dynamics. Moreover, they indicate that the foundations for the regulatory and enzymatic activities of this protein family have been set up very early during evolution

Bladder inflammatory transcriptome in response to tachykinins: Neurokinin 1 receptor-dependent genes and transcription regulatory elements

Directory of Open Access Journals (Sweden)

Dozmorov Igor

2007-05-01

Full Text Available Abstract Background Tachykinins (TK, such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs. Methods An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. Results The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF and inflammation (PAR-3, IL-1R, IL-6, α-NGF, TSP2. In the absence of

Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

LENUS (Irish Health Repository)

Gegenbauer, Kristina

2013-11-01

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis*

Science.gov (United States)

Yang, Yang; Wang, Chenji; Zhang, Pingzhao; Gao, Kun; Wang, Dejie; Yu, Hongxiu; Zhang, Ting; Jiang, Sirui; Hexige, Saiyin; Hong, Zehui; Yasui, Akira; Liu, Jun O.; Huang, Haojie; Yu, Long

2013-01-01

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression. PMID:23150668

Acute differential effects of milk-derived dietary proteins on postprandial lipaemia in obese non-diabetic subjects

DEFF Research Database (Denmark)

Holmer-Jensen, Jens; Hartvigsen, Merete; Mortensen, L.S.

2012-01-01

Postprandial lipaemia is an established risk factor for atherosclerosis. To investigate the acute effect of four milk-derived dietary proteins (alpha-lactalbumin, whey isolate, caseinoglycomacropeptide and whey hydrolysate) on postprandial lipaemia, we have conducted a randomized, acute, single...

Malnutrition in the acutely ill patient: is it more than just protein and ...

African Journals Online (AJOL)

Review Article: Malnutrition in the acutely ill patient: is it more than just protein and energy? 2011;24(3) ... illness/injury, significant mortality occurs after critically ill patients are discharged from ... a manner similar to that of administering an antibiotic or drug. .... delivery (median of 0.8 g/kg/day protein (after day 3) for the study.

Acute effects of heroin on emotions in heroin-dependent patients.

Science.gov (United States)

Blum, Julia; Gerber, Hana; Gerhard, Urs; Schmid, Otto; Petitjean, Sylvie; Riecher-Rössler, Anita; Wiesbeck, Gerhard A; Borgwardt, Stefan J; Walter, Marc

2013-01-01

Euphoria has been described in heroin-dependent individuals after heroin administration. However, affective disturbances and disorders are common in heroin dependence. The present study examined the acute effects of heroin on emotions in heroin-dependent patients. This randomized controlled crossover trial included 28 heroin-dependent patients (67.9% male, n = 19) in stable heroin-assisted treatment and 20 healthy controls. The patients were administered heroin or saline (placebo), the controls were administered saline. Data measuring mood, affects and heroin craving (BDI, AMRS, STAI, STAXI, and HCQ) were assessed before and 60 minutes after substance injection. Before substance injection, heroin-dependent patients showed significantly higher levels of anxiety and depression than healthy controls (p emotions, including craving, and a significant increase in emotional well-being (p emotions, once they had received heroin. Heroin dampens craving, negative emotions, and increases positive emotions. These findings indicate that heroin regulates emotions and underscore the clinical benefit of opioid substitution treatment for heroin-dependent patients. Copyright © American Academy of Addiction Psychiatry.

Characterization of Regulatory Dendritic Cells That Mitigate Acute Graft-versus-Host Disease in Older Mice Following Allogeneic Bone Marrow Transplantation

OpenAIRE

Scroggins, Sabrina M.; Olivier, Alicia K.; Meyerholz, David K.; Schlueter, Annette J.

2013-01-01

Despite improvements in human leukocyte antigen matching and pharmacologic prophylaxis, acute graft-versus-host disease (GVHD) is often a fatal complication following hematopoietic stem cell transplant (HSCT). Older HSCT recipients experience significantly increased morbidity and mortality compared to young recipients. Prophylaxis with syngeneic regulatory dendritic cells (DCreg) in young bone marrow transplanted (BMT) mice has been shown to decrease GVHD-associated mortality. To evaluate thi...

Cloning and expression of the translocator protein (18 kDa), voltage-dependent anion channel, and diazepam binding inhibitor in the gonad of largemouth bass (Micropterus salmoides) across the reproductive cycle.

Science.gov (United States)

Doperalski, Nicholas J; Martyniuk, Christopher J; Prucha, Melinda S; Kroll, Kevin J; Denslow, Nancy D; Barber, David S

2011-08-01

Cholesterol transport across the mitochondrial membrane is rate-limiting for steroidogenesis in vertebrates. Previous studies in fish have characterized expression of the steroidogenic acute regulatory protein, however the function and regulation of other genes and proteins involved in piscine cholesterol transport have not been evaluated. In the current study, mRNA sequences of the 18 kDa translocator protein (tspo; formerly peripheral benzodiazepine receptor), voltage-dependent anion channel (vdac), and diazepam binding inhibitor (dbi; also acyl-CoA binding protein) were cloned from largemouth bass. Gonadal expression was examined across reproductive stages to determine if expression is correlated with changes in steroid levels and with indicators of reproductive maturation. In testis, transcript abundance of tspo and dbi increased with reproductive maturation (6- and 23-fold maximal increase, respectively) and expression of tspo and dbi was positively correlated with reproductive stage, gonadosomatic index (GSI), and circulating levels of testosterone. Testis vdac expression was positively correlated with reproductive stage and GSI. In females, gonadal tspo and vdac expression was negatively correlated with GSI and levels of plasma testosterone and 17β-estradiol. Ovarian dbi expression was not correlated with indicators of reproductive maturation. These studies represent the first investigation of the steroidogenic role of tspo, vdac, and dbi in fish. Findings suggest that cholesterol transport in largemouth bass testis, but not in ovary, may be transcriptionally-regulated, however further investigation will be necessary to fully elucidate the role of these genes in largemouth bass steroidogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

Science.gov (United States)

Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

2016-03-04

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PROTEOSE INTOXICATIONS AND INJURY OF BODY PROTEIN : V. THE INCREASE IN NON-PROTEIN NITROGEN OF THE BLOOD IN ACUTE INFLAMMATORY PROCESSES AND ACUTE INTOXICATIONS.

Science.gov (United States)

Cooke, J V; Whipple, G H

1918-08-01

Sterile abscess formation in the dog is accompanied by a large increase in output of urinary nitrogen and also by a small but definite increase in the blood non-protein nitrogen. All this nitrogenous material of course is derived from body protein injury and autolysis. Septic inflammation in the dog (pleurisy, pneumonia, peritonitis, etc.) likewise shows a distinct rise in the blood non-protein nitrogen. This rise is not often so great as that frequently observed in the intoxication of intestinal obstruction. Many acute infections in man (septicemia, peritonitis, pneumonia, etc.) show a definite rise in the non-protein nitrogen and urea nitrogen of the blood; some cases show a very great rise above normal (over 100 mg. of non-protein nitrogen per 100 cc. of blood). There may be no anatomical change in the kidney beyond the familiar picture of cloudy swelling. This does not exclude the possibility of some transient functional derangement of the kidney epithelium. Certain obscure intoxications in man may show a considerable rise in the non-protein nitrogen of the blood, indicating a large amount of protein disintegration. These findings must be taken into account in any clinical analysis and interpretation of high non-protein nitrogen of the blood in pathological conditions.

Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

International Nuclear Information System (INIS)

Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

1987-01-01

C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

Immunophenotype and increased presence of CD4+CD25+ regulatory T cells in patients with acute lymphoblastic leukemia

OpenAIRE

WU, CUI-PING; QING, XI; WU, CUI-YUN; ZHU, HONG; ZHOU, HAI-YAN

2011-01-01

Acute lymphoblastic leukemia (ALL), cancer of the white blood cells, is a heterogeneous disease that mainly occurs due to the malignant cloning of original and naive lymphocytes. The aim of this study was to explore the immunophenotype, the percentage of CD4+CD25+ regulatory T cells (Tregs) and the expression of cytokines interleukin (IL)-2, IL-10 and TGF-β in patients with ALL. The immunophenotype and levels of CD4+CD25+ Tregs were detected using flow cytometry in the peripheral blood of 35 ...

Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

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Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-03-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

WHY DO THE ACUTE BEHAVIORAL EFFECTS OT TOLUENE IN RATS DEPEND ON THE ROUTE OF EXPOSURE?

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Despite evidence suggesting that the acute effects of organic solvents are related to their concentration in the brain, we have observed route-dependent differences in the acute behavioral effects of toluene. Whereas inhaled toluene disrupts the performance of rats on a visual si...

miRNA-dependent translational repression in the Drosophila ovary.

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John Reich

Full Text Available The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary.Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed.Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

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Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

Politically Induced Regulatory Risk and Independent Regulatory Agencies

OpenAIRE

Strausz, Roland

2015-01-01

Uncertainty in election outcomes generates politically induced regulatory risk. Political parties' risk attitudes towards such risk depend on a fluctuation effect that hurts both parties and an output--expansion effect that benefits at least one party. Notwithstanding the parties' risk attitudes, political parties have incentives to negotiate away all regulatory risk by pre-electoral bargaining. Efficient pre-electoral bargaining outcomes fully eliminate politically induced regulatory risk. P...

Lactose Induces Phenotypic and Functional Changes of Neutrophils and Macrophages to Alleviate Acute Pancreatitis in Mice

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Li-Long Pan

2018-04-01

Full Text Available Acute pancreatitis (AP is one common clinical acute abdominal disease, for which specific pharmacological or nutritional therapies remain elusive. Lactose, a macronutrient and an inducer of host innate immune responses, possesses immune modulatory functions. The current study aimed to investigate potential modulatory effects of lactose and the interplay between the nutrient and pancreatic immunity during experimentally induced AP in mice. We found that either prophylactic or therapeutic treatment of lactose time-dependently reduced the severity of AP, as evidenced by reduced pancreatic edema, serum amylase levels, and pancreatic myeloperoxidase activities, as well as by histological examination of pancreatic damage. Overall, lactose promoted a regulatory cytokine milieu in the pancreas and reduced infiltration of inflammatory neutrophils and macrophages. On acinar cells, lactose was able to suppress caerulein-induced inflammatory signaling pathways and to suppress chemoattractant tumor necrosis factor (TNF-α and monocyte chemotactic protein-1 production. Additionally, lactose acted on pancreas-infiltrated macrophages, increasing interleukin-10 and decreasing tumor necrosis factor alpha production. Notably, lactose treatment reversed AP-associated infiltration of activated neutrophils. Last, the effect of lactose on neutrophil infiltration was mimicked by a galectin-3 antagonist, suggesting a potential endogenous target of lactose. Together, the current study demonstrates an immune regulatory effect of lactose to alleviate AP and suggests its potential as a convenient, value-added therapeutic macronutrient to control AP, and lower the risk of its systemic complications.

Inhibitors Alter the Stochasticity of Regulatory Proteins to Force Cells to Switch to the Other State in the Bistable System.

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Jhang, Wun-Sin; Lo, Shih-Chiang; Yeh, Chen-Chao; Shu, Che-Chi

2017-06-30

The cellular behaviors under the control of genetic circuits are subject to stochastic fluctuations, or noise. The stochasticity in gene regulation, far from a nuisance, has been gradually appreciated for its unusual function in cellular activities. In this work, with Chemical Master Equation (CME), we discovered that the addition of inhibitors altered the stochasticity of regulatory proteins. For a bistable system of a mutually inhibitory network, such a change of noise led to the migration of cells in the bimodal distribution. We proposed that the consumption of regulatory protein caused by the addition of inhibitor is not the only reason for pushing cells to the specific state; the change of the intracellular stochasticity is also the main cause for the redistribution. For the level of the inhibitor capable of driving 99% of cells, if there is no consumption of regulatory protein, 88% of cells were guided to the specific state. It implied that cells were pushed, by the inhibitor, to the specific state due to the change of stochasticity.

Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

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Wu Yihe

2012-04-01

Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase - Pi-dependent pyrophosphorylase from bacteria

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Burnell Jim N

2010-01-01

Full Text Available Abstract Background Phosphoenolpyruvate synthetase (PEPS; EC 2.7.9.2 catalyzes the synthesis of phosphoenolpyruvate from pyruvate in Escherichia coli when cells are grown on a three carbon source. It also catalyses the anabolic conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis. A bioinformatics search conducted following the successful cloning and expression of maize leaf pyruvate, orthophosphate dikinase regulatory protein (PDRP revealed the presence of PDRP homologs in more than 300 bacterial species; the PDRP homolog was identified as DUF299. Results This paper describes the cloning and expression of both PEPS and DUF299 from E. coli and establishes that E. coli DUF299 catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PEPS. Conclusion This paper represents the first report of a bifunctional regulatory enzyme catalysing an ADP-dependent phosphorylation and a Pi-dependent pyrophosphorylation reaction in bacteria.

Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

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Marius Schild

2016-01-01

Full Text Available Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n=19 and untrained (SED, n=17 individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

Acute phase proteins as diagnostic markers in horses with colic

DEFF Research Database (Denmark)

Pihl, Tina; Scheepers, Elrien; Sanz, Macarena

2016-01-01

Objective – To investigate the diagnostic potential of the concentrations of acute-phase proteins serum amyloid A (SAA), haptoglobin (Hp), and fibrinogen in blood and peritoneal fluid (PF) for differentiating horses within flammatory colic (entero-colitis and peritonitis) from those with surgical...... of the model to correctly differentiate inflammatory from surgical colic was 86% determined as area under the receiver operating characteristic curve. Adding blood parameters (WBC, PCV, total plasma protein, lactate, SAA, Hp, and fibrinogen concentrations) to the logistic model based on clinical parameters...... revealed that only WBC and SAA and fibrinogen concentrations improved the model. With SAA included in the model no additional blood parameters improved the model, and the final model had an area under the curve of 90%. Addition of PF parameters (hemolysis, total protein concentration, WBC, SAA, or Hp...

Chronological protein synthesis in regenerating rat liver.

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He, Jinjun; Hao, Shuai; Zhang, Hao; Guo, Fuzheng; Huang, Lingyun; Xiao, Xueyuan; He, Dacheng

2015-07-01

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.

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Chen, Hanqing; Shen, Feng; Sherban, Alex; Nocon, Allison; Li, Yu; Wang, Hua; Xu, Ming-Jiang; Rui, Xianliang; Han, Jinyan; Jiang, Bingbing; Lee, Donghwan; Li, Na; Keyhani-Nejad, Farnaz; Fan, Jian-Gao; Liu, Feng; Kamat, Amrita; Musi, Nicolas; Guarente, Leonard; Pacher, Pal; Gao, Bin; Zang, Mengwei

2018-02-19

Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. The dysregulation of SIRT1

mRNA Expression of Interferon Regulatory Factors during Acute Rejection of Liver Transplants in Patients with Autoimmune Hepatitis.

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Nasiri, M; Geramizadeh, B; Nabavizadeh, S H; Male-Hosseini, S A; Karimi, M H; Saadat, I

2018-01-01

Interferon regulatory factors (IRFs) can play a critical role in the regulation of many facets of innate and adaptive immune responses through transcriptional activation of type I interferons, other proinflammatory cytokines, and chemokines. However, their roles in transplantation immunity still remain to be elucidated. To evaluate the time course of mRNA expression of all 9 members of IRFs family of transcription factors during liver allograft acute rejection. Blood samples of 19 patients with autoimmune hepatitis receiving liver transplants were collected on days 1, 3, 5, and 7 post-transplantation. The patients were followed for 6 months after transplantation and divided into two groups of acute rejection (AR) (n=4) and non-acute rejection (non-AR) (n=15). All of the studied transcription factors were down-regulated in AR-group on days 3, 5, and 7 post-transplantation compared to non-AR group. The mean±SEM IRF5 on day 7 post-transplantation was significantly (p=0.005) lower in AR-group than in non-AR group (0.7±0.21 vs . 1.91±0.27, respectively); expression of other IRFs family members was not significantly different between the two groups on days 3, 5, and 7 post-transplantation. IRF5 may have an important role during the acute rejection of liver transplants.

Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

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Rotwein Peter

2008-04-01

Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

Acute phase and transport protein synthesis in simulated infection in undernourished men using uniformly labelled Spirulina Platensis

International Nuclear Information System (INIS)

Reeds, P.J.; Opekun, A.; Jahoor, F.; Wong, W.W.; Klein, P.D.

1994-01-01

Although it has been known for many years that injury and infection lead to body nitrogen loss, the reason has remained obscure. In this paper, we develop the argument that the processes that are activated during infection demand the provision of specific amino acids which have to be supplied from body protein. In particular, we show that the positive acute phase proteins are very rich in the aromatic amino acids and the exaggerated use of these amino acids (phenylalanine, tryptophan and tyrosine) in acute phase protein synthesis lead to an endogenous ''amino acid imbalance'' which restricts the use of other amino acids for tissue protein synthesis. Minimally invasive protocols, involving the administration of 15 N and 13 C-labelled amino acids for studying whole body nitrogen turnover, amino acid oxidation and plasma protein synthesis are described. (author). 22 refs, 3 tabs

Stress-dependent changes in neuroinflammatory markers observed after common laboratory stressors are not seen following acute social defeat of the Sprague Dawley rat.

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Hueston, Cara M; Barnum, Christopher J; Eberle, Jaime A; Ferraioli, Frank J; Buck, Hollin M; Deak, Terrence

2011-08-03

Exposure to acute stress has been shown to increase the expression of pro-inflammatory cytokines in brain, blood and peripheral organs. However, the nature of the inflammatory response evoked by acute stress varies depending on the stressor used and species examined. The goal of the following series of studies was to characterize the consequences of social defeat in the Sprague Dawley (SD) rat using three different social defeat paradigms. In Experiments 1 and 2, adult male SD rats were exposed to a typical acute resident-intruder paradigm of social defeat (60 min) by placement into the home cage of a larger, aggressive Long Evans rat and brain tissue was collected at multiple time points for analysis of IL-1β protein and gene expression changes in the PVN, BNST and adrenal glands. In subsequent experiments, rats were exposed to once daily social defeat for 7 or 21 days (Experiment 3) or housed continuously with an aggressive partner (separated by a partition) for 7 days (Experiment 4) to assess the impact of chronic social stress on inflammatory measures. Despite the fact that social defeat produced a comparable corticosterone response as other stressors (restraint, forced swim and footshock; Experiment 5), acute social defeat did not affect inflammatory measures. A small but reliable increase in IL-1 gene expression was observed immediately after the 7th exposure to social defeat, while other inflammatory measures were unaffected. In contrast, restraint, forced swim and footshock all significantly increased IL-1 gene expression in the PVN; other inflammatory factors (IL-6, cox-2) were unaffected in this structure. These findings provide a comprehensive evaluation of stress-dependent inflammatory changes in the SD rat, raising intriguing questions regarding the features of the stress challenge that may be predictive of stress-dependent neuroinflammation. Copyright © 2011 Elsevier Inc. All rights reserved.

Use of activated protein C has no avail in the early phase of acute pancreatitis.

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Akay, Sinan; Ozutemiz, Omer; Yenisey, Cigdem; Simsek, Nilufer Genc; Yuce, Gul; Batur, Yucel

2008-01-01

Sepsis and acute pancreatitis have similar pathogenetic mechanisms that have been implicated in the progression of multiple organ failure. Drotrecogin alfa, an analogue of endogenous protein C, reduces mortality in clinical sepsis. Our objective was to evaluate the early therapeutic effects of activated protein C (APC) in a rat model of acute necrotizing pancreatitis. Acute necrotizing pancreatitis was induced by intraductal injection of 5% Na taurocholate. Hourly bolus injections of saline or recombinant human APC (drotrecogin alfa) was commenced via femoral venous catheter four hours after the induction of acute pancreatitis. The experiment was terminated nine hours after pancreatitis induction. Animals in group one (n=20) had a sham operation while animals in group two (n=20) received saline and animals in group three (n=20) received drotrecogin alfa boluses after acute pancreatitis induction. Pancreatic tissue for histopathologic scores and myeloperoxidase, glutathione reductase, glutathione peroxidase, and catalase activities were collected, and blood for serum amylase, urea, creatinine, and interleukin-6 measurements was withdrawn. Serum amylase activity was significantly lower in the APC treated group than the untreated group (17,435+/-432 U/L vs. 27,426+/-118 U/L, respectively). While the serum interleukin-6 concentration in the APC untreated group was significantly lower than the treated group (970+/-323 pg/mL vs. 330+/-368 pg/mL, respectively). In the early phase of acute pancreatitis, drotrecogin alfa treatment did not result in a significant improvement in oxidative and inflammatory parameters or renal functions.

Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

NARCIS (Netherlands)

Nielsen, B.H.; Jacobsen, S.; Andersen, P.H.; Niewold, T.A.; Heegaard, P.M.H.

2004-01-01

The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were

The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

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Ratti Antonia

2007-12-01

Full Text Available Abstract Background CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR suggests a role in post-transcriptional regulation of CDK5R1 expression. Results The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs. The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. Conclusion Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.

ACUTE PHASE PROTEIN INCREASE IN HIGH ALTITUDE MOUNTAINEERS

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Tolga Saka

Full Text Available ABSTRACT Introduction: Many middle-aged Turks go hiking in mountains to breathe some fresh air or to maintain fitness. Objective: This study investigated the effects of regular high altitude mountain climbing on the metabolic and hematological responses of mountaineers. Methods: Hematological and biochemical parameters were studied, as well as some hormonal values of 21 mountaineers and 16 healthy age-matched sedentary volunteers. Results: The neutrophil to lymphocyte ratio (NLR was significantly lower (p<0.04 in mountaineers compared with the sedentary group. Total protein (p<0.001 and albumin (p<0.001 were lower, while the levels of ferritin (p<0.04, creatine (p<0.03 and creatine phosphokinase (p<0.01 were higher in mountaineers. Other hematological and biochemical parameters, i.e., erythrocytes, leukocytes, hemoglobin and hematocrit, did not change significantly. Conclusion: Our results show that regular exposure to high altitude increased the serum levels of some acute phase proteins with anti-inflammatory properties.

Structural basis of divergent cyclin-dependent kinase activation by Spy1/RINGO proteins

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McGrath, Denise A.; Fifield, Bre-Anne; Marceau, Aimee H.; Tripathi, Sarvind; Porter, Lisa A.; Rubin, Seth M. (UCSC); (Windsor)

2017-06-30

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changes to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.

Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus

NARCIS (Netherlands)

Sedelnikova, S.E.; Smits, S.H.J.; Leonard, P.M.; Brinkman, A.B.; Oost, van der J.; Rafferty, J.B.

2001-01-01

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in

Protein-energy malnutrition developing after global brain ischemia induces an atypical acute-phase response and hinders expression of GAP-43.

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Smith, Shari E; Figley, Sarah A; Schreyer, David J; Paterson, Phyllis G

2014-01-01

Protein-energy malnutrition (PEM) is a common post-stroke problem. PEM can independently induce a systemic acute-phase response, and pre-existing malnutrition can exacerbate neuroinflammation induced by brain ischemia. In contrast, the effects of PEM developing in the post-ischemic period have not been studied. Since excessive inflammation can impede brain remodeling, we investigated the effects of post-ischemic malnutrition on neuroinflammation, the acute-phase reaction, and neuroplasticity-related proteins. Male, Sprague-Dawley rats were exposed to global forebrain ischemia using the 2-vessel occlusion model or sham surgery. The sham rats were assigned to control diet (18% protein) on day 3 after surgery, whereas the rats exposed to global ischemia were assigned to either control diet or a low protein (PEM, 2% protein) diet. Post-ischemic PEM decreased growth associated protein-43, synaptophysin and synaptosomal-associated protein-25 immunofluorescence within the hippocampal CA3 mossy fiber terminals on day 21, whereas the glial response in the hippocampal CA1 and CA3 subregions was unaltered by PEM. No systemic acute-phase reaction attributable to global ischemia was detected in control diet-fed rats, as reflected by serum concentrations of alpha-2-macroglobulin, alpha-1-acid glycoprotein, haptoglobin, and albumin. Acute exposure to the PEM regimen after global brain ischemia caused an atypical acute-phase response. PEM decreased the serum concentrations of albumin and haptoglobin on day 5, with the decreases sustained to day 21. Serum alpha-2-macroglobulin concentrations were significantly higher in malnourished rats on day 21. This provides the first direct evidence that PEM developing after brain ischemia exerts wide-ranging effects on mechanisms important to stroke recovery.

P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia

NARCIS (Netherlands)

de Vries, EGE; van Putten, WLJ; Verdonck, LF; Ossenkoppele, GJ; Verhoef, GEG; Vellenga, E

Despite treatment with intensive chemotherapy, a considerable number of patients with acute myeloid leukemia (AML) die from their disease due to the occurrence of resistance. Overexpression of the transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP) 1 has been identified

Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl-XL Protein▿

Science.gov (United States)

Tan, Ying-Xim; Tan, Timothy H. P.; Lee, Marvin J.-R.; Tham, Puay-Yoke; Gunalan, Vithiagaran; Druce, Julian; Birch, Chris; Catton, Mike; Fu, Nai Yang; Yu, Victor C.; Tan, Yee-Joo

2007-01-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments. PMID:17428862

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

Science.gov (United States)

Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-01-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

[Examination of acute phase proteins concentrations in children with allergic rhinitis].

Science.gov (United States)

Steiner, Iwona; Sobieska, Magdalena; Pucher, Beata; Grzegorowski, Michał; Samborski, Włodzimierz

2006-01-01

Allergic rhinitis is an inflammatory disorder of upper respiratory tract (about 15 per cent of the population in industrialized countries suffer from this condition), characterized by frequent sneezing and a runny or stuffy nose sometimes accompanied by watery eyes. As the most common allergic condition, allergic rhinitis affects people of all ages. Boys are twice as likely to get allergic rhinitis than girls. The median age of onset of the condition is 10 years old, meaning that equal numbers of children develop the condition before and after age 10. Symptoms usually appear in childhood first and then lessen by the age of 30 or 40. Seasonal allergic rhinitis usually results from tree, grass or weed pollen. With this type of rhinitis, symptoms will decrease with the arrival of cold weather. Perennial allergic rhinitis can cause year-round symptoms. This allergic reaction is the result of indoor irritants such as feathers, mold spores, animal dander (hair and skin shed by pets) or dust mites. It is often aggravated by a food allergy, the most common being an allergy to milk. Acute phase proteins (APP) belong to the most ancient part of the unspecific immunity and contribute markedly to the keeping of homeostasis. As much as 30 various proteins are for the moment regarded as APP. Being multifunctional regulators and effectors APP stay in multiple relations to practically all types of cells and molecules. Among APP following functional groups may be described: transport proteins (transferrin, ceruloplasmin and haptoglobin), clotting factors (fibrinogen), antiproteases (alpha1-antitrypsin, alpha1-antichymotrypsin, alpha2-macroglobulin), complement components (C3, C4) and several proteins of hardly known function, like C-reactive protein (CRP), serum amyloid A, acid alpha1-glycoprotein (AGP) and others. From a group of 32 children, aged from 5 to 14 years, with symptoms of seasonal allergic rhinitis, and from a control group of 10 healthy children sex and age matched

Regulatory T Cells in Post-stroke Immune Homeostasis.

Science.gov (United States)

Liesz, Arthur; Kleinschnitz, Christoph

2016-08-01

The secondary neuroinflammatory response has come into focus of experimental stroke research. Immunological mechanisms after acute stroke are being investigated in the hope to identify novel and druggable pathways that contribute to secondary infarct growth after stroke. Among a variety of neuroimmunological events after acute brain ischemia, including microglial activation, brain leukocyte invasion, and secretion of pro-inflammatory factors, lymphocytes have been identified as the key leukocyte subpopulation driving the neuroinflammatory response and contributing to stroke outcome. Several studies have shown that pro-inflammatory lymphocyte subpopulations worsen stroke outcome and that inhibiting their invasion to the injured brain is neuroprotective. In contrast to the effector functions of pro-inflammatory lymphocytes, regulatory T cells (Treg) are critically involved in maintaining immune homeostasis and have been characterized as disease-limiting protective cells in several inflammatory conditions, particularly in primary inflammatory diseases of the central nervous system (CNS). However, due to the complex function of regulatory cells in immune homeostasis and disease, divergent findings have been described for the role of Treg in stroke models. Emerging evidence suggests that this discrepancy arises from potentially differing functions of Treg depending on the predominant site of action within the neurovascular unit and the surrounding inflammatory milieu. This article will provide a comprehensive review of current findings on Treg in brain ischemia models and discuss potential reasons for the observed discrepancies.

Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila

DEFF Research Database (Denmark)

Bantignies, Frédéric; Roure, Virginie; Comet, Itys

2011-01-01

In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes...... of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related Drosophila virilis species and they are part of a large gene...

Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

Science.gov (United States)

Kalveram, Birte; Ikegami, Tetsuro

2013-04-01

Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

DNA-dependent protein kinase (DAN-PK), a key enzyme in the re-ligation of DNA double-strand breaks

International Nuclear Information System (INIS)

Hennequin, C.; Averbeck, D.

1999-01-01

Repair pathways of DNA are now defined and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEH) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEj implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridization in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catalytic subunit (DNA-PK cs ) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immuno-deficient (scid) mice are deficient in DNA-PK cs : this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylate Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival. (authors)

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

Directory of Open Access Journals (Sweden)

Kristina Kirschner

2015-03-01

Full Text Available The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage and chronically activated (in senescent or pro-apoptotic conditions p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

Energy Technology Data Exchange (ETDEWEB)

Takaoka, Yuki [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Kawamoto, Seiji, E-mail: skawa@hiroshima-u.ac.jp [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Katayama, Akiko [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Nakano, Toshiaki [Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (China); Yamanaka, Yasushi; Takahashi, Miki [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Shimada, Yayoi; Chiang, Kuei-Chen [Kazusa Institute for Drug Discovery, Josai International University, Kisarazu (Japan); Ohmori, Naoya [Kazusa Institute for Drug Discovery, Josai International University, Kisarazu (Japan); Faculty of Nursing, Josai International University, Togane (Japan); Aki, Tsunehiro [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Goto, Takeshi; Sato, Shuji [Kazusa Institute for Drug Discovery, Josai International University, Kisarazu (Japan); Faculty of Nursing, Josai International University, Togane (Japan); Goto, Shigeru [Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (China); Iwao Hospital, Yufuin (Japan); Chen, Chao-Long [Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (China); Ono, Kazuhisa [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan)

2013-02-08

Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4{sup +}Foxp3{sup +} Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

International Nuclear Information System (INIS)

Takaoka, Yuki; Kawamoto, Seiji; Katayama, Akiko; Nakano, Toshiaki; Yamanaka, Yasushi; Takahashi, Miki; Shimada, Yayoi; Chiang, Kuei-Chen; Ohmori, Naoya; Aki, Tsunehiro; Goto, Takeshi; Sato, Shuji; Goto, Shigeru; Chen, Chao-Long; Ono, Kazuhisa

2013-01-01

Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4 + Foxp3 + Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings

PGC-1α and exercise intensity dependent adaptations in mouse skeletal muscle

DEFF Research Database (Denmark)

Brandt, Nina; Dethlefsen, Maja Munk; Bangsbo, Jens

2017-01-01

The aim of the present study was to examine the role of PGC-1α in intensity dependent exercise and exercise training-induced metabolic adaptations in mouse skeletal muscle. Whole body PGC-1α knockout (KO) and littermate wildtype (WT) mice performed a single treadmill running bout at either low...... intensity dependent increases in LC3I and LC3II protein and intensity independent decrease in p62 protein in skeletal muscle late in recovery and increased LC3II with exercise training independent of exercise intensity and volume in WT mice. Furthermore, acute exercise and exercise training did not increase...... LC3I and LC3II protein in PGC-1α KO. In addition, exercise-induced mRNA responses of PGC-1α isoforms were intensity dependent. In conclusion, these findings indicate that exercise intensity affected autophagy markers differently in skeletal muscle and suggest that PGC-1α regulates both acute...

