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Sample records for mammalian p21-activated kinase-ii

  1. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

    PubMed

    Civiero, Laura; Cirnaru, Maria Daniela; Beilina, Alexandra; Rodella, Umberto; Russo, Isabella; Belluzzi, Elisa; Lobbestael, Evy; Reyniers, Lauran; Hondhamuni, Geshanthi; Lewis, Patrick A; Van den Haute, Chris; Baekelandt, Veerle; Bandopadhyay, Rina; Bubacco, Luigi; Piccoli, Giovanni; Cookson, Mark R; Taymans, Jean-Marc; Greggio, Elisa

    2015-12-01

    Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.

  2. P21 activated kinases

    PubMed Central

    Rane, Chetan K; Minden, Audrey

    2014-01-01

    The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer. PMID:24658305

  3. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  4. p21-activated kinase signaling in breast cancer

    PubMed Central

    Gururaj, Anupama E; Rayala, Suresh K; Kumar, Rakesh

    2005-01-01

    The p21-activated kinases signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. A wealth of information has accumulated at an impressive pace in the recent past, both with regard to previously identified targets for p21-activated kinases that regulate the actin cytoskeleton and cellular stress pathways and with regard to newly identified targets and their role in cancer. Emerging data also provide new clues towards a previously unappreciated link between these various cellular processes. The present review attempts to provide a quick tutorial to the reader about the evolving significance of p21-activated kinases and small GTPases in breast cancer, using information from mouse models, tissue culture studies, and human materials. PMID:15642175

  5. Signaling, Regulation, and Specificity of the Type II p21-activated Kinases*

    PubMed Central

    Ha, Byung Hak; Morse, Elizabeth M.; Turk, Benjamin E.; Boggon, Titus J.

    2015-01-01

    The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases. PMID:25855792

  6. Regulation of macropinocytosis by p21-activated kinase-1.

    PubMed

    Dharmawardhane, S; Schürmann, A; Sells, M A; Chernoff, J; Schmid, S L; Bokoch, G M

    2000-10-01

    The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

  7. Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens.

    PubMed

    John Von Freyend, Simona; Kwok-Schuelein, Terry; Netter, Hans J; Haqshenas, Gholamreza; Semblat, Jean-Philippe; Doerig, Christian

    2017-04-21

    Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

  8. Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens

    PubMed Central

    John von Freyend, Simona; Kwok-Schuelein, Terry; Netter, Hans J.; Haqshenas, Gholamreza; Semblat, Jean-Philippe; Doerig, Christian

    2017-01-01

    Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases. PMID:28430160

  9. Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase

    DTIC Science & Technology

    2002-06-01

    AD Award Number: DAMDl7-01-1-0149 TITLE: Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase PRINCIPAL...Tamoxifen Dependent Interaction Between the DAMD17-00-1-0114 Estrogen Receptor and a Novel P21 Activated Kinase 6. AUTHOR(S) Steven P. Balk, M.D., Ph.D. 7...Z, Karas RH, nisms of androgen receptor activation and function. J Mendelsohn ME, Shaul PW 1999 Estrogen receptor a Steroid Biochem Mol Biol 69:307

  10. A major second messenger mediator of Electrophorus electricus electric tissue is CaM kinase II.

    PubMed

    Gotter, A L; Kaetzel, M A; Dedman, J R

    1997-09-01

    Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca2+ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca(2+)-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca2+/calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca(2+)-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes.

  11. The role of p21-activated kinases in hepatocellular carcinoma metastasis

    PubMed Central

    2014-01-01

    The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed. PMID:25093037

  12. p21-Activated Kinase 1 Plays a Critical Role in Cellular Activation by Nef

    PubMed Central

    Fackler, Oliver T.; Lu, Xiaobin; Frost, Jeffrey A.; Geyer, Matthias; Jiang, Bing; Luo, Wen; Abo, Arie; Alberts, Arthur S.; Peterlin, B. Matija

    2000-01-01

    The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef. PMID:10713183

  13. Group II p21-activated kinases as therapeutic targets in gastrointestinal cancer

    PubMed Central

    Shao, Yang-Guang; Ning, Ke; Li, Feng

    2016-01-01

    P21-activated kinases (PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group I (PAK1-3) and group II (PAK4-6). Focus is currently shifting from group I PAKs to group II PAKs. Group II PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group II PAKs have become popular potential drug target candidates. However, few group II PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and “drug-like” properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group II PAKs, the importance of group II PAKs in the development and progression of gastrointestinal cancer, and small-molecule inhibitors of group II PAKs for the treatment of cancer. PMID:26811660

  14. p21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils

    PubMed Central

    Martyn, Kendra D.; Kim, Moon-Ju; Quinn, Mark T.; Dinauer, Mary C.; Knaus, Ulla G.

    2005-01-01

    The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays an instrumental role in host defense and contributes to microbicial killing by releasing highly reactive oxygen species. This multicomponent enzyme is composed of membrane and cytosolic components that assemble in the plasma membrane or phagolysosome. While the guanosine S′-triphosphatase (GTPase) Rac2 has been shown to be a critical regulator of NADPH oxidase activity and assembly, the role of its effector, p21-activated kinase (Pak), in oxidase function has not been well defined. Using HIV-1 Tat-mediated protein transduction of Pak inhibitory domain, we show here that Pak activity is indeed required for efficient superoxide generation in intact neutrophils. Furthermore, we show that Pak translocates to the plasma membrane upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation and colocalizes with translocated p47phox and with p22phox, a subunit of flavocytochrome b558. Although activated Pak phosphorylated several essential serine residues in the C-terminus of p47phox, direct binding to p47phox was not observed. In contrast, active Pak bound directly to p22phox, suggesting flavocytochrome b was the oxidase-associated membrane target of this kinase and this association may facilitate further phosphorylation of p47phox in the assembling NADPH oxidase complex. PMID:16099876

  15. Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase, PAK6

    PubMed Central

    Gao, Jia; Ha, Byung Hak; Lou, Hua Jane; Morse, Elizabeth M.; Zhang, Rong; Calderwood, David A.; Turk, Benjamin E.; Boggon, Titus J.

    2013-01-01

    The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies. PMID:24204982

  16. Redundant Canonical and Noncanonical Caenorhabditis elegans p21-Activated Kinase Signaling Governs Distal Tip Cell Migrations

    PubMed Central

    Peters, Eldon C.; Gossett, Andrea J.; Goldstein, Bob; Der, Channing J.; Reiner, David J.

    2013-01-01

    p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events. PMID:23390595

  17. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

    PubMed

    Luo, Ting; Xu, Yanhua; Hoffman, Trevor L; Zhang, Tailin; Schilling, Thomas; Sargent, Thomas D

    2007-04-01

    Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

  18. P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells.

    PubMed

    Babagana, Mahamat; Johnson, Sydney; Slabodkin, Hannah; Bshara, Wiam; Morrison, Carl; Kandel, Eugene S

    2017-01-04

    BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti-cancer agents, BRAF-inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi-insensitivity in many BRAF-mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi-resistance. We explored the role of p21-activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi-resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto-oncogenes increased BRAFi-tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK- and BRAF-inhibitors synergistically affected intrinsically BRAFi-resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well-known resistance mechanisms.

  19. Entamoeba histolytica RacC Selectively Engages p21-Activated Kinase Effectors

    PubMed Central

    2015-01-01

    Rho family GTPases modulate actin cytoskeleton dynamics by signaling through multiple effectors, including the p21-activated kinases (PAKs). The intestinal parasite Entamoeba histolytica expresses ∼20 Rho family GTPases and seven isoforms of PAK, two of which have been implicated in pathogenesis-related processes such as amoebic motility and invasion and host cell phagocytosis. Here, we describe two previously unstudied PAK isoforms, EhPAK4 and EhPAK5, as highly specific effectors of EhRacC. A structural model based on 2.35 Å X-ray crystallographic data of a complex between EhRacCQ65L·GTP and the EhPAK4 p21 binding domain (PBD) reveals a fairly well-conserved Rho/effector interface despite deviation of the PBD α-helix. A structural comparison with EhRho1 in complex with EhFormin1 suggests likely determinants of Rho family GTPase signaling specificity in E. histolytica. These findings suggest a high degree of Rho family GTPase diversity and specificity in the single-cell parasite E. histolytica. Because PAKs regulate pathogenesis-related processes in E. histolytica, they may be valid pharmacologic targets for anti-amoebiasis drugs. PMID:25529118

  20. A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization

    PubMed Central

    Cau, Julien; Faure, Sandrine; Comps, Michel; Delsert, Claude; Morin, Nathalie

    2001-01-01

    Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions. PMID:11733543

  1. Resveratrol regulates neuronal glucose uptake and insulin sensitivity via P21-activated kinase 2 (PAK2).

    PubMed

    Varshney, Pallavi; Dey, Chinmoy Sankar

    2017-04-01

    We have recently reported P21-activated kinase 2 (PAK2), a serine/threonine kinase as a negative regulator of neuronal glucose uptake and insulin sensitivity. Resveratrol (RSV), a natural polyphenol with anti-oxidative, anti-inflammatory and anti-diabetic properties, regulates PAK2 activity in HepG2 and ESC-B5 cell apoptosis. However, regulation of PAK2 by RSV in neuronal insulin signaling pathway, if any, is still unknown. In the present study, RSV treatment significantly increased PAK2 activity under insulin-sensitive and insulin-resistant condition, along with a marked decrease in glucose uptake in differentiated N2A cells. Pretreatment with AMPK inhibitor, followed by RSV treatment resulted in reduction in PAK2 activity whereas glucose uptake showed an increase. However, pretreatment with Akt inhibitor and then RSV exposure significantly increased PAK2 activity, with a corresponding decrease in glucose uptake. RSV treatment increased AMPK activity and decreased Akt activity. In conclusion, RSV negatively regulates neuronal glucose uptake and insulin sensitivity via PAK2. Copyright © 2017. Published by Elsevier Inc.

  2. p21-activated kinase 4 regulates mitotic spindle positioning and orientation

    PubMed Central

    Bompard, Guillaume; Morin, Nathalie

    2012-01-01

    During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex.1 Spindle orientation is also regulated by integrin-mediated cell adhesion2 and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell.3 Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis.   In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial.4 Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division.5 In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation. PMID:22960742

  3. p21-activated kinase 4 regulates mitotic spindle positioning and orientation.

    PubMed

    Bompard, Guillaume; Morin, Nathalie

    2012-01-01

    During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex. Spindle orientation is also regulated by integrin-mediated cell adhesion and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell. Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial. Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division. In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation.

  4. Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation

    PubMed Central

    Aix, Esther; Gutiérrez-Gutiérrez, Óscar; Sánchez-Ferrer, Carlota; Aguado, Tania

    2016-01-01

    The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc−/−) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc−/− newborns but rescued in G3 Terc−/−/p21−/− mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts. PMID:27241915

  5. LASS5 Interacts with SDHB and Synergistically Represses p53 and p21 Activity

    PubMed Central

    Jiang, Z.; Li, F.; Wan, Y.; Han, Z.; Yuan, W.; Cao, L.; Deng, Y.; Peng, X.; Chen, F.; Fan, X.; Liu, X.; Dai, G.; Wang, Y.; Zeng, Q.; Shi, Y.; Zhou, Z.; Chen, Y.; Xu, W.; Luo, S.; Chen, S.; Ye, X.; Mo, X.; Wu, X.; Li, Y.