COMK ENCODES THE COMPETENCE TRANSCRIPTION FACTOR, THE KEY REGULATORY PROTEIN FOR COMPETENCE DEVELOPMENT IN BACILLUS-SUBTILIS

NARCIS (Netherlands)

VANSINDEREN, D; LUTTINGER, A; KONG, LY; DUBNAU, D; VENEMA, G; HAMOEN, L

comK is a positive autoregulatory gene occupying a central position in the com petence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late

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

Directory of Open Access Journals (Sweden)

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

Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia

NARCIS (Netherlands)

A. Holleman (Amy); M.L. den Boer (Monique); K.M. Kazemier (Karin); H.B. Beverloo (Berna); A.R.M. von Bergh (Anne); G.E. Janka-Schaub (Gritta); R. Pieters (Rob)

2005-01-01

textabstractDrug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7,

WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System.

Science.gov (United States)

Herrou, Julien; Czyż, Daniel M; Willett, Jonathan W; Kim, Hye-Sook; Chhor, Gekleng; Babnigg, Gyorgy; Kim, Youngchang; Crosson, Sean

2016-04-01

The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes

Impact of protein uptake and degradation on recombinant protein secretion in yeast

DEFF Research Database (Denmark)

Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

2014-01-01

Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

Cyclin dependent kinase inhibitor 2A/B gene deletions are markers of poor prognosis in Indian children with acute lymphoblastic leukemia.

Science.gov (United States)

Agarwal, Manisha; Bakhshi, Sameer; Dwivedi, Sadanand N; Kabra, Madhulika; Shukla, Rashmi; Seth, Rachna

2018-06-01

Cyclin dependent kinase inhibitor 2A/B (CDKN2A/B) genes are implicated in many malignancies including acute lymphoblastic leukemia (ALL). These tumor suppressor genes, with a key regulatory role in cell cycle are located on chromosome 9p21.3. Previous studies involving CDKN2A/B gene deletions have shown mixed associations with survival outcome in childhood ALL. Hundred and four newly diagnosed children with ALL (1-14 years) were enrolled in this study. Genomic DNA from pretreatment bone marrow/peripheral blood samples of these children was investigated for copy number alterations in CDKN2A/B genes using multiplex ligation dependent probe amplification assay. Immunophenotype subtyping and cytogenetic and molecular analysis of ALL was performed at start of induction chemotherapy in all children. Children were monitored for response to prednisolone (Day 8), complete morphological remission, and minimal residual disease at the end of induction. The minimum postinduction follow-up period was 6 months. CDKN2A/B deletions were seen in 19.8% (18/91) of B lineage acute lymphoblastic leukemia (B-ALL) and 38.5% (5/13) of T lineage acute lymphoblastic leukemia (T-ALL). Monoallelic CDKN2A/B deletions were found in 61.1% of total deletions in B-ALL while all the children with T-ALL harbored biallelic deletions. The prevalence of CDKN2A/B gene deletions was found to be significantly higher in older children (P = 0.002), in those with higher leukocyte count (P = 0.037), and in National Cancer Institute high risk group patients (P = 0.001) in the B-ALL subgroup. Hazard ratio was significantly high for children with CDKN2A/B deletions in total cohort (P = 0.004). Children with CDKN2A/B deletion had significantly lesser event free survival (P = 0.03). CDKN2A/B deletions were significantly more prevalent in T-ALL subgroup and were found to have higher hazard ratio and lesser event free survival in total cohort in our study. © 2018 Wiley Periodicals, Inc.

Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6

Energy Technology Data Exchange (ETDEWEB)

Penrose, Harrison; Heller, Sandra; Cable, Chloe [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States); Makboul, Rania [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States); Pathology Department, Assiut University, Assiut (Egypt); Chadalawada, Gita; Chen, Ying [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States); Crawford, Susan E. [Department of Pathology, Saint Louis University School of Medicine, 1402 South Grand Blvd, Saint Louis, MO 63104 (United States); Savkovic, Suzana D., E-mail: ssavkovi@tulane.edu [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States)

2016-01-15

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.

Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6

International Nuclear Information System (INIS)

Penrose, Harrison; Heller, Sandra; Cable, Chloe; Makboul, Rania; Chadalawada, Gita; Chen, Ying; Crawford, Susan E.; Savkovic, Suzana D.

2016-01-01

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.

Regulatory network of GATA3 in pediatric acute lymphoblastic leukemia.

Science.gov (United States)

Hou, Qianqian; Liao, Fei; Zhang, Shouyue; Zhang, Duyu; Zhang, Yan; Zhou, Xueyan; Xia, Xuyang; Ye, Yuanxin; Yang, Hanshuo; Li, Zhaozhi; Wang, Leiming; Wang, Xi; Ma, Zhigui; Zhu, Yiping; Ouyang, Liang; Wang, Yuelan; Zhang, Hui; Yang, Li; Xu, Heng; Shu, Yang

2017-05-30

GATA3 polymorphisms were reported to be significantly associated with susceptibility of pediatric B-lineage acute lymphoblastic leukemia (ALL), by impacting on GATA3 expression. We noticed that ALL-related GATA3 polymorphism located around in the tissue-specific enhancer, and significantly associated with GATA3 expression. Although the regulatory network of GATA3 has been well reported in T cells, the functional status of GATA3 is poorly understood in B-ALL. We thus conducted genome-wide gene expression association analyses to reveal expression associated genes and pathways in nine independent B-ALL patient cohorts. In B-ALL patients, 173 candidates were identified to be significantly associated with GATA3 expression, including some reported GATA3-related genes (e.g., ITM2A) and well-known tumor-related genes (e.g., STAT4). Some of the candidates exhibit tissue-specific and subtype-specific association with GATA3. Through overexpression and down-regulation of GATA3 in leukemia cell lines, several reported and novel GATA3 regulated genes were validated. Moreover, association of GATA3 expression and its targets can be impacted by SNPs (e.g., rs4894953), which locate in the potential GATA3 binding motif. Our findings suggest that GATA3 may be involved in multiple tumor-related pathways (e.g., STAT/JAK pathway) in B-ALL to impact leukemogenesis through epigenetic regulation.

Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function

NARCIS (Netherlands)

Sander, L.E.; Sackett, S.D.; Dierssen, U.; Beraza, N.; Linke, R.; Müller, M.R.; Blander, J.M.; Tacke, F.; Trautwein, C.

2010-01-01

Acute-phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and antiinflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined.

Hepatic acute phase proteins control innate immune responses during infection by promoting myeloid derived suppressor cell function

NARCIS (Netherlands)

Sander, Leif E.; Dutton Sackett, Sara; Dierssen, Uta; Beraza, Naiara; Linke, Reinhold P.; Muller, Michael; Magarian Blander, Julie; Tacke, Frank; Trautwein, Christian

2010-01-01

Acute phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and anti-inflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined.

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

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Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

Impact of Diet Supplemented by Coconut Milk on Corticosterone and Acute Phase Protein Level under High Stocking Density

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Majid SHAKERI

2016-05-01

Full Text Available The purpose of this study was to investigate effects of coconut milk supplementation on corticosterone and acute phase protein level under high stocking density. A total 300 Cobb 500 male chicks were placed in cages and stocked as 10 birds/cage (normal stocking density and 15 birds/cage (high stocking density. The treatments were as (i control diet and stocked at 10 and 15 birds/cage (ii control diet + 3% coconut milk from 1-42 day and stocked at 10 and 15 birds/cage (iii control diet + 5% coconut milk from 1-42 day and stocked at 10 and 15 birds/cage. On day 42, 20 birds per treatment were slaughtered to collect blood samples. The results showed higher level of corticosterone and acute phase protein level in control diet compare to other supplemented diets with coconut milk. In conclusion, coconut milk decreased the level of corticosterone and acute phase protein when chicks were subjected to high stocking density.

Protein (multi-)location prediction: using location inter-dependencies in a probabilistic framework

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2014-01-01

Motivation Knowing the location of a protein within the cell is important for understanding its function, role in biological processes, and potential use as a drug target. Much progress has been made in developing computational methods that predict single locations for proteins. Most such methods are based on the over-simplifying assumption that proteins localize to a single location. However, it has been shown that proteins localize to multiple locations. While a few recent systems attempt to predict multiple locations of proteins, their performance leaves much room for improvement. Moreover, they typically treat locations as independent and do not attempt to utilize possible inter-dependencies among locations. Our hypothesis is that directly incorporating inter-dependencies among locations into both the classifier-learning and the prediction process can improve location prediction performance. Results We present a new method and a preliminary system we have developed that directly incorporates inter-dependencies among locations into the location-prediction process of multiply-localized proteins. Our method is based on a collection of Bayesian network classifiers, where each classifier is used to predict a single location. Learning the structure of each Bayesian network classifier takes into account inter-dependencies among locations, and the prediction process uses estimates involving multiple locations. We evaluate our system on a dataset of single- and multi-localized proteins (the most comprehensive protein multi-localization dataset currently available, derived from the DBMLoc dataset). Our results, obtained by incorporating inter-dependencies, are significantly higher than those obtained by classifiers that do not use inter-dependencies. The performance of our system on multi-localized proteins is comparable to a top performing system (YLoc+), without being restricted only to location-combinations present in the training set. PMID:24646119

Protein (multi-)location prediction: using location inter-dependencies in a probabilistic framework.

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Simha, Ramanuja; Shatkay, Hagit

2014-03-19

Knowing the location of a protein within the cell is important for understanding its function, role in biological processes, and potential use as a drug target. Much progress has been made in developing computational methods that predict single locations for proteins. Most such methods are based on the over-simplifying assumption that proteins localize to a single location. However, it has been shown that proteins localize to multiple locations. While a few recent systems attempt to predict multiple locations of proteins, their performance leaves much room for improvement. Moreover, they typically treat locations as independent and do not attempt to utilize possible inter-dependencies among locations. Our hypothesis is that directly incorporating inter-dependencies among locations into both the classifier-learning and the prediction process can improve location prediction performance. We present a new method and a preliminary system we have developed that directly incorporates inter-dependencies among locations into the location-prediction process of multiply-localized proteins. Our method is based on a collection of Bayesian network classifiers, where each classifier is used to predict a single location. Learning the structure of each Bayesian network classifier takes into account inter-dependencies among locations, and the prediction process uses estimates involving multiple locations. We evaluate our system on a dataset of single- and multi-localized proteins (the most comprehensive protein multi-localization dataset currently available, derived from the DBMLoc dataset). Our results, obtained by incorporating inter-dependencies, are significantly higher than those obtained by classifiers that do not use inter-dependencies. The performance of our system on multi-localized proteins is comparable to a top performing system (YLoc+), without being restricted only to location-combinations present in the training set.

Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

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Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

2014-03-01

Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. Copyright © 2014 Elsevier Inc. All rights reserved.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

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Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

2013-12-17

Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

Time-dependent sorption of two novel fungicides in soils within a regulatory framework.

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Gulkowska, Anna; Buerge, Ignaz J; Poiger, Thomas; Kasteel, Roy

2016-12-01

Convincing experimental evidence suggests increased sorption of pesticides on soil over time, which, so far, has not been considered in the regulatory assessment of leaching to groundwater. Recently, Beulke and van Beinum (2012) proposed a guidance on how to conduct, analyse and use time-dependent sorption studies in pesticide registration. The applicability of the recommended experimental set-up and fitting procedure was examined for two fungicides, penflufen and fluxapyroxad, in four soils during a 170 day incubation experiment. The apparent distribution coefficient increased by a factor of 2.5-4.5 for penflufen and by a factor of 2.5-2.8 for fluxapyroxad. The recommended two-site, one-rate sorption model adequately described measurements of total mass and liquid phase concentration in the calcium chloride suspension and the calculated apparent distribution coefficient, passing all prescribed quality criteria for model fit and parameter reliability. The guidance is technically mature regarding the experimental set-up and parameterisation of the sorption model for the two moderately mobile and relatively persistent fungicides under investigation. These parameters can be used for transport modelling in soil, thereby recognising the existence of the experimentally observed, but in the regulatory leaching assessment of pesticides not yet routinely considered phenomenon of time-dependent sorption. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity.

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Recarte-Pelz, Pedro; Tàssies, Dolors; Espinosa, Gerard; Hurtado, Begoña; Sala, Núria; Cervera, Ricard; Reverter, Joan Carles; de Frutos, Pablo García

2013-03-12

Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population. Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples. Plasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients. The present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE.

Mutational robustness of gene regulatory networks.

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Aalt D J van Dijk

Full Text Available Mutational robustness of gene regulatory networks refers to their ability to generate constant biological output upon mutations that change network structure. Such networks contain regulatory interactions (transcription factor-target gene interactions but often also protein-protein interactions between transcription factors. Using computational modeling, we study factors that influence robustness and we infer several network properties governing it. These include the type of mutation, i.e. whether a regulatory interaction or a protein-protein interaction is mutated, and in the case of mutation of a regulatory interaction, the sign of the interaction (activating vs. repressive. In addition, we analyze the effect of combinations of mutations and we compare networks containing monomeric with those containing dimeric transcription factors. Our results are consistent with available data on biological networks, for example based on evolutionary conservation of network features. As a novel and remarkable property, we predict that networks are more robust against mutations in monomer than in dimer transcription factors, a prediction for which analysis of conservation of DNA binding residues in monomeric vs. dimeric transcription factors provides indirect evidence.

Structural study of surfactant-dependent interaction with protein

Energy Technology Data Exchange (ETDEWEB)

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

2015-06-24

Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

Cis-regulatory RNA elements that regulate specialized ribosome activity.

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Xue, Shifeng; Barna, Maria

2015-01-01

Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5'UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.

The evolutionary capacitor HSP90 buffers the regulatory effects of mammalian endogenous retroviruses.

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Hummel, Barbara; Hansen, Erik C; Yoveva, Aneliya; Aprile-Garcia, Fernando; Hussong, Rebecca; Sawarkar, Ritwick

2017-03-01

Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

C-reactive protein testing in patients with acute rhinosinusitis leads to a reduction in antibiotic use

DEFF Research Database (Denmark)

Llor, Carl; Bjerrum, Lars; Arranz, Javier

2012-01-01

OBJECTIVE: To evaluate the effect of C-reactive protein (CRP) testing on the antibiotic prescribing in patients with acute rhinosinusitis. METHODS: Audit-based study carried out in primary care centres in Spain. GPs registered episodes of rhinosinusitis during 3-week period before and after...... an intervention. Two types of intervention were considered: full intervention group (FIG) consisting in individual feedback based on results from the first registry, courses in rational antibiotic prescribing, guidelines, patient information leaflets, workshops on rapid tests and use of the CRP test. GPs...... in the partial intervention group (PIG) underwent all the above intervention except for the workshop and they did not have access to CRP. Multilevel logistic regression analysis was performed considering the prescription of antibiotics as the dependent variable. RESULTS: Two hundred and ten physicians were...

Emergence of the acute-phase protein hemopexin in jawed vertebrates

OpenAIRE

Dooley, Helen; Buckingham, E. Bryan; Criscitiello, Michael F.; Flajnik, Martin F.

2010-01-01

When released from damaged erythrocytes free heme not only provides a source of iron for invading bacteria but is also highly toxic due to its ability to catalyze free radical formation. Hemopexin (Hx) binds free heme with very high affinity and thus protects against heme toxicity, sequesters heme from pathogens, and helps conserve valuable iron. Hx is also an acute-phase serum protein (APP), whose expression is induced by inflammation. To date Hx has been identified as far back in phylogeny ...

NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

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Habjan, Matthias; Pichlmair, Andreas; Elliott, Richard M; Overby, Anna K; Glatter, Timo; Gstaiger, Matthias; Superti-Furga, Giulio; Unger, Hermann; Weber, Friedemann

2009-05-01

Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

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Sato, Yuma; Watanabe, Shumpei; Fukuda, Yoshinari; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-03-15

Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV. IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

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Tao Zhang

2015-11-01

Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

Light-dependent roles of the G-protein α subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei

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Kubicek Christian P

2009-09-01

Full Text Available Abstract Background The filamentous ascomycete Hypocrea jecorina (anamorph Trichoderma reesei is primarily known for its efficient enzymatic machinery that it utilizes to decompose cellulosic substrates. Nevertheless, the nature and transmission of the signals initiating and modulating this machinery are largely unknown. Heterotrimeric G-protein signaling represents one of the best studied signal transduction pathways in fungi. Results Analysis of the regulatory targets of the G-protein α subunit GNA1 in H. jecorina revealed a carbon source and light-dependent role in signal transduction. Deletion of gna1 led to significantly decreased biomass formation in darkness in submersed culture but had only minor effects on morphology and hyphal apical extension rates on solid medium. Cellulase gene transcription was abolished in Δgna1 on cellulose in light and enhanced in darkness. However, analysis of strains expressing a constitutively activated GNA1 revealed that GNA1 does not transmit the essential inducing signal. Instead, it relates a modulating signal with light-dependent significance, since induction still required the presence of an inducer. We show that regulation of transcription and activity of GNA1 involves a carbon source-dependent feedback cycle. Additionally we found a function of GNA1 in hydrophobin regulation as well as effects on conidiation and tolerance of osmotic and oxidative stress. Conclusion We conclude that GNA1 transmits a signal the physiological relevance of which is dependent on both the carbon source as well as the light status. The widespread consequences of mutations in GNA1 indicate a broad function of this Gα subunit in appropriation of intracellular resources to environmental (especially nutritional conditions.

Partial regulatory T cell depletion prior to acute feline immunodeficiency virus infection does not alter disease pathogenesis.

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S Rochelle Mikkelsen

2011-02-01

Full Text Available Feline immunodeficiency virus (FIV infection in cats follows a disease course similar to HIV-1, including a short acute phase characterized by high viremia, and a prolonged asymptomatic phase characterized by low viremia and generalized immune dysfunction. CD4(+CD25(hiFoxP3(+ immunosuppressive regulatory T (Treg cells have been implicated as a possible cause of immune dysfunction during FIV and HIV-1 infection, as they are capable of modulating virus-specific and inflammatory immune responses. Additionally, the immunosuppressive capacity of feline Treg cells has been shown to be increased during FIV infection. We have previously shown that transient in vivo Treg cell depletion during asymptomatic FIV infection reveals FIV-specific immune responses suppressed by Treg cells. In this study, we sought to determine the immunological influence of Treg cells during acute FIV infection. We asked whether Treg cell depletion prior to infection with the highly pathogenic molecular clone FIV-C36 in cats could alter FIV pathogenesis. We report here that partial Treg cell depletion prior to FIV infection does not significantly change provirus, viremia, or CD4(+ T cell levels in blood and lymphoid tissues during the acute phase of disease. The effects of anti-CD25 mAb treatment are truncated in cats acutely infected with FIV-C36 as compared to chronically infected cats or FIV-naïve cats, as Treg cell levels were heightened in all treatment groups included in the study within two weeks post-FIV infection. Our findings suggest that the influence of Treg cell suppression during FIV pathogenesis is most prominent after Treg cells are activated in the environment of established FIV infection.

Partial regulatory T cell depletion prior to acute feline immunodeficiency virus infection does not alter disease pathogenesis.

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Mikkelsen, S Rochelle; Long, Julie M; Zhang, Lin; Galemore, Erin R; VandeWoude, Sue; Dean, Gregg A

2011-02-25

Feline immunodeficiency virus (FIV) infection in cats follows a disease course similar to HIV-1, including a short acute phase characterized by high viremia, and a prolonged asymptomatic phase characterized by low viremia and generalized immune dysfunction. CD4(+)CD25(hi)FoxP3(+) immunosuppressive regulatory T (Treg) cells have been implicated as a possible cause of immune dysfunction during FIV and HIV-1 infection, as they are capable of modulating virus-specific and inflammatory immune responses. Additionally, the immunosuppressive capacity of feline Treg cells has been shown to be increased during FIV infection. We have previously shown that transient in vivo Treg cell depletion during asymptomatic FIV infection reveals FIV-specific immune responses suppressed by Treg cells. In this study, we sought to determine the immunological influence of Treg cells during acute FIV infection. We asked whether Treg cell depletion prior to infection with the highly pathogenic molecular clone FIV-C36 in cats could alter FIV pathogenesis. We report here that partial Treg cell depletion prior to FIV infection does not significantly change provirus, viremia, or CD4(+) T cell levels in blood and lymphoid tissues during the acute phase of disease. The effects of anti-CD25 mAb treatment are truncated in cats acutely infected with FIV-C36 as compared to chronically infected cats or FIV-naïve cats, as Treg cell levels were heightened in all treatment groups included in the study within two weeks post-FIV infection. Our findings suggest that the influence of Treg cell suppression during FIV pathogenesis is most prominent after Treg cells are activated in the environment of established FIV infection.

The onset of the progression of acute phase response mechanisms induced by extreme impacts can be followed by the decrease in blood levels of positive acute phase proteins.

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Larina, Olga; Bekker, Anna

Studies performed at space flights and earth-based simulation models detected the plasma indices of acute phase reaction (APR), i.e. the increase of APR cytokine mediators and alterations in the production of blood acute phase proteins (APP) at the initial stages of adaptation to altered gravity conditions. Acute phase response is the principal constituent of the functional activity of innate immunity system. Changes in plasma APPs contents are considered to serve the restoration of homeostasis state. According to trends of their concentration shifts at the evolving of acute phase reaction APPs are denoted as positive, neutral, or negative. Plasma concentrations of positive acute phase proteins α1-acid glycoprotein (α1-AGP), α1-antitrypsin (α1-AT), and neutral α2-macroglobulin (α2-M) were measured in human study at 12-hour antiorthostatic position (AOP) with 15° head down tilt and hypoxia experiments at 14% oxygen in pressure chamber. Both of these impacts were shown to produce alterations in the APP levels indicative for acute phase response. Nevertheless, in AOP experiment noticeable decrease in α1-AGP concentration occurred by hour 12, and even more pronounced decline of α1-AGP and α1-AT were found on hypoxia hours 12 and 36. Acute phase proteins α1-AGP and α2-M possess the features of proteinase inhibitors. This function is implemented by the formation of complexes with the molecules of proteolytic enzymes which subsequently are removed from the blood flow. Transient decrease in plasma concentrations of protease inhibitors on early phases of APR development was reported to result from the growth of plasma protease activity due to cathepsin release from activated leukocytes, which had not yet been compensated by enhanced APP synthesis. Being a carrier protein for positively charged and neutral substances, α1-AGP shows pronounced elevation in its blood content during APR development. As assumed, it is required for the transportation of the increased

IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

Science.gov (United States)

Wiertsema, Selma P; Corscadden, Karli J; Mowe, Eva N; Zhang, Guicheng; Vijayasekaran, Shyan; Coates, Harvey L; Mitchell, Timothy J; Thomas, Wayne R; Richmond, Peter C; Kirkham, Lea-Ann S

2012-01-01

Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply) and 3 NTHi (P4, P6 and PD) proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion) and 63 healthy age-matched controls, using a newly developed multiplex bead assay. Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

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Selma P Wiertsema

Full Text Available BACKGROUND: Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. METHODS: We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply and 3 NTHi (P4, P6 and PD proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion and 63 healthy age-matched controls, using a newly developed multiplex bead assay. RESULTS: Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. CONCLUSIONS: Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

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Wang, Hui; Tian, Chan; Sun, Jing; Chen, Li-Na; Lv, Yan; Yang, Xiao-Dong; Xiao, Kang; Wang, Jing; Chen, Cao; Shi, Qi; Shao, Qi-Xiang; Dong, Xiao-Ping

2017-08-01

Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrP Sc ; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrP Sc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrP Sc . The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

Vitamin K-dependent carboxylation of pulmonary surfactant-associated proteins

International Nuclear Information System (INIS)

Rannels, S.R.; Gallaher, K.J.; Wallin, R.; Rannels, D.E.

1987-01-01

Rat type II pneumocytes expressed vitamin K-dependent carboxylase activity that incorporated 14 CO 2 into microsomal protein precursors of molecular weights similar to those of surfactant-associated proteins (SAP). Compared to carboxylated precursor proteins present in the liver, these molecules appeared to be unique to the lung. Antibodies raised against purified rat surfactant reacted with SAP resolved by NaDodSO 4 /PAGE and with surfactant-containing lamellar bodies in type II pneumocyte cytoplasm. NaDodSO 4 /PAGE of microsomal proteins, after carboxylase-catalyzed incorporation of 14 CO 2 , demonstrated radiolabeled, immunoreactive products identical to SAP. The presence of γ-carboxyglutamic acid in these proteins was confirmed by HPLC analysis of SAP hydrolysates. Furthermore, lung carboxylase activity and SAP matured over similar time courses during fetal lung development. These results show that SAP are carboxylated by type II cells via a vitamin K-dependent pathway analogous to that for hepatic carboxylation of clotting factors. Further analogy to the clotting system suggest that γ-carboxyglutamic acid residues in SAP polypeptides play a role in Ca 2+ binding and thus in the known requirements for both cation and SAP in the physiological function of pulmonary surfactant

Identification of Redox and Glucose-Dependent Txnip Protein Interactions

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Benjamin J. Forred

2016-01-01

Full Text Available Thioredoxin-interacting protein (Txnip acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S. Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip.

Rev and Rex proteins of human complex retroviruses function with the MMTV Rem-responsive element

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Dudley Jaquelin P

2009-02-01

Full Text Available Abstract Background Mouse mammary tumor virus (MMTV encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV, human T-cell leukemia virus (HTLV, and human endogenous retrovirus type K (HERV-K. In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE. Results MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export. Conclusion These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.

Region-specific differences in bioenergetic proteins and protein response to acute high fat diet in brains of low and high capacity runner rats.

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Gan, Li; Ma, Delin; Li, Min; Yang, Fu-Chen; Rogers, Robert S; Wheatley, Joshua L; Koch, Lauren G; Britton, Steven L; Thyfault, John P; Geiger, Paige C; Stanford, John A

2018-05-01

Aerobic capacity is a strong predictor of mortality. Low capacity runner (LCR) rats exhibit reduced mitochondrial function in peripheral organs. A high fat diet (HFD) can worsen metabolic phenotype in LCR rats. Little is known about metabolic changes in the brains of these rats, however. This study examined protein markers of mitochondrial function and metabolism as a function of aerobic running capacity and an acute HFD in four brain regions: the striatum, hippocampus, hypothalamus, and substantia nigra. After 3 days HFD or chow diets, we measured peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1-α), nuclear respiratory factors 1 (Nrf-1), mitochondrial transcription factor A (TFAM), and phosphorylated (activated) AMP-activated protein kinase (p-AMPK) protein levels in the four brain regions. LCR rats exhibited lower levels of mitochondrial proteins (PGC1-α, Nrf-1, TFAM), and greater p-AMPK, in striatum, but not in the other brain regions. Mitochondrial protein levels were greater in HFD LCR striatum, while p-AMPK was lower in this group. Markers of lower mitochondrial biogenesis and increased metabolic demand were limited to the LCR striatum, which nevertheless maintained the capacity to respond to an acute HFD challenge. Copyright © 2018 Elsevier B.V. All rights reserved.

Variation in the sodium-dependent vitamin C transporter 2 gene is associated with risk of acute coronary syndrome among women.

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Christine Dalgård

Full Text Available BACKGROUND: Vitamin C is associated with a lower risk of coronary heart disease possibly due to its anti-oxidative effects, beneficial effects on endothelial function and importance in collagen synthesis. The sodium-dependent vitamin C transporter 2 is responsible for the transport of vitamin C into various cells and malfunction of this protein leads to reduced vitamin C in tissue, including the arterial wall. We tested the hypothesis that candidate variations rs6139591 and rs1776964 in the gene coding for sodium-dependent vitamin C transporter 2 are associated with development of acute coronary syndrome. DESIGN: In the Danish Diet, Cancer and Health cohort study, we performed a case-cohort study among 57,053 subjects aged 50-64 years. RESULTS: During a mean follow-up period of 6.4 years, we identified 936 cases and randomly selected a sub-cohort (n = 1,580 with full information on genotypes and covariates. Using Cox proportional hazard models, we found that women with the rs6139591 TT genotype and a lower than median dietary vitamin C intake had a higher risk of acute coronary syndrome compared with those with the CC genotype (adjusted HR 5.39, 95% confidence interval, 2.01-14.50. We also observed a not as strong but positive although inconsistent association for women at a higher than median intake of vitamin C rich food. For the rs1776964 polymorphism, we found a higher risk (adjusted HR 3.45, 95% CI, 1.16-10.28 among TT-homozygous women with higher than median vitamin C intake compared with the CC genotype and low vitamin C intake. Among men, weaker and non-significant associations were observed for both polymorphisms. CONCLUSION: Genetic variation in the sodium-dependent vitamin C transporter 2 is associated with risk of incident acute coronary syndrome in women. The genotype effects may not be fully compensated by a higher intake of vitamin C rich food.

Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory.

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Figueiredo, Luciana Silva; de Freitas, Betânia Souza; Garcia, Vanessa Athaíde; Dargél, Vinícius Ayub; Köbe, Luiza Machado; Kist, Luiza Wilges; Bogo, Maurício Reis; Schröder, Nadja

2016-11-01

Alterations of brain iron levels have been observed in a number of neurodegenerative disorders. We have previously demonstrated that iron overload in the neonatal period results in severe and persistent memory deficits in the adulthood. Protein degradation mediated by the ubiquitin-proteasome system (UPS) plays a central regulatory role in several cellular processes. Impairment of the UPS has been implicated in the pathogenesis of neurodegenerative disorders. Here, we examined the effects of iron exposure in the neonatal period (12th-14th day of postnatal life) on the expression of proteasome β-1, β-2, and β-5 subunits, and ubiquitinated proteins in brains of 15-day-old rats, to evaluate the immediate effect of the treatment, and in adulthood to assess long-lasting effects. Two different memory types, emotionally motivated conditioning and object recognition were assessed in adult animals. We found that iron administered in the neonatal period impairs both emotionally motivated and recognition memory. Polyubiquitinated protein levels were increased in the hippocampus, but not in the cortex, of adult animals treated with iron. Gene expression of subunits β1 and β5 was affected by age, being higher in the early stages of development in the hippocampus, accompanied by an age-related increase in polyubiquitinated protein levels in adults. In the cortex, gene expression of the three proteasome subunits was significantly higher in adulthood than in the neonatal period. These findings suggest that expression of proteasome subunits and activity are age-dependently regulated. Iron exposure in the neonatal period produces long-lasting harmful effects on the UPS functioning, which may be related with iron-induced memory impairment.

The climate impacts of bioenergy systems depend on market and regulatory policy contexts.

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Lemoine, Derek M; Plevin, Richard J; Cohn, Avery S; Jones, Andrew D; Brandt, Adam R; Vergara, Sintana E; Kammen, Daniel M

2010-10-01

Biomass can help reduce greenhouse gas (GHG) emissions by displacing petroleum in the transportation sector, by displacing fossil-based electricity, and by sequestering atmospheric carbon. Which use mitigates the most emissions depends on market and regulatory contexts outside the scope of attributional life cycle assessments. We show that bioelectricity's advantage over liquid biofuels depends on the GHG intensity of the electricity displaced. Bioelectricity that displaces coal-fired electricity could reduce GHG emissions, but bioelectricity that displaces wind electricity could increase GHG emissions. The electricity displaced depends upon existing infrastructure and policies affecting the electric grid. These findings demonstrate how model assumptions about whether the vehicle fleet and bioenergy use are fixed or free parameters constrain the policy questions an analysis can inform. Our bioenergy life cycle assessment can inform questions about a bioenergy mandate's optimal allocation between liquid fuels and electricity generation, but questions about the optimal level of bioenergy use require analyses with different assumptions about fixed and free parameters.