    2017-01-01

    Longevity Assurance 5 (LASS5), a member of the LASS/Ceramide Synthases family, synthesizes C16-ceramide and is implicated in tumor biology. However, its precise role is not yet well understood. A yeast two-hybrid screen was performed using a human cDNA library to identify potential LASS5-interaction partners. One identified clone encodes succinate dehydrogenase subunit B (SDHB). Mammalian two-hybrid assays showed that LASS5 interacts with SDHB, and the result was also confirmed by GST pull-down and co-immunoprecipitation assays. The C-terminal fragment of SDHB was required for the interaction. LASS5 and SDHB were co-localized in COS-7 cells. LASS5 and SDHB expressions were found to be up-regulated in neuroglioma tissue. Transfection assays showed that LASS5 or SDHB expression repressed p53 or p21 reporter activity, respectively. Simultaneous LASS5 and SDHB expression resulted in stronger repression of p53 and p21 reporter activity, suggesting that LASS5 and SDHB interaction may synergistically affect transcriptional regulation of p53 and p21. Our data provide new molecular insights into potential roles of LASS5 and SDHB in tumor biology. PMID:27280497

  6. Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

    SciTech Connect

    Germane, Katherine L.; Spiller, Benjamin W.

    2011-09-20

    Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

  7. The Cbl proteins are binding partners for the Cool/Pix family of p21-activated kinase-binding proteins.

    PubMed

    Flanders, James A; Feng, Qiyu; Bagrodia, Shubha; Laux, Maria T; Singavarapu, Avinash; Cerione, Richard A

    2003-08-28

    Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21-activated kinase (PAK). Using the yeast two-hybrid screen, we identified Cbl-b as a Cool family binding partner. We co-immunoprecipitated endogenous Cool and Cbl-b from a variety of breast cancer cell lines. The Cool-Cbl-b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins. Expression of Cbl-b effectively blocks the ability of Cool-2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl-b acts as a negative regulator for signaling activities requiring PAK activation.

  8. Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain.

    PubMed

    Kim, Duk-Joong; Choi, Chang-Ki; Lee, Chan-Soo; Park, Mee-Hee; Tian, Xizhe; Kim, Nam Doo; Lee, Kee-In; Choi, Joong-Kwon; Ahn, Jin Hee; Shin, Eun-Young; Shin, Injae; Kim, Eung-Gook

    2016-04-29

    p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.

  9. Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.

    PubMed

    Albin, Stephanie D; Davis, Graeme W

    2004-08-04

    Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

  10. CaM Kinase II-dependent pathophysiological signalling in endothelial cells.

    PubMed

    Cai, Hua; Liu, Depei; Garcia, Joe G N

    2008-01-01

    Calcium/calmodulin-dependent protein kinase II (CaM Kinase II) is a known modulator of cardiac pathophysiology. The present review uniquely focuses on novel CaM Kinase II-mediated endothelial cell signalling which, under pathophysiological conditions, may indirectly modulate cardiac functions via alterations in endothelial or endocardial responses. CaM Kinase II has four different isoforms and various splicing variants for each isoform. The endothelial cell CaM Kinase II isoforms are sensitive to KN93 and a threonine 286-mutated inhibitory peptide. In macrovascular endothelial cells derived from aortas, CaM Kinase II mediates redox-sensitive upregulation of endothelial nitric oxide synthase (eNOS) gene expression by hydrogen peroxide (H2O2) and oscillatory shear stress, and a rapid activation of eNOS in response to bradykinin. In endothelial cells derived from lung microvessels, CaM Kinase II mediates barrier dysfunction, particularly when activated by thrombin. In brain capillary endothelial cells, CaM Kinase II lies upstream of voltage-gated potassium channels and hypoxia-induced cell swelling. In both macrovascular and microvascular endothelial cells, CaM Kinase II mediates actin cytoskeleton reorganization via distinct p38 MAPK/HSP27 and ERK1/2/MLCK signalling pathways, respectively. Although understanding of endothelium-specific CaM Kinase II signalling is nascent, data accumulated so far have demonstrated a potentially significant role of CaM Kinase II in endothelial cell pathophysiology.

  11. Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

    PubMed Central

    Mira, Jean-Paul; Benard, Valerie; Groffen, John; Sanders, Luraynne C.; Knaus, Ulla G.

    2000-01-01

    Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth. PMID:10618392

  12. Novel p21-Activated Kinase 4 (PAK4) Allosteric Modulators Overcome Drug Resistance and Stemness in Pancreatic Ductal Adenocarcinoma.

    PubMed

    Aboukameel, Amro; Muqbil, Irfana; Senapedis, William; Baloglu, Erkan; Landesman, Yosef; Shacham, Sharon; Kauffman, Michael; Philip, Philip A; Mohammad, Ramzi M; Azmi, Asfar S

    2017-01-01

    The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases and is found to be overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells but not in normal human pancreatic ductal epithelia (HPDE). Gene copy number amplification studies in PDAC patient cohorts confirmed PAK4 amplification making it an attractive therapeutic target in PDAC. We investigated the antitumor activity of novel PAK4 allosteric modulators (PAM) on a panel of PDAC cell lines and chemotherapy-resistant flow-sorted PDAC cancer stem cells (CSC). The toxicity and efficacy of PAMs were evaluated in multiple subcutaneous mouse models of PDAC. PAMs (KPT-7523, KPT-7189, KPT-8752, KPT-9307, and KPT-9274) show antiproliferative activity in vitro against different PDAC cell lines while sparing normal HPDE. Cell growth inhibition was concurrent with apoptosis induction and suppression of colony formation in PDAC. PAMs inhibited proliferation and antiapoptotic signals downstream of PAK4. Co-immunoprecipitation experiments showed disruption of PAK4 complexes containing vimentin. PAMs disrupted CSC spheroid formation through suppression of PAK4. Moreover, PAMs synergize with gemcitabine and oxaliplatin in vitro KPT-9274, currently in a phase I clinical trial (clinicaltrials.gov; NCT02702492), possesses desirable pharmacokinetic properties and is well tolerated in mice with the absence of any signs of toxicity when 200 mg/kg daily is administered either intravenously or orally. KPT-9274 as a single agent showed remarkable antitumor activity in subcutaneous xenograft models of PDAC cell lines and CSCs. These proof-of-concept studies demonstrated the antiproliferative effects of novel PAMs in PDAC and warrant further clinical investigations. Mol Cancer Ther; 16(1); 76-87. ©2016 AACR.

  13. p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.

    PubMed

    DeSantiago, Jaime; Bare, Dan J; Xiao, Lei; Ke, Yunbo; Solaro, R John; Banach, Kathrin

    2014-02-01

    Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca(2+) handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1(-/-)) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca(2+) transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1(-/-) VMs during 15 min of simulated ischemia. However, Pak1(-/-) VMs exhibited an exaggerated increase in [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) overload in Pak1(-/-) VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1(-/-) VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47(phox-/-)) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1(-/-) VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca(2+) overload in Pak1(-/-) VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Functional integrity of the t-tubular system in cardiomyocytes depends on p21-activated kinase 1

    PubMed Central

    DeSantiago, Jaime; Bare, Dan J; Ke, Yunbo; Sheehan, Katherine A.; Solaro, R. John; Banach, Kathrin

    2013-01-01

    p21-activated kinase (Pak1), a serine-threonine protein kinase, regulates cytoskeletal dynamics and cell motility. Recent experiments further demonstrate that loss of Pak1 results in exaggerated hypertrophic growth in response to pathophysiological stimuli. Calcium (Ca) signaling plays an important role in the regulation of transcription factors involved in hypertrophic remodeling. Here we aimed to determine the role of Pak1 in cardiac excitation-contraction coupling (ECC). Ca transients were recorded in isolated, ventricular myocytes (VMs) from WT and Pak1−/− mice. Pak1−/− Ca transients had a decreased amplitude, prolonged rise time and delayed recovery time. Di-8-ANNEPS staining revealed a decreased t-tubular density in Pak1−/− VMs that coincided with decreased cell capacitance and increased dis-synchrony of Ca induced Ca release (CICR) at individual release units. These changes were not observed in atrial myocytes of Pak1−/− mice where the t-tubular system is only sparsely developed. Experiments in cultured rabbit VMs supported a role of Pak1 in the maintenance of the t-tubular structure. T-tubular density in rabbit VMs significantly decreased within 24h of culture. This was accompanied by a decrease of the Ca transient amplitude and a prolongation of its rise time. However, overexpression of constitutively active Pak1 in VMs attenuated the structural remodeling as well as changes in ECC. The results provide significant support for a prominent role of Pak1 activity not only in the functional regulation of ECC but for the structural maintenance of the t-tubular system whose remodeling is an integral feature of hypertrophic remodeling. PMID:23612118

  15. p21-activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes

    PubMed Central

    DeSantiago, Jaime; Bare, Dan J; Xiao, Lei; Ke, Yunbo; Solaro, R. John; Banach, Kathrin

    2014-01-01

    Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca2+ handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1-/-) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca2+ transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1-/- VMs during 15 min of simulated ischemia. However, Pak1-/- VMs exhibited an exaggerated increase in [Ca2+]i, which resulted in spontaneous Ca2+ release events and waves. The Ca2+ overload in Pak1-/- VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1-/- VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47phox-/-) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1-/- VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca2+ overload in Pak1-/- VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca2+ overload under conditions where no significant changes in excitation-contraction coupling are yet evident. PMID:24380729

  16. p-21-Activated kinase 1 mediates gastrin-stimulated proliferation in the colorectal mucosa via multiple signaling pathways.

    PubMed

    Huynh, Nhi; Yim, Mildred; Chernoff, Jonathan; Shulkes, Arthur; Baldwin, Graham S; He, Hong

    2013-03-15

    Gastrins, including amidated (Gamide) and glycine-extended (Ggly) forms, function as growth factors for the gastrointestinal mucosa. The p-21-activated kinase 1 (PAK1) plays important roles in growth factor signaling networks that control cell motility, proliferation, differentiation, and transformation. PAK1, activated by both Gamide and Ggly, mediates gastrin-stimulated proliferation and migration, and activation of β-catenin, in gastric epithelial cells. The aim of this study was to investigate the role of PAK1 in the regulation by gastrin of proliferation in the normal colorectal mucosa in vivo. Mucosal proliferation was measured in PAK1 knockout (PAK1 KO) mice by immunohistochemistry. The expression of phosphorylated and unphosphorylated forms of the signaling molecules PAK1, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT), and the expression of β-catenin and its downstream targets c-Myc and cyclin D1, were measured in gastrin knockout (Gas KO) and PAK1 KO mice by Western blotting. The expression and activation of PAK1 are decreased in Gas KO mice, and these decreases are associated with reduced activation of ERK, AKT, and β-catenin. Proliferation in the colorectal mucosa of PAK1 KO mice is reduced, and the reduction is associated with reduced activation of ERK, AKT, and β-catenin. In compensation, antral gastrin mRNA and serum gastrin concentrations are increased in PAK1 KO mice. These results indicate that PAK1 mediates the stimulation of colorectal proliferation by gastrins via multiple signaling pathways involving activation of ERK, AKT, and β-catenin.

  17. In vivo yeast cell morphogenesis is regulated by a p21-activated kinase in the human pathogen Penicillium marneffei.

    PubMed

    Boyce, Kylie J; Schreider, Lena; Andrianopoulos, Alex

    2009-11-01

    Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37 degrees C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37 degrees C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37 degrees C. Loss of pakB also leads to the inappropriate production of yeast cells at 25 degrees C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.

  18. Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth

    PubMed Central

    Murray, Brion W.; Guo, Chuangxing; Piraino, Joseph; Westwick, John K.; Zhang, Cathy; Lamerdin, Jane; Dagostino, Eleanor; Knighton, Daniel; Loi, Cho-Ming; Zager, Michael; Kraynov, Eugenia; Popoff, Ian; Christensen, James G.; Martinez, Ricardo; Kephart, Susan E.; Marakovits, Joseph; Karlicek, Shannon; Bergqvist, Simon; Smeal, Tod

    2010-01-01

    Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (Kd = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers. PMID:20439741

  19. Group I p21-activated kinases regulate thyroid cancer cell migration and are overexpressed and activated in thyroid cancer invasion.

    PubMed

    McCarty, Samantha K; Saji, Motoyasu; Zhang, Xiaoli; Jarjoura, David; Fusco, Alfredo; Vasko, Vasyl V; Ringel, Matthew D

    2010-12-01

    p21-activated kinases (PAKs) are a family of serine/threonine kinases that regulate cytoskeletal dynamics and cell motility. PAKs are subdivided into group I (PAKs 1-3) and group II (PAKs 4-6) on the basis of structural and functional characteristics. Based on prior gene expression data that predicted enhanced PAK signaling in the invasive fronts of aggressive papillary thyroid cancers (PTCs), we hypothesized that PAKs functionally regulate thyroid cancer cell motility and are activated in PTC invasive fronts. We examined PAK isoform expression in six human thyroid cancer cell lines (BCPAP, KTC1, TPC1, FTC133, C643, and SW1746) by quantitative reverse transcription-PCR and western blot. All cell lines expressed PAKs 1-4 and PAK6 mRNA and PAKs 1-4 protein; PAK6 protein was variably expressed. Samples from normal and malignant thyroid tissues also expressed PAKs 1-4 and PAK6 mRNA; transfection with the group I (PAKs 1-3) PAK-specific p21 inhibitory domain molecular inhibitor reduced transwell filter migration by ∼50% without altering viability in all cell lines (P<0.05). BCPAP and FTC133 cells were transfected with PAK1, PAK2, or PAK3-specific small interfering RNA (siRNA); only PAK1 siRNA reduced migration significantly for both cell lines. Immunohistochemical analysis of seven invasive PTCs demonstrated an increase in PAK1 and pPAK immunoactivity in the invasive fronts versus the tumor center. In conclusion, PAK isoforms are expressed in human thyroid tissues and cell lines. PAK1 regulates thyroid cancer cell motility, and PAK1 and pPAK levels are increased in PTC invasive fronts. These data implicate PAKs as regulators of thyroid cancer invasion.

  20. Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

    PubMed

    Chu, Ji; Pham, Ngoc T; Olate, Nicole; Kislitsyna, Karina; Day, Mary-Clare; LeTourneau, Phillip A; Kots, Alexander; Stewart, Randolph H; Laine, Glen A; Cox, Charles S; Uray, Karen

    2013-01-11

    Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

  1. p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase.

    PubMed

    Barrows, Douglas; Schoenfeld, Sarah M; Hodakoski, Cindy; Silkov, Antonina; Honig, Barry; Couvillon, Anthony; Shymanets, Aliaksei; Nürnberg, Bernd; Asara, John M; Parsons, Ramon

    2015-11-27

    Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP3 or Gβγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP3 and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.

  2. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  3. p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase*

    PubMed Central

    Barrows, Douglas; Schoenfeld, Sarah M.; Hodakoski, Cindy; Silkov, Antonina; Honig, Barry; Couvillon, Anthony; Shymanets, Aliaksei; Nürnberg, Bernd; Asara, John M.; Parsons, Ramon

    2015-01-01

    Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP3 or Gβγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP3 and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated. PMID:26438819

  4. Identification of Novel Gene Targets and Functions of p21-Activated Kinase 1 during DNA Damage by Gene Expression Profiling

    PubMed Central

    Motwani, Mona; Li, Da-Qiang; Horvath, Anelia; Kumar, Rakesh

    2013-01-01

    P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1

  5. Targeting of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Colbran, Roger J

    2004-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types. PMID:14653781

  6. Casein kinase II inhibition induces apoptosis in pancreatic cancer cells.

    PubMed

    Hamacher, Rainer; Saur, Dieter; Fritsch, Ralph; Reichert, Maximilian; Schmid, Roland M; Schneider, Günter

    2007-09-01

    Pancreatic cancer is one of the most common causes of cancer death in western civilization. The five-year survival rate is below 1% and of the 10% of patients with resectable disease only around one-fifth survives 5 years. Survival rates have not changed much during the last 20 years, demonstrating the inefficacy of current available therapies. To improve the prognosis of pancreatic cancer, there is the need to develop effective non-surgical treatment for this disease. The protein kinase casein kinase II (CK2) is a ubiquitously expressed serine-threonine kinase and its activity is enhanced in all human tumors examined so far. The contribution of CK2 to the tumor maintenance of pancreatic cancer has not been investigated. To investigate the function of CK2 in pancreatic cancer cells we used the CK2 specific inhibitors 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole and Apigenin. Furthermore, we interfered with CK2 expression using CK2 specific siRNAs. Interfering with CK2 function led to a reduction of pancreatic cancer cell viability, which was due to caspase-dependent apoptosis. The induction of apoptosis correlated with a reduced NF-kappaB-dependent transcriptional activity. This study validates CK2 as a molecular drug target in a preclinical in vitro model of pancreatic cancer.

  7. Testing the Role of p21-Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease

    DTIC Science & Technology

    2016-06-01

    2014 - 5/14/ 2017 10.0% DOD $190,195 1.20 calendar Testing the Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically...Chernoff) 2/1/2016 - 1/31/ 2017 5.0% BCA $92,593 0.60 calendar Dissecting a Breast Cancer Amplicon using CRISPR/Cas The major goal of this...Boumber, UNMCC) 9/15/2015 - 9/14/ 2017 2.0% DOD No Salary 0.24 calendar Msi2 Regulates the Aggressiveness of Non-Small Cell Lung Cancer (NSCLC

  8. Myc oncoproteins are phosphorylated by casein kinase II.

    PubMed Central

    Lüscher, B; Kuenzel, E A; Krebs, E G; Eisenman, R N

    1989-01-01

    Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus. Images PMID:2663470

  9. Depletion of casein kinase II by antisense oligonucleotide prevents neuritogenesis in neuroblastoma cells.

    PubMed Central

    Ulloa, L; Díaz-Nido, J; Avila, J

    1993-01-01

    Casein kinase II is a multifunctional protein kinase which has been implicated in the regulation of cell growth and differentiation. This enzyme is much more abundant in neurons than in any other cell type. The treatment of neuroblastoma cells with an antisense oligodeoxyribonucleotide which specifically results in the depletion of casein kinase II catalytic subunits blocks neuritogenesis. Accordingly, this enzyme may perform an essential role during neurite growth in developing neurons. Casein kinase II depletion induced by antisense oligodeoxyribonucleotide is accompanied by a site-specific dephosphorylation of microtubule-associated protein MAP1B (also referred to as MAP5, MAP1.X or MAP1.2), which is paralleled by a release of MAP1B from microtubules. We therefore propose that phosphorylation by casein kinase II may be required for the proper MAP1B functioning in the promotion of the assembly of microtubules which constitute the cytoskeletal scaffolding of growing axon-like neurites. Images PMID:8467810

  10. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  11. A protein related to p21-activated kinase (PAK) that is involved in neurogenesis in the Drosophila adult central nervous system.

    PubMed

    Melzig, J; Rein, K H; Schäfer, U; Pfister, H; Jäckle, H; Heisenberg, M; Raabe, T

    1998-11-05

    Brains are organized by the developmental processes generating them. The embryonic neurogenic phase of Drosophila melanogaster has been studied in detail at the genetic, cellular and molecular level. In contrast, much of what is known of postembryonic brain development has been gathered by neuroanatomical and gene expression studies. The molecular mechanisms underlying cellular diversity and structural organisation in the adult brain, such as the establishment of the correct neuroblast number, the spatial and temporal control of neuroblast proliferation, cell fate determination, and the generation of the precise pattern of neuronal connectivity, are largely unknown. In a screen for viable mutations affecting adult central brain structures, we isolated the mushroom bodies tiny (mbt) gene of Drosophila, which encodes a protein related to p21-activated kinase (PAK). We show that mutations in mbt primarily interfere with the generation or survival of the intrinsic cells (Kenyon cells) of the mushroom body, a paired neuropil structure in the adult brain involved in learning and memory.

  12. Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.

    PubMed Central

    Butler, L S; Silva, A J; Abeliovich, A; Watanabe, Y; Tonegawa, S; McNamara, J O

    1995-01-01

    Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory. Images Fig. 2 PMID:7624331

  13. Crystal structure of the N-terminal SH3 domain of mouse {beta}PIX, p21-activated kinase-interacting exchange factor

    SciTech Connect

    Li Xiaofeng; Liu Xueqi; Sun Fei; Gao Jia; Zhou Hongwei; Gao, George F.; Bartlam, Mark; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn

    2006-01-06

    The mouse {beta}PIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3{sub 2}21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact {beta}-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse {beta}PIX-SH3 domain binding the way in which the {beta}PIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse {beta}PIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse {beta}PIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of {beta}PIX-SH3 domain resulting in its multiple biological functions.

  14. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  15. Inhibition of group 1 p21-activated kinases suppresses pancreatic stellate cell activation and increases survival of mice with pancreatic cancer.

    PubMed

    Yeo, Dannel; Phillips, Phoebe; Baldwin, Graham S; He, Hong; Nikfarjam, Mehrdad

    2017-05-01

    Pancreatic cancer remains one of the most lethal of all solid tumors. Pancreatic stellate cells (PSCs) are primarily responsible for the fibrosis that constitutes the stroma and p21-activated kinase 1 (PAK1) may have a role in signalling pathways involving PSCs. This study aimed to examine the role of PAK1 in PSCs and in the interaction of PSCs with pancreatic cancer cells. Human PSCs were isolated using the modified outgrowth method. The effect of inhibiting PAK1 with group 1 PAK inhibitor, FRAX597, on cell proliferation and apoptosis in vitro was measured by thymidine incorporation and annexin V assays, respectively. The effect of depleting host PAK1 on the survival of mice with pancreatic Pan02 cell tumors was evaluated using PAK1 knockout (KO) mice. PAK1 was expressed in isolated PSCs. FRAX597 reduced the activation of PSCs, inhibited PSC proliferation, and increased PSC apoptosis at least in partial by inhibiting PAK1 activity. The decreased expression and activity of PAK1 in PAK1 KO mice tumors was associated with an increased mouse survival. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis. Targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer.

  16. Group I p21-activated kinases facilitate Tax-mediated transcriptional activation of the human T-cell leukemia virus type 1 long terminal repeats

    PubMed Central

    2013-01-01

    Background Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and tropical spastic paraparesis. HTLV-1 encodes transactivator protein Tax that interacts with various cellular factors to modulate transcription and other biological functions. Additional cellular mediators of Tax-mediated transcriptional activation of HTLV-1 long terminal repeats (LTR) remain to be identified and characterized. Results In this study, we investigated the regulatory role of group I p21-activated kinases (Paks) in Tax-induced LTR activation. Both wild-type and kinase-dead mutants of Pak3 were capable of potentiating the activity of Tax to activate LTR transcription. The effect of Paks on the LTR was attributed to the N-terminal regulatory domain and required the action of CREB, CREB-regulating transcriptional coactivators (CRTCs) and p300/CREB-binding protein. Paks physically associated with Tax and CRTCs. Paks were recruited to the LTR in the presence of Tax. siRNAs against either Pak1 or Pak3 prevented the interaction of Tax with CRTC1 and the recruitment of Tax to the LTR. These siRNAs also inhibited LTR-dependent transcription in HTLV-1-transformed MT4 cells and in cells transfected with an infectious clone of HTLV-1. Conclusion Group I Paks augment Tax-mediated transcriptional activation of HTLV-1 LTR in a kinase-independent manner. PMID:23622267

  17. p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

    PubMed Central

    Tyagi, Nikhil; Marimuthu, Saravanakumar; Bhardwaj, Arun; Deshmukh, Sachin K.; Srivastava, Sanjeev K.; Singh, Ajay P.; McClellan, Steven; Carter, James E.; Singh, Seema

    2015-01-01

    Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24+/CD44+/EpCAM+) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24−/CD44−/EpCAM−) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemo-sensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC. PMID:26546043

  18. Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach.