Protein source and quality in therapeutic foods affect the immune response and outcome in severe acute malnutrition

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Protein is a vital component of therapeutic foods designed to treat severe acute malnutrition (SAM) in children; however there are still unknowns about the quality and quantity of the proteins to use in these foods. This review examines two recent studies investigating several different qualities an...

ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) is a new acute-phase protein isolated from cattle during experimental infection

DEFF Research Database (Denmark)

Pineiro, M.; Andres, M.; Iturralde, M.

2004-01-01

We have isolated from calf serum a protein with an apparent M, of 120,000. The protein was detected by using antibodies against major acute-phase protein in pigs with acute inflammation. The amino acid sequence of an internal fragment revealed that this protein is the bovine counterpart of ITIH4......, and Peptostreptococcus indolicus to induce an acute-phase reaction. All animals developed moderate to severe clinical mastitis and exhibited remarkable increases in ITIH4 concentration in serum (from 3 to 12 times the initial values, peaking at 48 to 72 h after infection) that correlated with the severity of the disease....... Animals with experimental infections with bovine respiratory syncytial virus (BRSV) also showed increases in ITIH4 concentration (from two- to fivefold), which peaked at around 7 to 8 days after inoculation. Generally, no response was seen after a second infection of the same animals with the virus...

A deeper look into transcription regulatory code by preferred pair distance templates for transcription factor binding sites

KAUST Repository

Kulakovskiy, Ivan V.

2011-08-18

Motivation: Modern experimental methods provide substantial information on protein-DNA recognition. Studying arrangements of transcription factor binding sites (TFBSs) of interacting transcription factors (TFs) advances understanding of the transcription regulatory code. Results: We constructed binding motifs for TFs forming a complex with HIF-1α at the erythropoietin 3\\'-enhancer. Corresponding TFBSs were predicted in the segments around transcription start sites (TSSs) of all human genes. Using the genome-wide set of regulatory regions, we observed several strongly preferred distances between hypoxia-responsive element (HRE) and binding sites of a particular cofactor protein. The set of preferred distances was called as a preferred pair distance template (PPDT). PPDT dramatically depended on the TF and orientation of its binding sites relative to HRE. PPDT evaluated from the genome-wide set of regulatory sequences was used to detect significant PPDT-consistent binding site pairs in regulatory regions of hypoxia-responsive genes. We believe PPDT can help to reveal the layout of eukaryotic regulatory segments. © The Author 2011. Published by Oxford University Press. All rights reserved.

Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis

DEFF Research Database (Denmark)

Eckersall, P D; Young, F J; Nolan, A M

2006-01-01

and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M......The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp...

Regulatory T cells in acute myelogenous leukemia: is it time for immunomodulation?

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Ustun, Celalettin; Miller, Jeffrey S; Munn, David H; Weisdorf, Daniel J; Blazar, Bruce R

2011-11-10

The microenviroment of acute myelogenous leukemia (AML) is suppressive for immune effector cells. Regulatory T cells (Tregs) have been recognized as a contributor factor and may be recruited and exploited by leukemic cells to evade immunesurveillance. Studies have shown that the frequencies of marrow and blood Tregs are greater in patients with AML than in control patients. Although increased Tregs have been associated with a decreased risk of GVHD after allogeneic HCT and hence may impede the graft-versus-tumor effect, recent findings indicate that that this may not be the case. Because there is a need to improve outcomes of standard treatment (chemotherapy with or without allogeneic HCT) in AML, targeting Tregs present an outstanding opportunity in AML because discoveries may apply throughout its treatment. Here, we review data on the roles of Tregs in mediating immune system-AML interactions. We focused on in vitro, animal, and observational human studies of Tregs in AML biology, development, prognosis, and therapy in different settings (eg, vaccination and HCT). Manipulation of Tregs or other types of immunomodulation may become a part of AML treatment in the future.

Protein Loop Dynamics Are Complex and Depend on the Motions of the Whole Protein

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Michael T. Zimmermann

2012-04-01

Full Text Available We investigate the relationship between the motions of the same peptide loop segment incorporated within a protein structure and motions of free or end-constrained peptides. As a reference point we also compare against alanine chains having the same length as the loop. Both the analysis of atomic molecular dynamics trajectories and structure-based elastic network models, reveal no general dependence on loop length or on the number of solvent exposed residues. Rather, the whole structure affects the motions in complex ways that depend strongly and specifically on the tertiary structure of the whole protein. Both the Elastic Network Models and Molecular Dynamics confirm the differences in loop dynamics between the free and structured contexts; there is strong agreement between the behaviors observed from molecular dynamics and the elastic network models. There is no apparent simple relationship between loop mobility and its size, exposure, or position within a loop. Free peptides do not behave the same as the loops in the proteins. Surface loops do not behave as if they were random coils, and the tertiary structure has a critical influence upon the apparent motions. This strongly implies that entropy evaluation of protein loops requires knowledge of the motions of the entire protein structure.

Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue

NARCIS (Netherlands)

Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

1992-01-01

The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

Science.gov (United States)

De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

2017-09-01

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Delay-independent stability of genetic regulatory networks.

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Wu, Fang-Xiang

2011-11-01

Genetic regulatory networks can be described by nonlinear differential equations with time delays. In this paper, we study both locally and globally delay-independent stability of genetic regulatory networks, taking messenger ribonucleic acid alternative splicing into consideration. Based on nonnegative matrix theory, we first develop necessary and sufficient conditions for locally delay-independent stability of genetic regulatory networks with multiple time delays. Compared to the previous results, these conditions are easy to verify. Then we develop sufficient conditions for global delay-independent stability for genetic regulatory networks. Compared to the previous results, this sufficient condition is less conservative. To illustrate theorems developed in this paper, we analyze delay-independent stability of two genetic regulatory networks: a real-life repressilatory network with three genes and three proteins, and a synthetic gene regulatory network with five genes and seven proteins. The simulation results show that the theorems developed in this paper can effectively determine the delay-independent stability of genetic regulatory networks.

Analysis of Protein Phosphorylation and Its Functional Impact on Protein-Protein Interactions via Text Mining of the Scientific Literature.

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Wang, Qinghua; Ross, Karen E; Huang, Hongzhan; Ren, Jia; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

2017-01-01

Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. Their activation or inactivation dictate downstream events: what substrates are modified and their subsequent impact (e.g., activation state, localization, protein-protein interactions (PPIs)). The biomedical literature continues to be the main source of evidence for experimental information about protein phosphorylation. Automatic methods to bring together phosphorylation events and phosphorylation-dependent PPIs can help to summarize the current knowledge and to expose hidden connections. In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

Science.gov (United States)

Kondo, Norihiko; Ishii, Yasuyuki; Son, Aoi; Sakakura-Nishiyama, Junko; Kwon, Yong-Won; Tanito, Masaki; Nishinaka, Yumiko; Matsuo, Yoshiyuki; Nakayama, Toshinori; Taniguchi, Masaru; Yodoi, Junji

2004-03-29

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

ATPase and GTPase Tangos Drive Intracellular Protein Transport.

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Shan, Shu-Ou

2016-12-01

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

International Nuclear Information System (INIS)

Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

2006-01-01

The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement

Helios expression in regulatory T cells promotes immunosuppression, angiogenesis and the growth of leukemia cells in pediatric acute lymphoblastic leukemia.

Science.gov (United States)

Li, Xue; Li, Dong; Huang, Xiaoyang; Zhou, Panpan; Shi, Qing; Zhang, Bing; Ju, Xiuli

2018-04-01

Regulatory T cells (Tregs) characterized by the transcription factor forkhead box P3 (FoxP3) are crucial for maintaining immune tolerance and preventing autoimmunity. However, FoxP3 does not function alone and Helios is considered a potential candidate for defining Treg subsets. In this study, we investigated the expression and function of Helios for identifying Tregs in childhood precursor B-cell acute lymphoblastic leukemia (pre-B ALL). Our results demonstrated that patients with pre-B ALL had a higher percentage of Helios + FoxP3 + CD4 + Tregs. And there was a positive correlation between the expression of Helios and the suppressive function of Tregs, the risk gradation of ALL. Helios in combination with CD4 and FoxP3 may be an effective way to detect functional Tregs in pre-B ALL by promoting the secretion of transforming growth factor (TGF)-β1. Furthermore, Helios + Tregs could regulate angiogenesis in the BM niche of pre-B ALL via the VEGFA/VEGFR2 pathway. We also found Helios + Tregs decreased apoptosis rate of nalm-6 cells by up-regulating the expression of anti-apoptosis protein Bcl-2. In summary, these data strongly imply the physiological importance of Helios expression in Tregs, and suggest that the manipulation of Helios may serve as a novel strategy for cancer immunotherapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Effects of Xiangqing Anodyne Spray on Treating Acute Soft-Tissue Injury Mainly Depend on Suppressing Activations of AKT and p38 Pathways

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Shudong Wang

2016-01-01

Full Text Available Objectives. In the present study we try to elucidate the mechanism of Xiangqing anodyne spray (XQAS effects on acute soft-tissue injury (STI. Methods. Acute STI model was established by hammer blow in the rat hind leg muscle. Within 8 hours, instantly after modeling and per 2-hour interval repeated topical applications with or without XQAS, CP or IH ethanol extracts spray (CPS and IHS were performed, respectively; muscle swelling rate and inflammation-related biochemical parameters, muscle histological observation, and mRNA and protein expression were then examined. Results. XQAS dose-dependently suppressed STI-caused muscle swelling, proinflammatory mediator productions, and oxidative stress as well as severe pathological changes in the injured muscle tissue. Moreover, CPS mainly by blocking p38 activation while IHS majorly by blocking AKT activation led to cytoplastic IκBα degradation with NF-κB p65 translocated into the nucleus. There are synergistic effects between CP and IH components in the XQAS on preventing from acute STI with suppressing IκBα degradation, NF-κB p65 translocation, and subsequent inflammation and oxidative stress-related abnormality. Conclusion. Marked effects of XQAS on treating acute STI are ascribed to strong anti-inflammatory and antioxidative actions with a reasonable combination of CP active components, blocking p38-NF-κB pathway activated, and IH active components, blocking AKT-NF-κB pathway activated.

Protein synthesis controls phosphate homeostasis.

Science.gov (United States)

Pontes, Mauricio H; Groisman, Eduardo A

2018-01-01

Phosphorus is an essential element assimilated largely as orthophosphate (Pi). Cells respond to Pi starvation by importing Pi from their surroundings. We now report that impaired protein synthesis alone triggers a Pi starvation response even when Pi is plentiful in the extracellular milieu. In the bacterium Salmonella enterica serovar Typhimurium , this response entails phosphorylation of the regulatory protein PhoB and transcription of PhoB-dependent Pi transporter genes and is eliminated upon stimulation of adenosine triphosphate (ATP) hydrolysis. When protein synthesis is impaired due to low cytoplasmic magnesium (Mg 2+ ), Salmonella triggers the Pi starvation response because ribosomes are destabilized, which reduces ATP consumption and thus free cytoplasmic Pi. This response is transient because low cytoplasmic Mg 2+ promotes an uptake in Mg 2+ and a decrease in ATP levels, which stabilizes ribosomes, resulting in ATP consumption and Pi increase, thus ending the response. Notably, pharmacological inhibition of protein synthesis also elicited a Pi starvation response in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae Our findings identify a regulatory connection between protein synthesis and Pi homeostasis that is widespread in nature. © 2018 Pontes and Groisman; Published by Cold Spring Harbor Laboratory Press.

THE INFLUENCE OF PATHOGENETIC THERAPY ON THE LEVER OF CYTOKINES IN PATIENTS WITH ACUTE BRUCELLOSIS

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N. I. Kovalevich

2016-01-01

Full Text Available The purpose of the study was to determine the level of proinflammatory cytokines: IL-12, IL-8 and IFNγ, neopterin and lipopolysaccharide-binding protein in the serum of patients with acute brucellosis before and after antibiotic therapy. The clinical data from 32 patients with laboratory-confirmed diagnosis — “acute brucellosis” admitted to the diagnosis, treatment and examination of occupational diseases brucellosis GBUZ SC “City Clinical Hospital No. 2”, the city of Stavropol were used in the study. The concentrations IL-12, IL-8, IFNγ cytokines and acute-phase proteins in serum was determined by ELISA. In the acute phase of brucellosis infection (before treatment had high levels of pro-inflammatory cytokines IL-8 and IFNγ, but despite holding a course of antibiotic treatment in the serum of patients with preserved high levels of IL-8, indicative of active inflammation in the absence of clinical manifestations. IL-12 level, a key cytokine in the initiation of lymphocyte-dependent immune response was lower than in the control group. Evaluation of the cytokine status (IL-8, IL-12, IL-18 and proteins of acute inflammation phase (neopterin and lipopolysaccharide-binding protein will provide valuable information for monitoring the effect of pharmacotherapy of acute brucellosis. Indicators of lipopolysaccharide-binding protein and neopterin in the serum of patients with brucellosis should be considered as a marker of inflammatory activity and as a predictor of outcome of acute brucellosis.

Involvement of activated leukocytes in the regulation of plasma levels of acute phase proteins in microgravity simulation experiments

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Larina, Olga; Bekker, Anna; Turin-Kuzmin, Alexey

2016-07-01

Earth-based studies of microgravity effects showed the induction of the mechanisms of acute phase reaction (APR). APR comprises the transition of stress-sensitive protein kinases of macrophages and other responsive cells into the active state and the phosphorylation of transcription factors which in turn stimulate the production of acute-phase reaction cytokines. Leukocyte activation is accompanied by the acceleration of the formation of oxygen radicals which can serve a functional indice of leukocyte cell state. The series of events at acute phase response result in selective changes in the synthesis of a number of secretory blood proteins (acute phase proteins, APPs) in liver cells thus contributing the recovery of homeostasis state in the organism. Earlier experiment with head-down tilt showed the increase in plasma concentrations of two cytokine mediators of acute phase response, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) being the outcome of the activation of producer cells, foremost, leukocytes. In experiment with 4-day dry immersion chemiluminescent (ChL) reply of the whole blood samples to a test stimulus were studied along with the measurements of plasma levels of APPs, namely, alpha1-antitrypsin (alpha1-AT), alpha1-acid glycoprotein (alpha1-AGP), alpha2-macroglobulin (alpha2-M), ceruloplasmin (Cer), haptoglobin (Hp), C3-complement component (C3), C-reactive protein (CRP). Eight individuals aged 21.2 ± 3.2 years were the test subjects in the investigation. Protein studies showed a noticeable increase in the mean plasma levels of all APPs measured in experiment thus producing the evidence of the activation of acute phase response mechanisms while individual patterns revealed variability during the immersion period. The overall trends were similar to these in the previous immersion series. The augment in the strength of signal in stimulated light emission tests was higher after 1- and 2-day of immersion exposure than before the

Commensal Lactobacillus Controls Immune Tolerance during Acute Liver Injury in Mice

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Nobuhiro Nakamoto

2017-10-01

Full Text Available Summary: Gut-derived microbial antigens trigger the innate immune system during acute liver injury. During recovery, regulatory immunity plays a role in suppressing inflammation; however, the precise mechanism underlying this process remains obscure. Here, we find that recruitment of immune-regulatory classical dendritic cells (cDCs is crucial for liver tolerance in concanavalin A-induced acute liver injury. Acute liver injury resulted in enrichment of commensal Lactobacillus in the gut. Notably, Lactobacillus activated IL-22 production by gut innate lymphoid cells and raised systemic IL-22 levels. Gut-derived IL-22 enhanced mucosal barrier function and promoted the recruitment of regulatory cDCs to the liver. These cDCs produced IL-10 and TGF-β through TLR9 activation, preventing further liver inflammation. Collectively, our results indicate that beneficial gut microbes influence tolerogenic immune responses in the liver. Therefore, modulation of the gut microbiota might be a potential option to regulate liver tolerance. : Nakamoto et.al. find that Lactobacillus accumulates in the gut and activates IL-22 production by innate lymphoid cells during acute liver injury. Gut-derived IL-22 contributes to liver tolerance via induction of regulatory DCs. Keywords: immune tolerance, dendritic cell, innate lymphoid cell, acute liver injury, interleukin-10, interleukin-22, microbiota, dysbiosis

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men.