    PubMed

    Al-Shboul, Othman; Mustafa, Ayman

    2015-06-01

    Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzyme-linked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress.

  19. p21-activated kinase 2 regulates HSPC cytoskeleton, migration, and homing via CDC42 activation and interaction with β-Pix

    PubMed Central

    Reddy, Pavankumar N. G.; Radu, Maria; Xu, Ke; Wood, Jenna; Harris, Chad E.; Chernoff, Jonathan

    2016-01-01

    Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (Pak2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating Pak2 functions in HSPCs are unknown. Here, we demonstrate that both Pak2 kinase activity and its interaction with the PAK-interacting exchange factor-β (β-Pix) are required to reconstitute defective Pak2Δ/Δ HSPC homing to the BM. Pak2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas Pak2 interaction with β-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in Pak2Δ/Δ HSPCs were rescued by wild-type (WT) Pak2 but not by a Pak2-kinase dead mutant (KD). Expression of a β-Pix interaction-defective mutant of Pak2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of Pak2, we found a paradoxical decrease in baseline activation of CDC42 in Pak2Δ/Δ HSPCs, which was rescued by expression of Pak2-WT but not by Pak2-KD; defective homing of Pak2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both Pak2 kinase activity and its interaction with β-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of Pak2 in HSPC migration and homing. PMID:26932803

  20. Hair Growth Promoting and Anticancer Effects of p21-activated kinase 1 (PAK1) Inhibitors Isolated from Different Parts of Alpinia zerumbet.

    PubMed

    Taira, Nozomi; Nguyen, Binh Cao Quan; Tawata, Shinkichi

    2017-01-14

    PAK1 (p21-activated kinase 1) is an emerging target for the treatment of hair loss (alopecia) and cancer; therefore, the search for PAK1 blockers to treat these PAK1-dependent disorders has received much attention. In this study, we evaluated the anti-alopecia and anticancer effects of PAK1 inhibitors isolated from Alpinia zerumbet (alpinia) in cell culture. The bioactive compounds isolated from alpinia were found to markedly promote hair cell growth. Kaempferol-3-O-β-d-glucuronide (KOG) and labdadiene, two of the isolated compounds, increased the proliferation of human follicle dermal papilla cells by approximately 117%-180% and 132%-226%, respectively, at 10-100 μM. MTD (2,5-bis(1E,3E,5E)-6-methoxyhexa-1,3,5-trien-1-yl)-2,5-dihydrofuran) and TMOQ ((E)-2,2,3,3-tetramethyl-8-methylene-7-(oct-6-en-1-yl)octahydro-1H-quinolizine) showed growth-promoting activity around 164% and 139% at 10 μM, respectively. The hair cell proliferation induced by these compounds was significantly higher than that of minoxidil, a commercially available treatment for hair loss. Furthermore, the isolated compounds from alpinia exhibited anticancer activity against A549 lung cancer cells with IC50 in the range of 67-99 μM. Regarding the mechanism underlying their action, we hypothesized that the anti-alopecia and anticancer activities of these compounds could be attributed to the inhibition of the oncogenic/aging kinase PAK1.

  1. Activation of cAMP signaling attenuates impaired hepatic glucose disposal in aged male p21-activated protein kinase-1 knockout mice.

    PubMed

    Chiang, Yu-Ting Alex; Ip, Wilfred; Shao, Weijuan; Song, Zhuolun Eric; Chernoff, Jonathan; Jin, Tianru

    2014-06-01

    p21-activated protein kinase-1 (Pak1) plays a role in insulin secretion and glucagon-like peptide-1 (GLP-1) production. Pak1(-/-) mice were found to carry a defect in ip pyruvate tolerance test (IPPTT), leading us to speculate whether Pak1 represses hepatic gluconeogenesis. We show here that the defect in IPPTT became more severe in aged Pak1(-/-) mice. In primary hepatocytes, 2,2'-dihydroxy-1,1'-dinaphthyldisulfide, a potent inhibitor of group I Paks, reduced basal glucose production (GP), attenuated forskolin- or glucagon-stimulated GP, and attenuated the stimulation of forskolin on the expression of Pck1 and G6pc. In addition, the capacity of primary hepatocytes isolated from Pak1(-/-) mice in GP at the basal level is significantly lower than that of the control littermates. These in vitro observations imply that the direct effect of Paks in hepatocytes is the stimulation of gluconeogenesis and that the impairment in IPPTT in Pak1(-/-) mice is due to the lack of Pak1 elsewhere. Consecutive ip injection of forskolin for 2 weeks increased gut proglucagon expression, associated with improved IPPTT in aged Pak1(-/-) mice and wild-type controls. In addition, administration of the DPP-IV (dipeptidyl peptidase-4) inhibitor sitagliptin for 1 week reversed the defect in IPPTT in aged Pak1(-/-) mice, associated with increased plasma GLP-1 levels. Our observations indicate a potential role of Pak1 in the gut/pancreas/liver axis in controlling glucose disposal and affirmed the therapeutic application of GLP-1 and DPP-IV inhibitors in attenuating hepatic gluconeogenesis.

  2. Transcriptional regulation of ataxia-telangiectasia and Rad3-related protein by activated p21-activated kinase-1 protects keratinocytes in UV-B-induced premalignant skin lesions.

    PubMed

    Beesetti, S; Mavuluri, J; Surabhi, R P; Oberyszyn, T M; Tober, K; Pitani, R S; Joseph, L D; Venkatraman, G; Rayala, S K

    2017-07-10

    Sun-induced skin lesions, in particular actinic keratosis, are generally considered as premalignant skin lesions that can progress into squamous cell carcinoma (SCC) and invasive SCC if left untreated. Therefore, understanding the molecular mechanisms by which the ultraviolet-B (UV-B)-exposed cells are being protected and the signaling pathways that promote the progression of certain premalignant skin lesions to malignant lesions will permit us to prevent or cure skin cancers. In the current study, we found that phospho-p21-activated kinase-1 (Pak1) and Pak1 expression was high in clinical samples of sunlight-induced premalignant skin lesions assessed by immunohistochemistry. Further, we observed that phospho-Pak1 and Pak1 levels are high in UV-B-exposed hairless SKH mouse model skin samples as compared with unexposed skin tissue. Our results from cell line and animal models showed that Pak1 is activated in response to UV-B radiation, and this activated Pak1 translocates from the cytoplasm to the nucleus. Inside the nucleus, Pak1 via C-Fos binds to a specific promoter region of DNA repair kinase ATR (ataxia-telangiectasia and Rad3-related protein) and acts as a transcriptional regulator of ATR. Results from our analysis showed that Pak1 overexpression, knockdown and Pak1 knockout cell line models showed that Pak1 confers protection to keratinocytes from UV-B-induced apoptosis and DNA damage via ATR. To our knowledge, this is the first study that evaluates the functional and clinical significance of a signaling molecule, Pak1, in sun-induced premalignant skin lesions and indicates that increased Pak1 activation and expression could serve as an early warning sign of progression toward non-melanoma skin cancer, if ignored.Oncogene advance online publication, 10 July 2017; doi:10.1038/onc.2017.218.

  3. Dissociation of Crk-associated substrate from the vimentin network is regulated by p21-activated kinase upon acetylcholine activation of airway smooth muscle

    PubMed Central

    Wang, Ruping; Li, Qing-Fen; Anfinogenova, Yana; Tang, Dale D.

    2006-01-01

    The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, stimulation of tracheal smooth muscle strips with acetylcholine (ACh) resulted in the increase in ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly) in association with force development. Activation with ACh also induced vimentin phosphorylation at Ser-56 as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips upon contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The decrease in distribution of CAS in cytoskeletal vimentin elicited by ACh was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble vimentin to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser-56 and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated the dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during acetylcholine activation of tracheal smooth muscle tissues. PMID:16997882

  4. P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

    PubMed Central

    Wang, Yiming; Gratzke, Christian; Tamalunas, Alexander; Wiemer, Nicolas; Ciotkowska, Anna; Rutz, Beata; Waidelich, Raphaela; Strittmatter, Frank; Liu, Chunxiao; Stief, Christian G.; Hennenberg, Martin

    2016-01-01

    Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under

  5. Aberrant calcium/calmodulin-dependent protein kinase II (CaMKII) activity is associated with abnormal dendritic spine morphology in the ATRX mutant mouse brain.

    PubMed

    Shioda, Norifumi; Beppu, Hideyuki; Fukuda, Takaichi; Li, En; Kitajima, Isao; Fukunaga, Kohji

    2011-01-05

    In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the sucrose-nonfermenting 2 family, cause several mental retardation disorders, including α-thalassemia X-linked mental retardation syndrome. We generated ATRX mutant mice lacking exon 2 (ATRX(ΔE2) mice), a mutation that mimics exon 2 mutations seen in human patients and associated with milder forms of retardation. ATRX(ΔE2) mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). Consistent with other mouse models of mental retardation, ATRX(ΔE2) mice exhibited longer and thinner dendritic spines compared with wild-type mice without changes in spine number. Interestingly, aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRX(ΔE2) mice. Increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-guanine nucleotide exchange factors (GEFs) T-cell lymphoma invasion and metastasis 1 (Tiam1) and kalirin-7, known substrates of CaMKII. We confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRX(ΔE2) mice. In cultured cortical neurons, PP1 inhibition by okadaic acid increased CaMKII-dependent Tiam1 and kalirin-7 phosphorylation. Together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes plus elevated Rac1-GEF/PAK signaling seen in ATRX(ΔE2) mice may contribute to mental retardation syndromes seen in human patients.

  6. p21-activated kinase 2 regulates HSPC cytoskeleton, migration, and homing via CDC42 activation and interaction with β-Pix.

    PubMed

    Reddy, Pavankumar N G; Radu, Maria; Xu, Ke; Wood, Jenna; Harris, Chad E; Chernoff, Jonathan; Williams, David A

    2016-04-21

    Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (Pak2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating Pak2 functions in HSPCs are unknown. Here, we demonstrate that both Pak2 kinase activity and its interaction with the PAK-interacting exchange factor-β (β-Pix) are required to reconstitute defective ITALIC! Pak2 (ITALIC! Δ/Δ)HSPC homing to the BM. Pak2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas Pak2 interaction with β-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs were rescued by wild-type (WT) Pak2 but not by a Pak2-kinase dead mutant (KD). Expression of a β-Pix interaction-defective mutant of Pak2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of Pak2, we found a paradoxical decrease in baseline activation of CDC42 in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs, which was rescued by expression of Pak2-WT but not by Pak2-KD; defective homing of ITALIC! Pak2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both Pak2 kinase activity and its interaction with β-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of Pak2 in HSPC migration and homing. © 2016 by The American Society of Hematology.