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Mitchell, Cameron J; Churchward-Venne, Tyler A; Parise, Gianni; Bellamy, Leeann; Baker, Steven K; Smith, Kenneth; Atherton, Philip J; Phillips, Stuart M

2014-01-01

Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n = 23) and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m²) underwent a primed constant infusion of L-[ring-¹³C₆] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (Pmuscle volume and acute rates of MPS measured over 1-3 h (r = 0.02), 3-6 h (r = 0.16) or the aggregate 1-6 h post-exercise period (r = 0.10). Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4E-BP1(Thr37/46) at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression.

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Miedlich, Susanne U; Taya, Manisha; Young, Melissa Rasar; Hammes, Stephen R

2017-06-15

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

In silico analysis, mapping of regulatory elements and corresponding dna-protein interaction in polyphenol oxidase gene promoter from different rice varieties

International Nuclear Information System (INIS)

Mahmood, T.; Rehman, M.; Aziz, E.

2015-01-01

Polyphenol oxidase (PPO) is an important enzyme that has positive impact regarding plant resistance against different biotic and abiotic stresses. In the present study PPO promoter from six different rice varieties was amplified and then analyzed for cis- and trans-acting elements. The study revealed a total of 79 different cis-acting regulatory elements including 11 elements restricted to only one or other variety. Among six varieties Pakhal-Basmati had highest number (5) of these elements, whereas C-622 and Rachna-Basmati have no such sequences. Rachna-Basmati, IR-36-Basmati and Kashmir- Basmati had 1, 2 and 3 unique elements, respectively. Different elementsrelated to pathogen, salt and water stresses were found, which may be helpful in controlling PPO activity according to changing environment. Moreover, HADDOCK was used to understand molecular mechanism of PPO regulation and it was found that DNA-protein interactions are stabilized by many potential hydrogen bonds. Adenine and arginine were the most reactive residues in DNA and proteins respectively.Structural comparison of different protein-DNA complexes show that even a highly conserved transcriptional factor can adopt different conformations when they contact a different DNA binding sequence, however their stable interactions depend on the number of hydrogen bonds formed and distance. (author)

PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer’s amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE

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Lahiri Debomoy K

2013-01-01

Full Text Available Abstract Background Alzheimer’s disease (AD is intimately tied to amyloid-β (Aβ peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE, at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones. Results EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2, nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF, and specificity protein 1 (SP1. These sites crossed a known single nucleotide polymorphism (SNP. EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis. Conclusions We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also

Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

International Nuclear Information System (INIS)

Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

2011-01-01

Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

Recurrent rewiring and emergence of RNA regulatory networks.

Science.gov (United States)

Wilinski, Daniel; Buter, Natascha; Klocko, Andrew D; Lapointe, Christopher P; Selker, Eric U; Gasch, Audrey P; Wickens, Marvin

2017-04-04

Alterations in regulatory networks contribute to evolutionary change. Transcriptional networks are reconfigured by changes in the binding specificity of transcription factors and their cognate sites. The evolution of RNA-protein regulatory networks is far less understood. The PUF (Pumilio and FBF) family of RNA regulatory proteins controls the translation, stability, and movements of hundreds of mRNAs in a single species. We probe the evolution of PUF-RNA networks by direct identification of the mRNAs bound to PUF proteins in budding and filamentous fungi and by computational analyses of orthologous RNAs from 62 fungal species. Our findings reveal that PUF proteins gain and lose mRNAs with related and emergent biological functions during evolution. We demonstrate at least two independent rewiring events for PUF3 orthologs, independent but convergent evolution of PUF4/5 binding specificity and the rewiring of the PUF4/5 regulons in different fungal lineages. These findings demonstrate plasticity in RNA regulatory networks and suggest ways in which their rewiring occurs.

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone ( Haliotis discus hannai Ino) and its acute response to thermal exposure

Science.gov (United States)

Li, Jiaqi; He, Qingguo; Sun, Hui; Liu, Xiao

2012-01-01

Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone ( Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

Changes in mitochondrial perilipin 3 and perilipin 5 protein content in rat skeletal muscle following endurance training and acute stimulated contraction.

Science.gov (United States)

Ramos, S V; Turnbull, P C; MacPherson, R E K; LeBlanc, P J; Ward, W E; Peters, S J

2015-04-01

What is the central question of this study? The aim was to determine whether mitochondrial protein content of perilipin 3 (PLIN3) and perilipin 5 (PLIN5) is increased following endurance training and whether mitochondrial PLIN5 protein is increased to a greater extent in endurance-trained rats when compared with sedentary rats following acute contraction. What is the main finding and its importance? Mitochondrial PLIN3 but not PLIN5 protein was increased in endurance-trained compared with sedentary rats, suggesting a mitochondrial role for PLIN3 due to chronic exercise. Contrary to our hypothesis, acute mitochondrial PLIN5 protein was similar in both sedentary and endurance-trained rats. Endurance training results in an increased association between skeletal muscle lipid droplets and mitochondria. This association is likely to be important for the expected increase in intramuscular fatty acid oxidation that occurs with endurance training. The perilipin family of lipid droplet proteins, PLIN(2-5), are thought to play a role in skeletal muscle lipolysis. Recently, results from our laboratory demonstrated that skeletal muscle mitochondria contain PLIN3 and PLIN5 protein. Furthermore, 30 min of stimulated contraction induces an increased mitochondrial PLIN5 content. To determine whether mitochondrial content of PLIN3 and PLIN5 is altered with endurance training, Sprague-Dawley rats were randomized into sedentary or endurance-trained groups for 8 weeks of treadmill running followed by an acute (30 min) sciatic nerve stimulation to induce lipolysis. Mitochondrial PLIN3 protein was ∼1.5-fold higher in red gastrocnemius of endurance-trained rats compared with sedentary animals, with no change in mitochondrial PLIN5 protein. In addition, there was an increase in plantaris intramuscular lipid storage. Acute electrically stimulated contraction in red gastrocnemius from sedentary and endurance-trained rats resulted in a similar increase of mitochondrial PLIN5 between

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

Science.gov (United States)

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Science.gov (United States)

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

Science.gov (United States)

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

Effects of glucocorticoid combined with antibiotics on serum infection indexes, acute phase proteins and stress hormones in patients with severe pneumonia

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Yang Yu

2017-10-01

Full Text Available Objective: To study the effects of glucocorticoid combined with antibiotics on serum infection indexes, acute phase proteins and stress hormones in patients with severe pneumonia. Methods: a total of 80 patients with severe pneumonia who were hospitalized between August 2014 and January 2017 were retrospectively analyzed and divided into the routine treatment group (n=46 who received conventional antibiotic therapy and the combined treatment group (n=34 who received glucocorticoid combined with antibiotic therapy, and the differences in infection indexes, acute proteins and stress hormones were compared between the two groups of patients before and after treatment. Results: The differences in serum levels of infection indexes, acute phase proteins and stress hormones were not statistically significant between the two groups before treatment. After 1 week of treatment, serum infection indexes CRP and PCT levels of observation group were lower than those of control group; serum acute phase proteins α1-AT, α1-AG and CER levels were lower than those of control group; serum stress hormones Cor, AngⅠ and AngⅡ levels were lower than those of control group. Conclusion: Glucocorticoid combined with antibiotics can effectively inhibit systemic infection and stress and optimize the illness in patients with severe pneumonia.

Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort.

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Jane E Salmon

2011-03-01

Full Text Available Pregnancy in women with systemic lupus erythematosus (SLE or antiphospholipid antibodies (APL Ab--autoimmune conditions characterized by complement-mediated injury--is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins--membrane cofactor protein (MCP, complement factor I (CFI, and complement factor H (CFH--in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%. Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.ClinicalTrials.gov NCT00198068.

Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

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Andreas Bitter

2015-01-01

Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

OpenAIRE

Kristie, T M; Roizman, B

1986-01-01

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

In vivo and in vitro expression of myeloid antigens on B-lineage acute lymphoblastic leukemia cells.

Science.gov (United States)

Hara, J; Kawa-Ha, K; Yumura-Yagi, K; Kurahashi, H; Tawa, A; Ishihara, S; Inoue, M; Murayama, N; Okada, S

1991-01-01

The expression of myeloid antigens has been extensively examined using two-color analysis in 43 children with B-lineage acute lymphoblastic leukemia (ALL). On pre-culture cells, CD33 expression was frequently observed in CD19+, CD10- B-precursor ALL, and CD14 was expressed only on the cells from B-precursor ALL expressing CD19, CD10 and CD20, and B-ALL. After 2 or 3 days of culture without TPA, CD13 emerged on the cells from 21 of 29 patients irrespective of the presence or the absence of fetal calf serum in the culture. Of four patients with CD10+ B-precursor ALL, which showed no expression of CD13 after culture, two had T-cell associated antigens. Whereas the addition of TPA to the culture enhanced the expression of CD13 on the cells from acute non-lymphocytic leukemia (ANLL), TPA reduced the expression of this antigen on B-precursor cells. These findings suggest that the regulatory mechanism of CD13 expression may be different between B-precursor ALL and ANLL. Co-culture with cycloheximide mostly abrogated the induction of CD13, suggesting that CD13 expression was mainly dependent on de novo protein synthesis.

Acute and persistent Mycobacterium tuberculosis infections depend on the thiol peroxidase TpX.

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Yanmin Hu

Full Text Available The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H(2O(2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-gamma activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2 knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models.

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

DEFF Research Database (Denmark)

Yde, C W; Olsen, B B; Meek, D

2008-01-01

25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

DEFF Research Database (Denmark)

McCord, Ronald A; Michishita, Eriko; Hong, Tao

2009-01-01

-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia

DEFF Research Database (Denmark)

Willer, Anton; Jakobsen, Janus Schou; Ohlsson, E

2015-01-01

orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general......Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which...... influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.Leukemia...

Therapeutic regulatory T-cell adoptive transfer ameliorates established murine chronic GVHD in a CXCR5-dependent manner

Science.gov (United States)

McDonald-Hyman, Cameron; Flynn, Ryan; Panoskaltsis-Mortari, Angela; Peterson, Nicholas; MacDonald, Kelli P. A.; Hill, Geoffrey R.; Luznik, Leo; Serody, Jonathan S.; Murphy, William J.; Maillard, Ivan; Munn, David H.; Turka, Laurence A.; Koreth, John; Cutler, Corey S.; Soiffer, Robert J.; Antin, Joseph H.; Ritz, Jerome

2016-01-01

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation. In cGVHD, alloreactive T cells and germinal center (GC) B cells often participate in GC reactions to produce pathogenic antibodies. Although regulatory T cells (Tregs) can inhibit GC reactions, Treg numbers are reduced in cGVHD, contributing to cGVHD pathogenesis. Here, we explored 2 means to increase Tregs in cGVHD: interleukin-2/monoclonal antibody (IL-2/mAb) complexes and donor Treg infusions. IL-2/mAb complexes given over 1 month were efficacious in expanding Tregs and treating established cGVHD in a multi-organ-system disease mouse model characterized by GC reactions, antibody deposition, and lung dysfunction. In an acute GVHD (aGVHD) model, IL-2/mAb complexes given for only 4 days resulted in rapid mortality, indicating IL-2/mAb complexes can drive conventional T-cell (Tcon)-mediated injury. In contrast, Treg infusions, which uniformly suppress aGVHD, increased Treg frequency and were effective in preventing the onset of, and treating, established cGVHD. Efficacy was dependent upon CXCR5-sufficient Tregs homing to, and inhibiting, GC reactions. These studies indicate that the infusion of Tregs, especially ones enriched for GC homing, may be desirable for cGVHD therapy. Although IL-2/mAb complexes can be efficacious in cGVHD, a cautious approach needs to be taken in settings in which aGVHD elements, and associated Tcon, are present. PMID:27385791

Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

International Nuclear Information System (INIS)

Watkins, D.T.

1991-01-01

The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

Nuclear Magnetic Resonance structural studies of peptides and proteins from the vaso-regulatory System

International Nuclear Information System (INIS)

Sizun, Philippe

1991-01-01

The aim of the present work is to show how Nuclear Magnetic Resonance (NMR) allows to determine the 3D structure of peptides and proteins in solution. A comparative study of peptides involved in the vaso-regulatory System (form small hormonal peptide to the 65 amido-acid protein hirudin) has allowed to design most efficient NMR 1D and 2D strategies. It rapidly appeared that the size of the peptide plays a key role in the structuration of the molecule, smallest peptides being weakly structured owing to the lack of cooperative effects. As the molecular size increases or if conformational locks are present (disulfide bridges) the probability of stable secondary structure increases. For the protein hirudin, a combination of ail available NMR parameters deduced form dedicated experiments (chemical shifts, coupling constants, overhauser effects, accessibility of amide protons) and molecular modelling under constraints allows a clear 3D structure to be proposed for this protein in solution. Finally, a comparative study of the experimental structures and of those deduced form prediction rules has shed light on the concept of structural predisposition, the latter being of high value for a better understanding of structure-activity relationships. (author) [fr

Bone Abnormalities in Mice with Protein Kinase A (PKA) Defects Reveal a Role of Cyclic AMP Signaling in Bone Stromal Cell-Dependent Tumor Development.

Science.gov (United States)

Liu, S; Shapiro, J M; Saloustros, E; Stratakis, C A

2016-11-01

Protein kinase A (PKA) is an important enzyme for all eukaryotic cells. PKA phosphorylates other proteins, thus, it is essential for the regulation of many diverse cellular functions, including cytoplasmic trafficking and signaling, organelle structure and mitochondrial oxidation, nuclear gene expression, the cell cycle, and cellular division. The PKA holoenzyme is composed of 2 regulatory and 2 catalytic subunits. Four regulatory (R1α, R1β, R2α, and R2β) and 4 catalytic subunits (Cα, Cβ, Cγ, and Prkx) have been identified, giving rise to mainly PKA-I (when the 2 regulatory subunits are either R1α or R1β), or PKA-II (when the 2 regulatory subunits are either R2α or R2β). Mutations in the PKA subunits can lead to altered total PKA activity or abnormal PKA-I to PKA-II ratio, leading to various abnormalities in both humans and mice. These effects can be tissue-specific. We studied the effect of PKA subunit defects on PKA activity and bone morphology of mice that were single or double heterozygous for null alleles of the various PKA subunit genes. Bone lesions including fibrous dysplasia, myxomas, osteo-sarcomas, -chondromas and -chondrosarcomas were found in these mice. Observational and molecular studies showed that these lesions were derived from bone stromal cells (BSCs). We conclude that haploinsufficiency for different PKA subunit genes affected bone lesion formation, new bone generation, organization, and mineralization in variable ways. This work identified a PKA subunit- and activity-dependent pathway of bone lesion formation from BSCs with important implications for understanding how cyclic AMP affects the skeleton and its tumorigenesis. © Georg Thieme Verlag KG Stuttgart · New York.

Glucose-lowering effect of whey protein depends upon clinical characteristics of patients with type 2 diabetes.