  7. Expression and phosphorylation of delta-CaM kinase II in cultured Alzheimer fibroblasts.

    PubMed

    Cavazzin, Chiara; Bonvicini, Cristian; Nocera, Annachiara; Racchi, Marco; Kasahara, Jiro; Tardito, Daniela; Gennarelli, Massimo; Govoni, Stefano; Racagni, Giorgio; Popoli, Maurizio

    2004-10-01

    Dysregulation of calcium homeostasis is among the major cellular alterations in Alzheimer's disease (AD). We studied Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), one of the major effectors regulating neuronal responses to changes in calcium fluxes, in cultured skin fibroblasts from subjects with sporadic AD. We found, by using PCR and Western analysis, that human fibroblasts express the delta-isoform of this kinase, and that CaM kinase II is the major Ca(2+)/calmodulin-dependent kinase in these cells. Protein expression level of the kinase was not significantly different in AD fibroblasts. However, the total activity of the kinase (stimulated by Ca(2+)/calmodulin) was significantly reduced in AD cell lines, whereas Ca(2+)-independent activity was significantly enhanced. The percent autonomy of the kinase (%Ca(2+)-independent/Ca(2+)-dependent activity) in AD cell lines was 62.8%, three-fold the corresponding percentage in control fibroblasts. The abnormal calcium-independent activity was not due to enhanced basal autophosphorylation of Thr(287). The observed abnormalities, if present in brain tissue, may be implicated either in dysfunction of neuroplasticity and cognitive functions or in dysregulation of cell cycle.

  8. Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin.

    PubMed Central

    Bogatcheva, N V; Vorotnikov, A V; Birukov, K G; Shirinsky, V P; Gusev, N B

    1993-01-01

    Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin. Images Figure 1 Figure 2 Figure 3 PMID:8452532

  9. Cross-Linking Proteins To Show Complex Formation: A Laboratory That Visually Demonstrates Calmodulin Binding to Calmodulin Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2003-01-01

    Presents a laboratory experiment demonstrating the binding of calcium/calmodulin to calmodulin kinase II, which is important in the metabolic and physiological activities of the cell. Uses SDS polyacrylamide gel electrophoresis (PAGE). (YDS)

  10. Cross-Linking Proteins To Show Complex Formation: A Laboratory That Visually Demonstrates Calmodulin Binding to Calmodulin Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2003-01-01

    Presents a laboratory experiment demonstrating the binding of calcium/calmodulin to calmodulin kinase II, which is important in the metabolic and physiological activities of the cell. Uses SDS polyacrylamide gel electrophoresis (PAGE). (YDS)

  11. Inactivation of Smad-Transforming Growth Factor β Signaling by Ca2+-Calmodulin-Dependent Protein Kinase II

    PubMed Central

    Wicks, Stephen J.; Lui, Stephen; Abdel-Wahab, Nadia; Mason, Roger M.; Chantry, Andrew

    2000-01-01

    Members of the transforming growth factor β (TGF-β) family transduce signals through Smad proteins. Smad signaling can be regulated by the Ras/Erk/mitogen-activated protein pathway in response to receptor tyrosine kinase activation and the gamma interferon pathway and also by the functional interaction of Smad2 with Ca2+-calmodulin. Here we report that Smad–TGF-β-dependent transcriptional responses are prevented by expression of a constitutively activated Ca2+-calmodulin-dependent protein kinase II (Cam kinase II). Smad2 is a target substrate for Cam kinase II in vitro at serine-110, -240, and -260. Cam kinase II induces in vivo phosphorylation of Smad2 and Smad4 and, to a lesser extent, Smad3. A phosphopeptide antiserum raised against Smad2 phosphoserine-240 reacted with Smad2 in vivo when coexpressed with Cam kinase II and by activation of the platelet-derived growth factor receptor, the epidermal growth factor receptor, HER2 (c-erbB2), and the TGF-β receptor. Furthermore, Cam kinase II blocked nuclear accumulation of a Smad2 and induced Smad2-Smad4 hetero-oligomerization independently of TGF-β receptor activation, while preventing TGF-β-dependent Smad2-Smad3 interactions. These findings provide a novel cross-talk mechanism by which Ca2+-dependent kinases activated downstream of multiple growth factor receptors antagonize cell responses to TGF-β. PMID:11027280

  12. Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth Manduca sexta.

    PubMed

    Lohr, Christian; Bergstein, Sandra; Hirnet, Daniela

    2007-01-01

    The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6-10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.

  13. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    PubMed

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  14. The Octopamine Receptor OAMB Mediates Ovulation via Ca2+/Calmodulin-Dependent Protein Kinase II in the Drosophila Oviduct Epithelium

    PubMed Central

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  15. The p21-activated kinase PAK is negatively regulated by POPX1 and POPX2, a pair of serine/threonine phosphatases of the PP2C family.

    PubMed

    Koh, Cheng-Gee; Tan, E-Jean; Manser, Edward; Lim, Louis

    2002-02-19

    The Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division. The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases (reviewed by [1]). The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known. We now report two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that efficiently inactivate PAK. POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX. The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK. Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42(V12). The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family.

  16. Calcium/calmodulin-dependent protein kinase II expression in motor neurons: effect of axotomy.

    PubMed

    Lund, L M; McQuarrie, I G

    1997-11-20

    Although Ca2+/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIalpha and IIbeta are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIbeta antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIalpha, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIalpha immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIalpha is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIalpha as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIbeta down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIalpha appears to support axonal regrowth.

  17. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  18. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  19. Long-term soluble Abeta1-40 activates CaM kinase II in organotypic hippocampal cultures.

    PubMed

    Tardito, Daniela; Gennarelli, Massimo; Musazzi, Laura; Gesuete, Raffaella; Chiarini, Stefania; Barbiero, Valentina Sara; Rydel, Russell E; Racagni, Giorgio; Popoli, Maurizio

    2007-09-01

    Recent findings suggested a role for soluble amyloid-beta (Abeta) peptides in Alzheimer's disease associated cognitive decline. We investigated the action of soluble, monomeric Abeta(1-40) on CaM kinase II, a kinase involved in neuroplasticity and cognition. We treated organotypic hippocampal cultures short-term (up to 4h) and long-term (5 days) with Abeta(1-40) (1nM-5microM). Abeta did not induce cell damage, apoptosis or synaptic loss. Short-term treatment down-regulated enzymatic activity of the kinase, by reducing its Thr(286) phosphorylation. In contrast, long-term treatment (1nM-microM) markedly and significantly up-regulated enzymatic activity, with peak stimulation at 10nM (three-fold). Up-regulation of activity was associated with increased expression of the alpha-isoform of CaM kinase II, increased phosphorylation at Thr(286) (activator residue) and decreased phosphorylation at Thr(305-306) (inhibitory residues). We investigated the effect of glutamate on CaM kinase II following exposure to 1 or 10nM Abeta(1-40). As previously reported, glutamate increased CaM kinase II activity. However, the glutamate effect was not altered by pretreatment of slices with Abeta. Short- and long-term Abeta treatment showed opposite effects on CaM kinase II, suggesting that long-term changes are an adaptation to the kinase early down-regulation. The marked effect of Abeta(1-40) on the kinase suggests that semi-physiological and slowly raising peptide concentrations may have a significant impact on synaptic plasticity in the absence of synaptic loss or neuronal cell death.

  20. Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.

    PubMed Central

    Manak, J R; Prywes, R

    1991-01-01

    Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation. Images PMID:2046671

  1. Bcl10 is phosphorylated on Ser138 by Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    Ishiguro, Kazuhiro; Ando, Takafumi; Goto, Hidemi; Xavier, Ramnik

    2007-03-01

    Ordered assembly of scaffold proteins Carma1-Bcl10-Malt1 determines NF-kappaB activation following T cell receptor (TCR) engagement. Carma1-Bcl10 interaction and the signaling pathway are controlled by Carma1 phosphorylation, which are induced by PKCtheta and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition to Carma1 phosphorylation, previous studies have demonstrated that Bcl10 is phosphorylated in the C-terminal Ser/Thr rich region following TCR engagement. However the kinases that phosphorylate Bcl10 are incompletely understood. Here we show that CaMKII phosphorylates Bcl10 on Ser138. Furthermore, a CaMKII inhibitor, KN93, and CaMKII siRNA substantially reduce Bcl10 phosphorylation induced by phorbol myristate acetate/ionomycin. S138A mutation prolongs Bcl10-induced NF-kappaB activation, suggesting that Bcl10 phosphorylation is involved in attenuation of NF-kappaB activation. These findings suggest that CaMKII modulates NF-kappaB activation via phosphorylating Bcl10 as well as Carma1.

  2. Calcium/calmodulin kinase II activity of hippocampus in kainate-induced epilepsy.

    PubMed Central

    Lee, M. C.; Ban, S. S.; Woo, Y. J.; Kim, S. U.

    2001-01-01

    This study investigated calcium/calmodulin kinase II (CaMKII) activity related to long-standing neuronal injury of the hippocampus in kainate (KA)-induced experimental temporal lobe epilepsy. Epileptic seizure was induced by injection of KA (1 microg/microL) dissolved in phosphate buffer (0.1 M, pH 7.4) into the left amygdala. Clinical seizures, histopathologic changes and CaMKII activity of the hippocampus were evaluated. Characteristic early limbic and late seizures were developed. Hippocampal CaMKII activity increased significantly 4 and 8 weeks after intra-amygdaloid injection of KA, when late seizures developed. The histopathologic changes of the hippocampus included swelling of neuronal cytoplasm with nuclear pyknosis and loss of neurons in CA3 during this period. The increased activity of CaMKII may correlate with appearance of distant damage in the hippocampus. The above results indicate that intra-amygdaloid injection of KA produces excitatory signals for ipsilateral CA3 neurons in the hippocampus and that subsequently increased levels of CaMKII in postsynaptic neurons induce neuronal injury via phosphorylation of N-methyl-D-aspartate type glutamate receptor. PMID:11641537

  3. A novel FGFR2 mutation in tyrosine kinase II domain, L617F, in Crouzon syndrome.

    PubMed

    Suh, Ye-Jin; Bae, Han-Sol; Choi, Jin-Young; Lee, Jong-Ho; Kim, Myung-Jin; Kim, Sukwha; Ryoo, Hyun-Mo; Baek, Seung-Hak

    2014-01-01

    The purposes of this study were to find a novel mutation of FGFR2 in Korean Crouzon syndrome patients and to identify the functional consequences of this mutation. The samples consisted of 16 Crouzon patients. Peripheral venous blood was collected from the patients. FGFR2 mutation screening was performed by direct PCR sequencing of all exons and part of the introns. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transcriptional activity, real-time PCR for the bone markers (osteocalcin and alkaline phosphatase), and Western blot for phosphorylated FGFR2 and ERK1/2-MAPK protein. Among 16 patients, 10 showed FGFR2 mutations that had already been reported elsewhere. A novel FGFR2 mutation associated with tyrosine kinase II (TK-II) domain, L617F, was found in one Crouzon syndrome patient by direct PCR sequencing. Presence of this mutation was confirmed using RFLP analysis. Runx2 transcriptional activity and expression of osteocalcin and alkaline phosphatase significantly increased in L617F-transfected cells compared to wild-type cells. FGFR2 autophosphorylation in L617F-transfected cells increased in 1% serum, but ERK1/2-MAPK protein was not activated. The FGFR2-L617F mutation associated with the TK domain is potentially related to premature suture closure in Crouzon syndrome patient. © 2013 Wiley Periodicals, Inc.

  4. Calmodulin-dependent kinase II regulates osteoblast differentiation through regulation of Osterix.

    PubMed

    Choi, You Hee; Choi, Jun-Ha; Oh, Jae-Wook; Lee, Kwang-Youl

    2013-03-08

    Osterix (Osx), a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. However, the precise molecular signaling mechanisms between Osterix and CaMKII are not known. In this study, we focused on the relationship between Osterix and CaMKII during osteoblast differentiation. We examined the role of the CaMKII pathway in the regulation of protein levels and its transcriptional activity on Osterix. We showed that CaMKII interacts with Osterix by increasing the protein levels and enhancing the transcriptional activity of Osterix. Conversely, CaMKII inhibitor KN-93 decreases the protein levels and increases the stability of Osterix. The siRNA-mediated knockdown of CaMKII decreased the protein levels and transcriptional activity of Osterix. These results suggest that Osterix is a novel target of CaMKII and the activity of Osterix can be modulated by a novel mechanism involving CaMKII during osteoblast differentiation.