Science.gov (United States)

Almario, Rogelio U; Buchan, Wendy M; Rocke, David M; Karakas, Sidika E

2017-01-01

Whey protein (WP) intake has been shown to reduce postprandial glycemia. Majority of WP research in type 2 diabetes (T2DM) involved acute challenge or weight loss studies. It is not known if WP supplementation can provide sustained glucose lowering. Our goal was to investigate the effects of WP on glycemia comprehensively by using continuous glucose monitoring (CGM) while avoiding the confounding effects of variable food intake through controlled feeding. This double-blinded and placebo (PL)-controlled study included 22 patients with T2DM patients (11 male, 11 female; age 57.1±12.6 years) on diet or metformin monotherapy. First, one serving (21 g) of WP was compared with PL in parallel-armed acute challenge studies. Next, in a crossover design, each patient underwent CGM twice, over 2 consecutive weeks, 3.5 days each week. Identical diets were provided by the study during both CGM periods. During the first CGM, one serving of either WP or PL was consumed before breakfast and another before dinner. During the second CGM, participants switched to the alternate supplement. Order of the supplements was randomized. During acute challenge studies, WP stimulated insulin and glucagon-like peptide (GLP)-1 secretion; suppressed ghrelin (all pObesity, hypertriglyceridemia and high fasting GLP-1 concentrations predicted increased glucose levels. Effects of WP supplementation on glycemia in T2DM depend on the baseline characteristics. Lower body weight, normal triglyceride and lower GLP-1 levels predict glucose lowering. In contrast, obesity, hypertriglyceridemia and high baseline GLP-1 predict increased glucose response.

Effect of acute beer ingestion on the liver: studies in female mice.

Science.gov (United States)

Kanuri, Giridhar; Wagnerberger, Sabine; Landmann, Marianne; Prigl, Eva; Hellerbrand, Claus; Bischoff, Stephan C; Bergheim, Ina

2015-04-01

The aim of the present study was to assess whether the effects of acute consumption of stout or pilsner beer on the liver differ from those of plain ethanol in a mouse model. Seven-week-old female C57BL/6J mice received either ethanol, stout or pilsner beer (ethanol content: 6 g/kg body weight) or isocaloric maltodextrin solution. Plasma alanine transaminase, markers of steatosis, lipogenesis, activation of the toll-like receptor-4 signaling cascade as well as lipid peroxidation and fibrogenesis in the liver were measured 12 h after acute ethanol or beer intake. Acute alcohol ingestion caused a marked ~11-fold increase in hepatic triglyceride accumulation in comparison to controls, whereas in mice exposed to stout and pilsner beer, hepatic triglyceride levels were increased only by ~6.5- and ~4-fold, respectively. mRNA expression of sterol regulatory element-binding protein 1c and fatty acid synthase in the liver did not differ between alcohol and beer groups. In contrast, expression of myeloid differentiation primary response gene 88, inducible nitric oxide synthases, but also the concentrations of 4-hydroxynonenal protein adducts, nuclear factor κB and plasminogen activator inhibitor-1 were induced in livers of ethanol treated mice but not in those exposed to the two beers. Taken together, our results suggest that acute ingestion of beer and herein especially of pilsner beer is less harmful to the liver than the ingestion of plain ethanol.

A gene regulatory network armature for T-lymphocyte specification

Energy Technology Data Exchange (ETDEWEB)

Fung, Elizabeth-sharon [Los Alamos National Laboratory

2008-01-01

Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.

Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

Directory of Open Access Journals (Sweden)

Natalia González

Full Text Available Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (reacquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate.

Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

Science.gov (United States)

Mitchison, A

1997-01-01

In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

Directory of Open Access Journals (Sweden)

Guangqi Li

Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

DEFF Research Database (Denmark)

Bless, N M; Huber-Lang, M; Guo, R F

2000-01-01

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES...... were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response....... Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti...

Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

DEFF Research Database (Denmark)

Nylandsted, J; Jäättelä, M; Hoffmann, E K

2004-01-01

Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...... that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume...... regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na...

Anti-regulatory T cells

DEFF Research Database (Denmark)

Andersen, Mads Hald

2017-01-01

responses to tumours or inhibiting autoimmunity development. However, recent studies report the discovery of self-reactive pro-inflammatory T cells—termed anti-regulatory T cells (anti-Tregs)—that target immune-suppressive cells. Thus, regulatory cells can now be defined as both cells that suppress immune...... reactions as well as effector cells that counteract the effects of suppressor cells and support immune reactions. Self-reactive anti-Tregs have been described that specifically recognize human leukocyte antigen-restricted epitopes derived from proteins that are normally expressed by regulatory immune cells......Our initial understanding of immune-regulatory cells was based on the discovery of suppressor cells that assure peripheral T-cell tolerance and promote immune homeostasis. Research has particularly focused on the importance of regulatory T cells (Tregs) for immune modulation, e.g. directing host...

Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

DEFF Research Database (Denmark)

Whiteford, James R; Xian, Xiaojie; Chaussade, Claire

2011-01-01

Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is ß1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine...

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

Science.gov (United States)

Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

2018-03-01

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

The Evolution of the Secreted Regulatory Protein Progranulin.

Science.gov (United States)

Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

2015-01-01

Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

Critical protein GAPDH and its regulatory mechanisms in cancer cells

International Nuclear Information System (INIS)

Zhang, Jin-Ying; Zhang, Fan; Hong, Chao-Qun; Giuliano, Armando E.; Cui, Xiao-Jiang; Zhou, Guang-Ji; Zhang, Guo-Jun; Cui, Yu-Kun

2015-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and posttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 (HIF-1), p53, nitric oxide (NO), and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications (PTMs) occurring in GAPDH in cancer cells result in new activities unrelated to the original glycolytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described

FBXW7 regulates glucocorticoid response in T-cell acute lymphoblastic leukaemia by targeting the glucocorticoid receptor for degradation.

Science.gov (United States)

Malyukova, A; Brown, S; Papa, R; O'Brien, R; Giles, J; Trahair, T N; Dalla Pozza, L; Sutton, R; Liu, T; Haber, M; Norris, M D; Lock, R B; Sangfelt, O; Marshall, G M

2013-04-01

Loss of function mutation in FBXW7, an E3 ubiquitin ligase, is associated with good prognosis and early glucocorticoid treatment response in childhood T-cell acute lymphoblastic leukemia (T-ALL) by unknown mechanisms. Here, we show that FBXW7 targets the glucocorticoid receptor α (GRα) for ubiquitylation and proteasomal degradation in a manner dependent on glycogen synthase kinase 3 β-mediated phsophorylation. FBXW7 inactivation caused elevated GRα levels, and enhanced the transcriptional response to glucocorticoids. There was significant enhancement of GR transcriptional responses in FBXW7-deficient cell lines and primary T-ALL samples, in particular, for those pro-apoptotic regulatory proteins, BIM and PUMA. Reduced FBXW7 expression or function promoted glucocorticoid sensitivity, but not sensitivity to other chemotherapeutic agents used in T-ALL. Moreover, this was a general feature of different cancer cell types. Taken together, our work defines GRα as a novel FBXW7 substrate and demonstrates that favorable patient prognosis in T-ALL is associated with FBXW7 mutations due to enhanced GRα levels and steroid sensitivity. These findings suggest that inactivation of FBXW7, a putative tumor suppressor protein, may create a synthetic lethal state in the presence of specific anticancer therapies.

Molecular chaperones in targeting misfolded proteins for ubiquitin-dependent degradation

DEFF Research Database (Denmark)

Kriegenburg, Franziska; Ellgaard, Lars; Hartmann-Petersen, Rasmus

2012-01-01

The accumulation of misfolded proteins presents a considerable threat to the health of individual cells and has been linked to severe diseases, including neurodegenerative disorders. Considering that, in nature, cells often are exposed to stress conditions that may lead to aberrant protein...... conformational changes, it becomes clear that they must have an efficient quality control apparatus to refold or destroy misfolded proteins. In general, cells rely on molecular chaperones to seize and refold misfolded proteins. If the native state is unattainable, misfolded proteins are targeted for degradation...... via the ubiquitin-proteasome system. The specificity of this proteolysis is generally provided by E3 ubiquitin-protein ligases, hundreds of which are encoded in the human genome. However, rather than binding the misfolded proteins directly, most E3s depend on molecular chaperones to recognize...

Regulatory Roles for Long ncRNA and mRNA

International Nuclear Information System (INIS)

Karapetyan, Armen R.; Buiting, Coen; Kuiper, Renske A.; Coolen, Marcel W.

2013-01-01

Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research

Regulatory Roles for Long ncRNA and mRNA

Energy Technology Data Exchange (ETDEWEB)

Karapetyan, Armen R.; Buiting, Coen; Kuiper, Renske A.; Coolen, Marcel W., E-mail: M.Coolen@gen.umcn.nl [Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, P.O. Box 9101, Nijmegen 6500 HB (Netherlands)

2013-04-26

Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.

Graphical analysis of pH-dependent properties of proteins predicted using PROPKA.

Science.gov (United States)

Rostkowski, Michał; Olsson, Mats H M; Søndergaard, Chresten R; Jensen, Jan H

2011-01-26

Charge states of ionizable residues in proteins determine their pH-dependent properties through their pKa values. Thus, various theoretical methods to determine ionization constants of residues in biological systems have been developed. One of the more widely used approaches for predicting pKa values in proteins is the PROPKA program, which provides convenient structural rationalization of the predicted pKa values without any additional calculations. The PROPKA Graphical User Interface (GUI) is a new tool for studying the pH-dependent properties of proteins such as charge and stabilization energy. It facilitates a quantitative analysis of pKa values of ionizable residues together with their structural determinants by providing a direct link between the pKa data, predicted by the PROPKA calculations, and the structure via the Visual Molecular Dynamics (VMD) program. The GUI also calculates contributions to the pH-dependent unfolding free energy at a given pH for each ionizable group in the protein. Moreover, the PROPKA-computed pKa values or energy contributions of the ionizable residues in question can be displayed interactively. The PROPKA GUI can also be used for comparing pH-dependent properties of more than one structure at the same time. The GUI considerably extends the analysis and validation possibilities of the PROPKA approach. The PROPKA GUI can conveniently be used to investigate ionizable groups, and their interactions, of residues with significantly perturbed pKa values or residues that contribute to the stabilization energy the most. Charge-dependent properties can be studied either for a single protein or simultaneously with other homologous structures, which makes it a helpful tool, for instance, in protein design studies or structure-based function predictions. The GUI is implemented as a Tcl/Tk plug-in for VMD, and can be obtained online at http://propka.ki.ku.dk/~luca/wiki/index.php/GUI_Web.

Salinity- and population-dependent genome regulatory response during osmotic acclimation in the killifish (Fundulus heteroclitus) gill.

Science.gov (United States)

Whitehead, Andrew; Roach, Jennifer L; Zhang, Shujun; Galvez, Fernando

2012-04-15

The killifish Fundulus heteroclitus is abundant in osmotically dynamic estuaries and it can quickly adjust to extremes in environmental salinity. We performed a comparative osmotic challenge experiment to track the transcriptomic and physiological responses to two salinities throughout a time course of acclimation, and to explore the genome regulatory mechanisms that enable extreme osmotic acclimation. One southern and one northern coastal population, known to differ in their tolerance to hypo-osmotic exposure, were used as our comparative model. Both populations could maintain osmotic homeostasis when transferred from 32 to 0.4 p.p.t., but diverged in their compensatory abilities when challenged down to 0.1 p.p.t., in parallel with divergent transformation of gill morphology. Genes involved in cell volume regulation, nucleosome maintenance, ion transport, energetics, mitochondrion function, transcriptional regulation and apoptosis showed population- and salinity-dependent patterns of expression during acclimation. Network analysis confirmed the role of cytokine and kinase signaling pathways in coordinating the genome regulatory response to osmotic challenge, and also posited the importance of signaling coordinated through the transcription factor HNF-4α. These genome responses support hypotheses of which regulatory mechanisms are particularly relevant for enabling extreme physiological flexibility.

Presenilin dependence of phospholipase C and protein kinase C signaling

DEFF Research Database (Denmark)

Dehvari, Nodi; Cedazo-Minguez, Angel; Isacsson, Ola

2007-01-01

-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha...

Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens

International Nuclear Information System (INIS)

Yotsui, Izumi; Serada, Satoshi; Naka, Tetsuji; Saruhashi, Masashi; Taji, Teruaki; Hayashi, Takahisa; Quatrano, Ralph S.; Sakata, Yoichi

2016-01-01

Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation

Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens

Energy Technology Data Exchange (ETDEWEB)

Yotsui, Izumi, E-mail: izumi.yotsui@riken.jp [Department of BioScience, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagayaku, Tokyo, 156-8502 (Japan); Serada, Satoshi, E-mail: serada@nibiohn.go.jp [Laboratory of Immune Signal, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka, 567-0085 (Japan); Naka, Tetsuji, E-mail: tnaka@nibiohn.go.jp [Laboratory of Immune Signal, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka, 567-0085 (Japan); Saruhashi, Masashi, E-mail: s13db001@mail.saitama-u.ac.jp [Department of BioScience, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagayaku, Tokyo, 156-8502 (Japan); Taji, Teruaki, E-mail: t3teruak@nodai.ac.jp [Department of BioScience, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagayaku, Tokyo, 156-8502 (Japan); Hayashi, Takahisa, E-mail: t4hayash@nodai.ac.jp [Department of BioScience, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagayaku, Tokyo, 156-8502 (Japan); Quatrano, Ralph S., E-mail: rsq@wustl.edu [Department of Biology, Washington University in St. Louis, St. Louis, MO, 63130-4899 (United States); Sakata, Yoichi, E-mail: sakata@nodai.ac.jp [Department of BioScience, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagayaku, Tokyo, 156-8502 (Japan)

2016-03-18

Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation

Changes of CSF-protein pattern in children with acute lymphoblastic leukemia during prophylactic CNS therapy (Berlin protocol)

International Nuclear Information System (INIS)

Siemes, H.; Rating, D.; Siegert, M.; Hanefeld, F.; Mueller, S.; Gadner, H.; Riehm, H.

1980-01-01

The cerebral spinal fluid (CSF)-protein profiles of ten children with previously untreated acute lymphoblastic leukemia (ALL) were investigated by agarose gel electrophoresis. The profiles were determined at diagnosis and during the fifth to eighth week of treatment when preventive therapy for central nervous system (CNS) leukemia (skull irradiation, intrathecal methotrexate (ithMTX) was administered. The profiles were compared with those obtained from a control group of 67 children and those from 42 patients with acute aseptic meningitis. The data from the latter group demonstrated the CSF-protein pattern of partial blood-CSF barrier (B-CSF-B) breakdown. The children with ALL showed no or only minor signs of a B-CSF-B impairment at diagnosis and after four weeks of systemic treatment. However, CSF changes indicative of a lesion of the B-CSF-B increased in all children continuously during CNS prophylaxis. The protein profile at the end of combined chemotherapy and radiotherapy was very similar to that in patients with acute aseptic meningitis. These observations point to neurotoxic side effects on the CNS barrier system with the combination of cranial radiation and ithMTX. A striking finding was restricted heterogeneity of gamma-globulin, observed in the CSF of nine out of the ten children with ALL before or during treatment. The significance of this abnormality is unknown

Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog-dependent

International Nuclear Information System (INIS)

Asselman, Jana; Shaw, Joseph R.; Glaholt, Stephen P.; Colbourne, John K.; De Schamphelaere, Karel A.C.

2013-01-01

Highlights: •Transcription patterns of 4 metallothionein isoforms in Daphnia pulex. •Under cadmium and copper stress these patterns are time-dependent. •Under cadmium and copper stress these patterns are homolog-dependent. •The results stress the complex regulation of metallothioneins. -- Abstract: Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1–mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species

Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog-dependent

Energy Technology Data Exchange (ETDEWEB)

Asselman, Jana, E-mail: jana.asselman@ugent.be [Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, Ghent (Belgium); Shaw, Joseph R.; Glaholt, Stephen P. [The School of Public and Environmental Affairs, Indiana University, Bloomington, IN (United States); Colbourne, John K. [School of Biosciences, The University of Birmingham, Birmingham (United Kingdom); De Schamphelaere, Karel A.C. [Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, Ghent (Belgium)

2013-10-15

Highlights: •Transcription patterns of 4 metallothionein isoforms in Daphnia pulex. •Under cadmium and copper stress these patterns are time-dependent. •Under cadmium and copper stress these patterns are homolog-dependent. •The results stress the complex regulation of metallothioneins. -- Abstract: Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1–mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species.

Native whey induces higher and faster leucinemia than other whey protein supplements and milk: A randomized controlled trial

Science.gov (United States)

Resistance exercise and protein intake are both strong stimuli for muscle protein synthesis. The potential for a protein to acutely increase muscle protein synthesis seems partly dependent on absorption kinetics and the amino acid composition. The aim of this double-blinded randomized cross-over stu...

Phospholipid-binding protein EhC2A mediates calcium-dependent translocation of transcription factor URE3-BP to the plasma membrane of Entamoeba histolytica.

Science.gov (United States)

Moreno, Heriberto; Linford, Alicia S; Gilchrist, Carol A; Petri, William A

2010-05-01

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca(2+)-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca(2+)-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca(2+). Recombinant and native EhC2A bound phospholipid liposomes in a Ca(2+)-dependent manner, with half-maximal binding occurring at 3.4 muM free Ca(2+). A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca(2+)-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca(2+) concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca(2+)-dependent anchoring of the transcription factor to the amebic plasma membrane.

Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

Energy Technology Data Exchange (ETDEWEB)

Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

2012-07-06

Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

International Nuclear Information System (INIS)

Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar

2012-01-01

Highlights: ► CFTR mutations produce cystic fibrosis. ► Chloride transport depends on the regulatory domain phosphorylation. ► Regulatory domain is intrinsically disordered. ► Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and β-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of α-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature

Dietary supplementation with aromatic amino acids increases protein synthesis in children wHh severe acute malnutrition

Science.gov (United States)

Although 2 earlier studies reported that aromatic amino acid (AAA) supplementation of children with severe acute malnutrition (SAM) improved whole-body protein anabolism during the early postadmission (maintenance) phase of rehabilitation, it is not known whether this positive effect was maintained ...

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-01-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet ?-cell

OpenAIRE

Kowluru, Anjaneyulu

2008-01-01

Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet ?-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the ?-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prok...

Acute phase proteins in cattle after exposure to complex stress

DEFF Research Database (Denmark)

Lomborg, S. R.; Nielsen, L. R.; Heegaard, Peter M. H.

2008-01-01

Abstract Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels...... concentrations of SAA and haptoglobin increased significantly in response to the stressors (P...... in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin...

Network perturbation by recurrent regulatory variants in cancer.

Directory of Open Access Journals (Sweden)

Kiwon Jang

2017-03-01

Full Text Available Cancer driving genes have been identified as recurrently affected by variants that alter protein-coding sequences. However, a majority of cancer variants arise in noncoding regions, and some of them are thought to play a critical role through transcriptional perturbation. Here we identified putative transcriptional driver genes based on combinatorial variant recurrence in cis-regulatory regions. The identified genes showed high connectivity in the cancer type-specific transcription regulatory network, with high outdegree and many downstream genes, highlighting their causative role during tumorigenesis. In the protein interactome, the identified transcriptional drivers were not as highly connected as coding driver genes but appeared to form a network module centered on the coding drivers. The coding and regulatory variants associated via these interactions between the coding and transcriptional drivers showed exclusive and complementary occurrence patterns across tumor samples. Transcriptional cancer drivers may act through an extensive perturbation of the regulatory network and by altering protein network modules through interactions with coding driver genes.

Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

Science.gov (United States)

Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

2011-01-01

It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

Late protein synthesis-dependent phases in CTA long-term memory: BDNF requirement

Directory of Open Access Journals (Sweden)

Araceli eMartínez-Moreno

2011-09-01

Full Text Available It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memory when protein synthesis was inhibited. Our previous studies on the insular cortex (IC, a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA, have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis dependent in different time-windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 hours after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement.

Science.gov (United States)

Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F; Escobar, Martha L

2011-01-01

It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

Enhanced expression of a calcium-dependent protein kinase

Indian Academy of Sciences (India)

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements.

Science.gov (United States)

Bhagwat, Anand S; Roe, Jae-Seok; Mok, Beverly Y L; Hohmann, Anja F; Shi, Junwei; Vakoc, Christopher R

2016-04-19

The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Intake of tryptophan-enriched whey protein acutely enhances recall of positive loaded words in patients with multiple sclerosis.

Science.gov (United States)

Lieben, Cindy K; Blokland, Arjan; Deutz, Nicolaas E; Jansen, Willemijn; Han, Gang; Hupperts, Raymond M

2018-02-01

Multiple sclerosis (MS) has physiological and/or immunological characteristics that diminish serotonin metabolism, a neurotransmitter associated with affective and cognitive functions. The aim was examine the acute and dose-dependent effects of a dietary tryptophan (TRP) enrichment on affective and cognitive functions in MS patients. We hypothesized that increased dietary availability of the amino acid TRP enhances serotonin concentrations and improves neuropsychological functions. In a double-blind, placebo-controlled, crossover study, MS patients with (n = 15) and without (n = 17) depressed mood ingested a whey protein mixture with 4 different amounts of TRP. Mood states, total plasma TRP and plasma TRP/ΣLNAA ratio were measured during each test session and cognitive tasks were conducted three hours after dietary intake. A fast, transient and dose-dependent increase of total plasma TRP and TRP/ΣLNAA ratio was found. Ratings of negative mood decreased over time, independent of the TRP dose. Relative to whey-only, immediate word recall and delayed recognition improved after ingestion of the lowest added TRP dose and was mainly due to better recollection for positive loaded words. Executive functions were not affected by a difference in TRP availability. A moderate addition of TRP to whey protein enhances memory processes without improving the mood state in MS. ccmo-registration number is NL32316.096.10. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Regulatory signals for intestinal amino acid transporters and peptidases

International Nuclear Information System (INIS)

Ferraris, R.P.; Kwan, W.W.; Diamond, J.

1988-01-01

Dietary protein ultimately regulates many processes involved in protein digestion, but it is often unclear whether proteins themselves, peptides, or amino acids (AAs) are the proximate regulatory signal. Hence the authors compared several processes involved in protein digestion in mice adapted to one of three rations, identical except for containing 54% of either casein, a partial hydrolysate of casein, or a free AA mixture simulating a complete hydrolysate of casein. The authors measured brush-border uptakes of seven AAs that variously serve as substrates for four AA transporters, and brush-border and cytosolic activities of four peptidases. The three rations yielded essentially the same AA uptake rates. Peptidase activities tended to be lower on the AA ration than on the protein ration. In other studies, all three rations yielded the same rates of brush-border peptide uptake; protein is only modestly more effective than AAs at inducing synthesis of pancreatic proteases; and, depending on the animal species, protein is either much less or much more effective than AAs at stimulating release of cholecystokinin and hence of pancreatic enzymes. Thus the regulators of each process involved in protein digestion are not necessarily that process's substrate

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

Science.gov (United States)

Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

[C1q/tumor necrosis factor related protein 6 (CTRP6) is involved in gentamicin-induced acute kidney injury in rats].

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Li, Rong; Yang, Xiaoxia; Yu, Yan; Zhou, Meilan; Tian, Xiujuan; Feng, Shidong; Wang, Hanmin

2016-11-01

Objective To explore the role of the anti-inflammatory cytokine C1q/tumor necrosis factor related protein 6 (CTRP6) in gentamicin-induced acute kidney injury in rats. Methods SD rats were divided into 5 groups including control group, model group and the other 3 experimental groups. The rats in model group and experimental groups were subcutaneously injected with gentamicin at the dose of 400 mg/(kg.d) for consecutive 2 days to induce acute renal injury. Two days before gentamicin injection, the rats in the 3 experimental groups were given pAd-CTRP6 at the doses of 0.5, 5 and 50 mg/kg, respectively. The serum levels of blood urea nitrogen (BUN) and creatinine (Cr) were respectively assayed with picric acid colorimetry and ultraviolet spectrophotometry; ELISA was used to detect serum CTRP6 content and the production of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the kidney homogenate; Western blotting was performed to detect the expressions of CTRP6, caspase-1 and pyrin domain containing 3 (NLRP3) proteins in the renal tissues of rats. Results Compared with control group, serum BUN and Cr contents increased in the model rats; the secretion of inflammatory factors IL-1β and TNF-α, as well as the expressions of caspase-1 and NLRP3 were also enhanced in the model group. Compared with the model group, serum BUN and Cr contents decreased in the experimental groups; the secretion of IL-1β and TNF-α, as well as the expressions of caspase-1 and NLRP3 were also attenuated in the experimental groups. Moreover, with the increase of the injection dosage of pAd-CTRP6, the suppressive effect was gradually strengthened. Conclusion CTRP6 can attenuate gentamicin-induced acute renal injury in rats in a dose-dependent manner.

Happiness as a Buffer of the Association Between Dependence and Acute Tobacco Abstinence Effects in African American Smokers.

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Liautaud, Madalyn M; Leventhal, Adam M; Pang, Raina D

2017-09-27

African-American (AA) smokers are at disproportionate risk of tobacco dependence, utilizing smoking to regulate stress, and poor cessation outcomes. Positive emotional traits may function as coping factors that buffer the extent to which dependence increases vulnerability to adverse responses to acute tobacco abstinence (i.e., tobacco withdrawal). This laboratory study examined subjective happiness (SH; dispositional orientation towards frequent and intense positive affect [PA] and life satisfaction) as a moderator of the relation between tobacco dependence and subjective and behavioral abstinence effects among AA smokers. AA smokers (N=420, 39.0% female) completed self-report measures of tobacco dependence and SH followed by two counterbalanced experimental sessions (non-abstinent vs. 16-hr abstinent) involving self-report measures of composite withdrawal, urge to smoke, and mood, and a behavioral smoking task in which participants could: (a) earn money to delay smoking reinstatement, and (b) subsequently purchase cigarettes to smoke. Tobacco dependence was positively associated with increased abstinence effects in composite withdrawal, urge to smoke, PA, and latency to smoking reinstatement (pssmokers. The current study contributes to a growing body of literature examining the potentially advantageous role of positive emotional traits to smokers. We do so by identifying a relatively understudied psychological construct within tobacco research-subjective happiness-that may suppress the extent to which more severe tobacco dependence increases risk for subjective withdrawal-related distress during acute smoking abstinence in African American smokers. In doing so, the study provides a primer for future targeting of subjective happiness and other positive emotional traits as means to understand and treat acute tobacco abstinence effects among dependent African American smokers. © The Author 2017. Published by Oxford University Press on behalf of the Society for

Muscle glycogen metabolism changes in rats fed early postnatal a fructose-rich diet after maternal protein malnutrition: effects of acute physical exercise at the maximal lactate steady-state intensity.

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Cambri, Lucieli T; Ribeiro, Carla; Botezelli, José D; Ghezzi, Ana C; Mello, Maria Ar

2014-01-01

The objective was to evaluate the muscle glucose metabolism in rats fed a fructose-rich diet after fetal protein malnutrition, at rest and after acute physical exercise at maximal lactate steady-state intensity. The male offspring born of mothers fed on a balanced or low-protein diet were split in four groups until 60 days: Balanced (B): balanced diet during the whole period; Balanced/Fructose (BF): balanced diet in utero and fructose-rich diet after birth; Low protein/Balanced (LB): low-protein diet in utero and balanced diet after birth; Low protein/Fructose (LF): low protein diet in utero and fructose-rich diet after birth. Acute physical exercise reduced the muscle glycogen concentrations in all groups, although the LF group showed higher concentrations at rest. There was no difference among the groups in the glucose uptake and oxidation rates in the isolated soleus muscle neither at rest nor after acute exercise. However, glycogen synthesis was higher in the LF muscle than in the others at rest. Acute physical exercise increased glycogen synthesis in all groups, and the LF group showed the highest values. The fructose-rich diet administered in rats after fetal protein malnutrition alters muscle glycogen concentrations and glycogen synthesis in the rest and after acute exercise at maximal lactate steady-state intensity.

Down-regulation of outer membrane proteins by noncoding RNAs: unraveling the cAMP-CRP- and sigmaE-dependent CyaR-ompX regulatory case

DEFF Research Database (Denmark)

Johansen, Jesper; Eriksen, Maiken; Kallipolitis, Birgitte

2008-01-01

is sufficient to trigger the envelope stress response. Recent work indicates that small Hfq-binding RNAs play a major role in maintaining envelope homeostasis and, so far, two sigma(E)-dependent small noncoding RNAs (sRNAs), MicA and RybB, have been shown to facilitate rapid removal of multiple omp transcripts......The sigma(E) (extracytoplasmic stress response sigma factor in Escherichia coli) signaling system of Gram-negative bacteria plays an essential role in the maintenance of the extracytoplasmic compartment. Upon induction of this system, approximately 100 genes are up-regulated. The majority...... is also up-regulated, directly or indirectly, by sigma(E). In addition, this work identified MicA as a factor that cooperates in the negative control of ompX expression. The conservation of CyaR, MicA, RybB, and their targets suggests that the omp mRNA-sRNA regulatory network is an integral part...

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes

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Serrano, Pauline N. [Department of Chemistry, University of California, Davis CA 95616 USA; Wang, Hongxin [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; Crack, Jason C. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Prior, Christopher [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Hutchings, Matthew I. [School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ UK; Thomson, Andrew J. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Kamali, Saeed [University of Tennessee Space Institute, Tullahome TN 37388-9700 USA; Yoda, Yoshitaka [Research and Utilization Division, SPring-8/JASRI, 1-1-1 Kouto, Sayo Hyogo 679-5198 Japan; Zhao, Jiyong [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Hu, Michael Y. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Alp, Ercan E. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Oganesyan, Vasily S. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Le Brun, Nick E. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Cramer, Stephen P. [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA

2016-10-25

The reaction of protein-bound iron–sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe₂(NO)₄(Cys)₂]) and Roussin's Black Salt (RBS, [Fe₄(NO)₇S₃]. In the latter case, the absence of ³²S/³⁴S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain*

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Scruggs, Sarah B.; Reisdorph, Rick; Armstrong, Mike L.; Warren, Chad M.; Reisdorph, Nichole; Solaro, R. John; Buttrick, Peter M.

2010-01-01

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were

Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

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Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

2013-01-01

Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816

Report of the European Society of Cardiology Cardiovascular Round Table regulatory workshop update of the evaluation of new agents for the treatment of acute coronary syndrome

DEFF Research Database (Denmark)

Bueno, Héctor; de Graeff, Pieter; Richard-Lordereau, Isabelle

2016-01-01

syndrome (ACS) to achieve consistency with current knowledge in the field. This manuscript summarizes the key output from a collaborative workshop, organized by the Cardiovascular Round Table and the European Affairs Committee of the European Society of Cardiology, involving clinicians, academic...... researchers, trialists, European and US regulators, and pharmaceutical industry researchers. Specific questions in four key areas were selected as priorities for changes in regulatory guidance: patient selection, endpoints, methodologic issues and issues related to the research for novel agents. Patients......Regulatory authorities interpret the results of randomized controlled trials according to published principles. The European Medicines Agency (EMA) is planning a revision of the 2000 and 2003 guidance documents on clinical investigation of new medicinal products for the treatment of acute coronary...

Regulatory Roles for Long ncRNA and mRNA

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Marcel W. Coolen

2013-04-01

Full Text Available Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2 with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.

The PP2A Regulatory Subunit Tap46, a Component of the TOR Signaling Pathway, Modulates Growth and Metabolism in Plants[W

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Ahn, Chang Sook; Han, Jeong-A; Lee, Ho-Seok; Lee, Semi; Pai, Hyun-Sook

2011-01-01

Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway. PMID:21216945

Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

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Yazmín Ramiro-Cortés

Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.