  5. Ca2+/calmodulin-dependent protein kinase II function in vascular remodelling.

    PubMed

    Singer, Harold A

    2012-03-15

    Vascular smooth muscle (VSM) undergoes a phenotypic switch in response to injury, a process that contributes to pathophysiological vascular wall remodelling. VSM phenotype switching is a consequence of changes in gene expression, including an array of ion channels and pumps affecting spatiotemporal features of intracellular Ca(2+) signals. Ca(2+) signalling promotes vascular wall remodelling by regulating cell proliferation, motility, and/or VSM gene transcription, although the mechanisms are not clear. In this review, the functions of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in VSM phenotype switching and synthetic phenotype function are considered. CaMKII isozymes have complex structural and autoregulatory properties. Vascular injury in vivo results in rapid changes in CaMKII isoform expression with reduced expression of CaMKIIγ and upregulation of CaMKIIδ in medial wall VSM. SiRNA-mediated suppression of CaMKIIδ or gene deletion attenuates VSM proliferation and consequent neointimal formation. In vitro studies support functions for CaMKII in the regulation of cell proliferation, motility and gene expression via phosphorylation of CREB1 and HDACIIa/MEF2 complexes. These studies support the concept, and provide potential mechanisms, whereby Ca(2+) signalling through CaMKIIδ promotes VSM phenotype transitions and vascular remodelling.

  6. Ca2+/calmodulin-dependent protein kinase II function in vascular remodelling

    PubMed Central

    Singer, Harold A

    2012-01-01

    Vascular smooth muscle (VSM) undergoes a phenotypic switch in response to injury, a process that contributes to pathophysiological vascular wall remodelling. VSM phenotype switching is a consequence of changes in gene expression, including an array of ion channels and pumps affecting spatiotemporal features of intracellular Ca2+ signals. Ca2+ signalling promotes vascular wall remodelling by regulating cell proliferation, motility, and/or VSM gene transcription, although the mechanisms are not clear. In this review, the functions of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in VSM phenotype switching and synthetic phenotype function are considered. CaMKII isozymes have complex structural and autoregulatory properties. Vascular injury in vivo results in rapid changes in CaMKII isoform expression with reduced expression of CaMKIIγ and upregulation of CaMKIIδ in medial wall VSM. SiRNA-mediated suppression of CaMKIIδ or gene deletion attenuates VSM proliferation and consequent neointimal formation. In vitro studies support functions for CaMKII in the regulation of cell proliferation, motility and gene expression via phosphorylation of CREB1 and HDACIIa/MEF2 complexes. These studies support the concept, and provide potential mechanisms, whereby Ca2+ signalling through CaMKIIδ promotes VSM phenotype transitions and vascular remodelling. PMID:22124148

  7. Regulation of gastrointestinal motility by Ca2+/calmodulin-stimulated protein kinase II.

    PubMed

    Perrino, Brian A

    2011-06-15

    Gastrointestinal (GI) motility ultimately depends upon the contractile activity of the smooth muscle cells of the tunica muscularis. Integrated functioning of multiple tissues and cell types, including enteric neurons and interstitial cells of Cajal (ICC) is necessary to generate coordinated patterns of motor activity that control the movement of material through the digestive tract. The neurogenic mechanisms that govern GI motility patterns are superimposed upon intrinsic myogenic mechanisms regulating smooth muscle cell excitability. Several mechanisms regulate smooth muscle cell responses to neurogenic inputs, including the multifunctional Ca(2+)/calmodulin-stimulated protein kinase II (CaMKII). CaMKII can be activated by Ca(2+) transients from both extracellular and intracellular sources. Prolonging the activities of Ca(2+)-sensitive K(+) channels in the plasma membrane of GI smooth muscle cells is an important regulatory mechanism carried out by CaMKII. Phospholamban (PLN) phosphorylation by CaMKII activates the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), increasing both the rate of Ca(2+) clearance from the myoplasm and the frequency of localized Ca(2+) release events from intracellular stores. Overall, CaMKII appears to moderate GI smooth muscle cell excitability. Finally, transcription factor activities may be facilitated by the neutralization of HDAC4 by CaMKII phosphorylation, which may contribute to the phenotypic plasticity of GI smooth muscle cells.

  8. Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation.

    PubMed

    Kanatsu, Yoshinori; Chen, Nai Hong; Mitoma, Junya; Nakagawa, Tetsuto; Hirabayashi, Yoshio; Higashi, Hideyoshi

    2012-07-01

    Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.

  9. Dissecting cooperative calmodulin binding to CaM kinase II: a detailed stochastic model.

    PubMed

    Byrne, Michael J; Putkey, John A; Waxham, M Neal; Kubota, Yoshihisa

    2009-12-01

    Calmodulin (CaM) is a major Ca(2+) binding protein involved in two opposing processes of synaptic plasticity of CA1 pyramidal neurons: long-term potentiation (LTP) and depression (LTD). The N- and C-terminal lobes of CaM bind to its target separately but cooperatively and introduce complex dynamics that cannot be well understood by experimental measurement. Using a detailed stochastic model constructed upon experimental data, we have studied the interaction between CaM and Ca(2+)-CaM-dependent protein kinase II (CaMKII), a key enzyme underlying LTP. The model suggests that the accelerated binding of one lobe of CaM to CaMKII, when the opposing lobe is already bound to CaMKII, is a critical determinant of the cooperative interaction between Ca(2+), CaM, and CaMKII. The model indicates that the target-bound Ca(2+) free N-lobe has an extended lifetime and may regulate the Ca(2+) response of CaMKII during LTP induction. The model also reveals multiple kinetic pathways which have not been previously predicted for CaM-dissociation from CaMKII.

  10. Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro.

    PubMed Central

    Ackerman, P; Glover, C V; Osheroff, N

    1985-01-01

    The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA. Images PMID:2987912

  11. Kinetics of inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole on calf thymus casein kinase II.

    PubMed

    Zandomeni, R O

    1989-09-01

    The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a specific inhibitor for RNA polymerase II transcription in vivo and in vitro [Tamm + Sehgal (1978) Adv. Virus Res. 22, 187-258; Zandomeni & Weinmann (1984) J. Biol. Chem. 259, 14804-14811]. The effect on RNA polymerase II-specific transcription seems to be mediated by its inhibition of nuclear casein kinase II [Zandomeni, Carrera-Zandomeni, Shugar & Weinmann (1986) J. Biol. Chem. 261, 3414-3419]. Inhibition studies indicated that DRB acted as a mixed-type inhibitor with respect to casein and as a competitive inhibitor with respect to the nucleotide phosphate donor substrates. The DRB inhibition constant is 7 microM for the calf thymus casein kinase II, with regard to both ATP and GTP.

  12. Modulation of calcium-mediated inactivation of ionic currents by Ca2+/calmodulin-dependent protein kinase II.

    PubMed Central

    Sakakibara, M; Alkon, D L; DeLorenzo, R; Goldenring, J R; Neary, J T; Heldman, E

    1986-01-01

    Iontophoretic injection of Ca2+ causes reduction of I0A (an early rapidly activating and inactivating K+ current) and I0C (a late Ca2+-dependent K+ current) measured across the isolated type B soma membrane (Alkon et al., 1984, 1985; Alkon and Sakakibara, 1984, 1985). Similarly, voltage-clamp conditions which cause elevation of [Ca2+]i are followed by reduction of I0A and I0C lasting 1-3 min. Iontophoretic injection of highly purified Ca2+/CaM-dependent protein kinase II (CaM kinase II) isolated from brain tissue (Goldenring et al., 1983) enhanced and prolonged this Ca2+-mediated reduction of I0A and I0C. ICa2+, a voltage-dependent Ca2+ current, also showed some persistent reduction under these conditions. Iontophoretic injection of heat-inactivated enzyme had no effect. Agents that inhibit or block Ca2+/CaM-dependent phosphorylation produced increased I0A and I0C amplitudes and prevented the effects of CaM kinase II injection. The results reported here and in other studies implicate Ca2+-stimulated phosphorylation in the regulation of type B soma ionic currents. PMID:2427133

  13. Function of cGMP-dependent protein kinase II in volume load-induced diuresis.

    PubMed

    Schramm, Andrea; Schinner, Elisabeth; Huettner, Johannes P; Kees, Frieder; Tauber, Philipp; Hofmann, Franz; Schlossmann, Jens

    2014-10-01

    Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3% of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50% in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.

  14. Reduced Arrhythmia Inducibility with Calcium/Calmodulin-Dependent Protein Kinase II Inhibition in Heart Failure Rabbits

    PubMed Central

    Hoeker, Gregory S.; Hanafy, Mohamed A.; Oster, Robert A.; Bers, Donald M.; Pogwizd, Steven M.

    2015-01-01

    Rationale Calcium/calmodulin-dependent protein kinase II (CaMKII) is activated in heart failure (HF) and can contribute to arrhythmias induced by β-adrenergic receptor-mediated sarcoplasmic reticulum calcium leak. Objective To evaluate the effect of CaMKII inhibition on ventricular tachycardia (VT) induction in conscious HF and naïve rabbits. Methods and Results Nonischemic HF was induced by aortic insufficiency and constriction. Electrocardiograms were recorded in rabbits pretreated with vehicle (saline) or the CaMKII inhibitor KN-93 (300 μg/kg); VT was induced by infusion of increasing doses of norepinephrine (NE, 1.56-25 μg/kg/min) in naïve (n = 8) and HF (n = 7) rabbits. With saline, median VT dose threshold in HF was 6.25 versus 12.5 μg/kg/min NE in naïve rabbits (p = 0.06). Pretreatment with KN-93 significantly increased VT threshold in HF and naïve rabbits (median = 25 μg/kg/min, p < 0.05 versus saline for both groups). Mean cycle length of VT initiation was shorter in HF (221 ± 20 ms) than naïve (296 ± 23 ms, p < 0.05) rabbits with saline; this difference was not significant after treatment with KN-93. Conclusions KN-93 significantly reduced arrhythmia inducibility and slowed initiation of VT, suggesting that CaMKII inhibition may have antiarrhythmic effects in the failing human heart. PMID:26650851

  15. Increased calcium/calmodulin-dependent protein kinase II activity by morphine-sensitization in rat hippocampus.

    PubMed

    Kadivar, Mehdi; Farahmandfar, Maryam; Ranjbar, Faezeh Esmaeli; Zarrindast, Mohammad-Reza

    2014-07-01

    Repeated exposure to drugs of abuse, such as morphine, elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect long-lasting changes in some of the important molecules involved in memory processing such as calcium/calmodulin-dependent protein kinase II (CaMKII). In the present study, we investigated the effect of morphine sensitization on mRNA expression of α and β isoforms and activity of CaMKII in the hippocampus of male rats. Animals were treated for 3 days with saline or morphine (20mg/kg) and following a washout period of 5 days, a challenge dose of morphine (5mg/kg) were administered. The results indicate that morphine administration in pre-treated animals produces behavioral sensitization, as determined by significant increase in locomotion and oral stereotypy behavior. In addition, repeated morphine treatment increased mRNA expression of both α and β isoforms of CaMKII in the hippocampus. The present study also showed that induction of morphine sensitization significantly increased both Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent activities of CaMK II in the rat hippocampus. However, acute administration of morphine (5mg/kg) did not alter either α and β CaMKII mRNA expression or CaMKII activity in the hippocampus. The stimulation effects of morphine sensitization on mRNA expression and activity of CaMKII were completely abolished by administration of naloxone, 30min prior to s.c. injections of morphine (20mg/kg/day×3 days). Our data demonstrated that induction of morphine sensitization could effectively modulate the activity and the mRNA expression of CaMKII in the hippocampus and this effect of morphine was exerted by the activation of opioid receptors.

  16. Calmodulin kinase II is required for fight or flight sinoatrial node physiology.

    PubMed

    Wu, Yuejin; Gao, Zhan; Chen, Biyi; Koval, Olha M; Singh, Madhu V; Guan, Xiaoqun; Hund, Thomas J; Kutschke, William; Sarma, Satyam; Grumbach, Isabella M; Wehrens, Xander H T; Mohler, Peter J; Song, Long-Sheng; Anderson, Mark E

    2009-04-07

    The best understood "fight or flight" mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by beta-adrenergic receptor (betaAR) agonist stimulation. HCN4 conducts an inward "pacemaker" current (I(f)) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I(f)-independent pathways are critical to SAN fight or flight responses. The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) is a downstream signal in the betaAR pathway that activates Ca(2+) homeostatic proteins in ventricular myocardium. Mice with genetic, myocardial and SAN cell CaMKII inhibition have significantly slower HRs than controls during stress, leading us to hypothesize that CaMKII actions on SAN Ca(2+) homeostasis are critical for betaAR agonist responses in SAN. Here we show that CaMKII mediates ISO HR increases by targeting SAN cell Ca(2+) homeostasis. CaMKII inhibition prevents ISO effects on SAN Ca(2+) uptake and release from intracellular sarcoplasmic reticulum (SR) stores that are necessary for increasing DDR. CaMKII inhibition has no effect on the ISO response in SAN cells when SR Ca(2+) release is disabled and CaMKII inhibition is only effective at slowing HRs during betaAR stimulation. These studies show the tightly coupled, but previously unanticipated, relationship of CaMKII to the betaAR pathway in fight or flight physiology and establish CaMKII as a critical signaling molecule for physiological HR responses to catecholamines.

  17. Ca(2+)/Calmodulin-Dependent Protein Kinase II in Vascular Smooth Muscle.

    PubMed

    Saddouk, F Z; Ginnan, R; Singer, H A

    2017-01-01

    Ca(2+)-dependent signaling pathways are central regulators of differentiated vascular smooth muscle (VSM) contractile function. In addition, Ca(2+) signals regulate VSM gene transcription, proliferation, and migration of dedifferentiated or "synthetic" phenotype VSM cells. Synthetic phenotype VSM growth and hyperplasia are hallmarks of pervasive vascular diseases including hypertension, atherosclerosis, postangioplasty/in-stent restenosis, and vein graft failure. The serine/threonine protein kinase Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous mediator of intracellular Ca(2+) signals. Its multifunctional nature, structural complexity, diversity of isoforms, and splice variants all characterize this protein kinase and make study of its activity and function challenging. The kinase has unique autoregulatory mechanisms, and emerging studies suggest that it can function to integrate Ca(2+) and reactive oxygen/nitrogen species signaling. Differentiated VSM expresses primarily CaMKIIγ and -δ isoforms. CaMKIIγ isoform expression correlates closely with the differentiated phenotype, and some studies link its function to regulation of contractile activity and Ca(2+) homeostasis. Conversely, synthetic phenotype VSM cells primarily express CaMKIIδ and substantial evidence links it to regulation of gene transcription, proliferation, and migration of VSM in vitro, and vascular hypertrophic and hyperplastic remodeling in vivo. CaMKIIδ and -γ isoforms have opposing functions at the level of cell cycle regulation, proliferation, and VSM hyperplasia in vivo. Isoform switching following vascular injury is a key step in promoting vascular remodeling. Recent availability of genetically engineered mice with smooth muscle deletion of specific isoforms and transgenics expressing an endogenous inhibitor protein (CAMK2N) has enabled a better understanding of CaMKII function in VSM and should facilitate future studies. © 2017 Elsevier Inc. All rights reserved.

  18. Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis.

    PubMed

    Mulekar, Jidnyasa Jayant; Bu, Qingyun; Chen, Fulu; Huq, Enamul

    2012-01-01

    Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  19. Behavioral modulation of neuronal calcium/calmodulin-dependent protein kinase II activity: differential effects on nicotine-induced spinal and supraspinal antinociception in mice.

    PubMed

    Damaj, M Imad

    2007-10-15

    Recent studies have implicated the involvement of Ca(2+)-dependent mechanisms, in particular calcium/calmodulin-dependent protein kinase II (CaM kinase II) in nicotine-induced antinociception using the tail-flick test. The spinal cord was suggested as a possible site of this involvement. The present study was undertaken to investigate the hypothesis that similar mechanisms exist for nicotine-induced antinociception in the hot-plate test, a response thought to be centrally mediated. In order to assess these mechanisms, i.c.v. administered CaM kinase II inhibitors were evaluated for their effects on antinociception produced by either i.c.v. or s.c. administration of nicotine in both tests. In addition, nicotine's analgesic effects were tested in mice lacking half of their CaM kinase II (CaM kinase II heterozygous) and compare it to their wild-type counterparts. Our results showed that although structurally unrelated CaM kinase II inhibitors blocked nicotine's effects in the tail-flick test in a dose-related manner, they failed to block the hot-plate responses. In addition, the antinociceptive effects of systemic nicotine in the tail-flick but not the hot-plate test were significantly reduced in CaM kinase II heterozygous mice. These observations indicate that in contrast to the tail-flick response, the mechanism of nicotine-induced antinociception in the hot-plate test is not mediated primarily via CaM kinase II-dependent mechanisms at the supraspinal level.

  20. Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum.

    PubMed

    Kim, Minkyung; Hennig, Grant W; Smith, Terence K; Perrino, Brian A

    2008-02-01

    Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.

  1. Sorting Nexin 27 Protein Regulates Trafficking of a p21-activated Kinase (PAK) Interacting Exchange Factor (β-Pix)-G Protein-coupled Receptor Kinase Interacting Protein (GIT) Complex via a PDZ Domain Interaction*

    PubMed Central

    Valdes, Julie L.; Tang, Jingrong; McDermott, Mark I.; Kuo, Jean-Cheng; Zimmerman, Seth P.; Wincovitch, Stephen M.; Waterman, Clare M.; Milgram, Sharon L.; Playford, Martin P.

    2011-01-01

    Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility. PMID:21926430

  2. Sorting nexin 27 protein regulates trafficking of a p21-activated kinase (PAK) interacting exchange factor (β-Pix)-G protein-coupled receptor kinase interacting protein (GIT) complex via a PDZ domain interaction.

    PubMed

    Valdes, Julie L; Tang, Jingrong; McDermott, Mark I; Kuo, Jean-Cheng; Zimmerman, Seth P; Wincovitch, Stephen M; Waterman, Clare M; Milgram, Sharon L; Playford, Martin P

    2011-11-11

    Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility.

  3. Basic fibroblast growth factor-induced translocation of p21-activated kinase to the membrane is independent of phospholipase C-gamma1 in the differentiation of PC12 cells.

    PubMed

    Shin, Kyung-Sun; Shin, Eun-Young; Lee, Chan-Soo; Quan, Song-Hua; Woo, Kyung-Nam; Soung, Nak-Kyun; Kwak, Sahng-June; Kim, Seung Ryul; Kim, Eung-Gook

    2002-05-31

    p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-gamma1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-gamma1 is functionally relevant for PAK targeting.

  4. Hemopoietic cell kinase (Hck) and p21-activated kinase 2 (PAK2) are involved in the down-regulation of CD1a lipid antigen presentation by HIV-1 Nef in dendritic cells.

    PubMed

    Shinya, Eiji; Shimizu, Masumi; Owaki, Atsuko; Paoletti, Samantha; Mori, Lucia; De Libero, Gennaro; Takahashi, Hidemi

    2016-01-01

    Dendritic cells (DCs) play a major role in in vivo pathogenesis of HIV-1 infection. Therefore, DCs may provide a promising strategy to control and eventually overcome the fatal infection. Especially, immature DCs express all CD1s, the non-MHC lipid antigen -presenting molecules, and HIV-1 Nef down-regulates CD1 expression besides MHC. Moreover, CD1d-restricted CD4(+) NKT cells are infected by HIV-1, reducing the number of these cells in HIV-1-infected individuals. To understand the exact role of DCs and CD1-mediated immune response during HIV-1 infection, Nef down-regulation of CD1a-restricted lipid/glycolipid Ag presentation in iDCs was analyzed. We demonstrated the involvement of the association of Nef with hemopoietic cell kinase (Hck) and p21-activated kinase 2 (PAK2), and that Hck, which is expressed strongly in iDCs, augmented this mutual interaction. Hck might be another therapeutic target to preserve the function of HIV-1 infected DCs, which are potential reservoirs of HIV-1 even after antiretroviral therapy.

  5. The P21-activated kinase expression pattern is different in non-small cell lung cancer and affects lung cancer cell sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors.

    PubMed

    Liu, Yang; Wang, Si; Dong, Qian-Ze; Jiang, Gui-Yang; Han, Yong; Wang, Liang; Wang, En-Hua

    2016-03-01

    Exploring methods for increasing epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) sensitivity has become a major focus in non-small cell lung cancer (NSCLC). Major downstream effectors of the Rho family small guanosine triphosphatases, P21-activated kinases (PAKs) activate the main signaling pathways downstream of EGFR and thus promote tumor cell proliferation. In this study, we explored the expression pattern of phosphorylated PAKs in NSCLC and their potential value as drug targets for treating cancer. The expression and prognostic significance of phosphorylated group I and II PAKs were evaluated in 182 patients with NSCLC. Immunohistochemical analysis revealed low group I PAK expression in normal lung tissues and increased expressed in the cytoplasm, particularly in lung squamous cell carcinoma. Abnormal group I PAK expression was associated with lymph node metastases and high tumor-node-metastases (TNM) stage in NSCLC patients and correlated with poor prognosis. We used group I PAK inhibitor (IPA3) to specifically decrease group I PAK activity in human lung cancer cell lines. Decreased group I PAK activity inhibited cell proliferation and combined IPA3 and EGFR-TKI (gefitinib) treatment inhibited cell proliferation in an obvious manner. Together, our results revealed the PAK expression pattern in NSCLC, and a role for group I PAK in cell proliferation, which provides evidence that decreased PAK activity may have a potential application as a molecular targeted therapy in advanced NSCLC.

  6. p21-Activated kinase 2 (PAK2) inhibits TGF-β signaling in Madin-Darby canine kidney (MDCK) epithelial cells by interfering with the receptor-Smad interaction.

    PubMed

    Yan, Xiaohua; Zhang, Junyu; Sun, Qinyu; Tuazon, Polygena T; Wu, Xiaoping; Traugh, Jolinda A; Chen, Ye-Guang

    2012-04-20

    TGF-β (transforming growth factor β) plays a variety of cellular functions mainly through the Smad pathway. Phosphorylation of the carboxyl SXS motif in R-Smads (Smad2 and Smad3) by the type I receptor TβRI is a key step for their activation. It has been reported that the serine/threonine kinase PAK2 (p21-activated kinase 2) can mediate TGF-β signaling in mesenchymal cells. Here, we report that PAK2 restricts TGF-β-induced Smad2/3 activation and transcriptional responsiveness in MDCK epithelial cells. Mechanistically, PAK2 associates with Smad2 and Smad3 in a kinase activity-dependent manner and blocks their activation. PAK2 phosphorylates Smad2 at Ser-417, which is adjacent to the L3 loop that contributes to the TβRI-R-Smad association. Consistently, substitution of Ser-417 with glutamic acid attenuates the interaction of Smad2 with TβRI. Together, our results indicate that PAK2 negatively modulate TGF-β signaling by attenuating the receptor-Smad interaction and thus Smad activation.

  7. p21-activated Kinase 2 (PAK2) Inhibits TGF-β Signaling in Madin-Darby Canine Kidney (MDCK) Epithelial Cells by Interfering with the Receptor-Smad Interaction*

    PubMed Central

    Yan, Xiaohua; Zhang, Junyu; Sun, Qinyu; Tuazon, Polygena T.; Wu, Xiaoping; Traugh, Jolinda A.; Chen, Ye-Guang

    2012-01-01

    TGF-β (transforming growth factor β) plays a variety of cellular functions mainly through the Smad pathway. Phosphorylation of the carboxyl SXS motif in R-Smads (Smad2 and Smad3) by the type I receptor TβRI is a key step for their activation. It has been reported that the serine/threonine kinase PAK2 (p21-activated kinase 2) can mediate TGF-β signaling in mesenchymal cells. Here, we report that PAK2 restricts TGF-β-induced Smad2/3 activation and transcriptional responsiveness in MDCK epithelial cells. Mechanistically, PAK2 associates with Smad2 and Smad3 in a kinase activity-dependent manner and blocks their activation. PAK2 phosphorylates Smad2 at Ser-417, which is adjacent to the L3 loop that contributes to the TβRI-R-Smad association. Consistently, substitution of Ser-417 with glutamic acid attenuates the interaction of Smad2 with TβRI. Together, our results indicate that PAK2 negatively modulate TGF-β signaling by attenuating the receptor-Smad interaction and thus Smad activation. PMID:22393057

  8. Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

    PubMed Central

    Shi, Juan; Geshi, Naomi; Takahashi, Shinichi; Kiyonaka, Shigeki; Ichikawa, Jun; Hu, Yaopeng; Mori, Yasuo; Ito, Yushi; Inoue, Ryuji

    2013-01-01

    The molecular mechanism underlying Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 μm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 μm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln855–Glu877) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr487 was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr487 (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr487 is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg8-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr487 may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells. PMID

  9. Particulate air pollution induces arrhythmia via oxidative stress and calcium calmodulin kinase II activation

    SciTech Connect

    Kim, Jin-Bae; Kim, Changsoo; Choi, Eunmi; Park, Sanghoon; Park, Hyelim; Pak, Hui-Nam; Lee, Moon-Hyoung; Shin, Dong Chun; Hwang, Ki-Chul; Joung, Boyoung

    2012-02-15

    Ambient particulate matter (PM) can increase the incidence of arrhythmia. However, the arrhythmogenic mechanism of PM is poorly understood. This study investigated the arrhythmogenic mechanism of PM. In Sprague–Dawley rats, QT interval was increased from 115.0 ± 14.0 to 142.1 ± 18.4 ms (p = 0.02) after endotracheal exposure of DEP (200 μg/ml for 30 min, n = 5). Ventricular premature contractions were more frequently observed after DEP exposure (100%) than baseline (20%, p = 0.04). These effects were prevented by pretreatment of N-acetylcysteine (NAC, 5 mmol/L, n = 3). In 12 Langendorff-perfused rat hearts, DEP infusion of 12.5 μg/ml for 20 min prolonged action potential duration (APD) at only left ventricular base increasing apicobasal repolarization gradients. Spontaneous early afterdepolarization (EAD) and ventricular tachycardia (VT) were observed in 8 (67%) and 6 (50%) hearts, respectively, versus no spontaneous triggered activity or VT in any hearts before DEP infusion. DEP-induced APD prolongation, EAD and VT were successfully prevented with NAC (5 mmol/L, n = 5), nifedipine (10 μmol/L, n = 5), and active Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) blockade, KN 93 (1 μmol/L, n = 5), but not by thapsigargin (200 nmol/L) plus ryanodine (10 μmol/L, n = 5) and inactive CaMKII blockade, KN 92 (1 μmol/L, n = 5). In neonatal rat cardiomyocytes, DEP provoked ROS generation in dose dependant manner. DEP (12.5 μg/ml) induced apoptosis, and this effect was prevented by NAC and KN 93. Thus, this study shows that in vivo and vitro exposure of PM induced APD prolongation, EAD and ventricular arrhythmia. These effects might be caused by oxidative stress and CaMKII activation. -- Highlights: ► The ambient PM consistently prolonged repolarization. ► The ambient PM induced triggered activity and ventricular arrhythmia. ► These effects were prevented by antioxidants, I{sub CaL} blockade and CaMKII blockade. ► The ambient PM can induce

  10. Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

    PubMed Central

    Sag, Can M.; Wadsack, Daniel P.; Khabbazzadeh, Sepideh; Abesser, Marco; Grefe, Clemens; Neumann, Kay; Opiela, Marie-Kristin; Backs, Johannes; Olson, Eric N.; Brown, Joan Heller; Neef, Stefan; Maier, Sebastian K.G.; Maier, Lars S.

    2010-01-01

    Background Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)δC mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 μm−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in

  11. Conditioned taste aversion and Ca/calmodulin-dependent kinase II in the parabrachial nucleus of rats.

    PubMed

    Krivanek, J

    2001-07-01

    Bielavska and colleagues (Bielavska, Sacchetti, Baldi, & Tassoni, 1999) have recently shown that KN-62, an inhibitor of calcium/calmodulin-dependent kinase II (CaCMK), induces conditioned taste aversion (CTA) when introduced into the parabrachial nucleus (PBN) of rats. The aim of the present report was to assess whether activity of CaCMK in the PBN is changed during CTA. We induced CTA in one group of rats by pairing saccharin consumption with an ip injection of lithium chloride. Another group of rats received lithium alone (without being paired with saccharin consumption) to test whether lithium has an effect on CaCMK in the PBN, independent of those effects due to training. In animals receiving CTA training, CaCMK activity in extracts of PBN was reduced by approximately 30% at the postacquisition intervals of 12, 24, and 48 h, compared to control animals receiving saccharin with saline injection. By 120 h after CTA training, no effect on CaCMK was present. At those postacquisition intervals showing CaCMK activity effects due to CTA, there were no effects attributable to lithium alone. Lithium alone produced only a short-lasting reduction in CaCMK activity (at 20 min a 30% decrease, at 60 min a 23% decrease; and at 6, 12, and 24 h no decrease). The time course of lithium-induced effects differed markedly from that of CTA training. All changes were Ca2+/- -dependent; we did not observe any changes in Ca-independent activity. CTA effects on CaCMK were selective for PBN, insofar as we did not observe any CTA effects on CaCMK in the visual cortex, a brain region unrelated to taste pathways. Since CTA produces a relatively long-lasting reduction in CaCMK activity (lasting 2 days or more) specifically in the PBN, which is critical a relay for taste information, the reduction of CaCMK activity may enable the consolidation of taste memory in an aversive situation.

  12. KN-93 inhibits IKr in mammalian cardiomyocytes

    PubMed Central

    Hegyi, Bence; Chen-Izu, Ye; Jian, Zhong; Shimkunas, Rafael; Izu, Leighton T.; Banyasz, Tamas

    2015-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 is widely used in multiple fields of cardiac research especially for studying the mechanisms of cardiomyopathy and cardiac arrhythmias. Whereas KN-93 is a potent inhibitor of CaMKII, several off-target effects have also been found in expression cell systems and smooth muscle cells, but there is no information on the KN93 side effects in mammalian ventricular myocytes. In this study we explore the effect of KN-93 on the rapid component of delayed rectifier potassium current (IKr) in the ventricular myocytes from rabbit and guinea pig hearts. Our data indicate that KN-93 exerts direct inhibitory effect on IKr that is not mediated via CaMKII. This off-target effect of KN93 should be taken into account when interpreting the data from using KN93 to investigate the role of CaMKII in cardiac function. PMID:26463508

  13. KN-93 inhibits IKr in mammalian cardiomyocytes.

    PubMed

    Hegyi, Bence; Chen-Izu, Ye; Jian, Zhong; Shimkunas, Rafael; Izu, Leighton T; Banyasz, Tamas

    2015-12-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 is widely used in multiple fields of cardiac research especially for studying the mechanisms of cardiomyopathy and cardiac arrhythmias. Whereas KN-93 is a potent inhibitor of CaMKII, several off-target effects have also been found in expression cell systems and smooth muscle cells, but there is no information on the KN93 side effects in mammalian ventricular myocytes. In this study we explore the effect of KN-93 on the rapid component of delayed rectifier potassium current (IKr) in the ventricular myocytes from rabbit and guinea pig hearts. Our data indicate that KN-93 exerts direct inhibitory effect on IKr that is not mediated via CaMKII. This off-target effect of KN93 should be taken into account when interpreting the data from using KN93 to investigate the role of CaMKII in cardiac function.

  14. Ablation of p21-activated kinase-1 in mice promotes isoproterenol-induced cardiac hypertrophy in association with activation of Erk1/2 and inhibition of protein phosphatase 2A.

    PubMed

    Taglieri, Domenico M; Monasky, Michelle M; Knezevic, Ivana; Sheehan, Katherine A; Lei, Ming; Wang, Xin; Chernoff, Jonathan; Wolska, Beata M; Ke, Yunbo; Solaro, R John

    2011-12-01

    Earlier investigations in our lab indicated an anti-adrenergic effect induced by activation of p21-activated kinase (Pak-1) and protein phosphatase 2A (PP2A). Our objective was to test the hypothesis that Pak-1/PP2A is a signaling cascade controlling stress-induced cardiac growth. We determined the effects of ablation of the Pak-1 gene on the response of the myocardium to chronic stress of isoproterenol (ISO) administration. Wild-type (WT) and Pak-1-knockout (Pak-1-KO) mice were randomized into six groups to receive either ISO, saline (CTRL), or ISO and FR180204, a selective inhibitor of Erk1/2. Echocardiography revealed that hearts of the Pak-1-KO/ISO group had increased LV fractional shortening, reduced LV chamber volume in diastole and systole, increased cardiac hypertrophy, and enhanced transmitral early filling deceleration time, compared to all other groups. The changes were associated with an increase in relative Erk1/2 activation in Pak-1-KO/ISO mice versus all other groups. ISO-induced cardiac hypertrophy and Erk1/2 activation in Pak-1-KO/ISO were attenuated when the selective Erk1/2 inhibitor FR180204 was administered. Immunoprecipitation showed an association between Pak-1, PP2A, and Erk1/2. Cardiac myocytes infected with an adenoviral vector expressing constitutively active Pak-1 showed a repression of Erk1/2 activation. p38 MAPK phosphorylation was decreased in Pak-1-KO/ISO and Pak-1-KO/CTRL mice compared to WT. Levels of phosphorylated PP2A were increased in ISO-treated Pak-1-KO mice, indicating reduced phosphatase activity. Maximum Ca(2+)-activated tension in detergent-extracted bundles of papillary fibers from ISO-treated Pak-1-KO mice was higher than in all other groups. Analysis of cTnI phosphorylation indicated that compared to WT, ISO-induced phosphorylation of cTnI was blunted in Pak-1-KO mice. Active Pak-1 is a natural inhibitor of Erk1/2 and a novel anti-hypertrophic signaling molecule upstream of PP2A. 2011 Elsevier Ltd. All rights reserved.

  15. Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

    PubMed

    Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

    1998-08-15

    Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

  16. Calcium/calmodulin dependent protein kinase II regulates the phosphorylation of cyclic AMP-responsive element-binding protein of spinal cord in rats following noxious stimulation.

    PubMed

    Fang, Li; Wu, Jing; Zhang, Xuan; Lin, Qing; Willis, William D

    2005-02-01

    We have previously reported that intradermal capsaicin injection causes the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats. The present study was designed to investigate the role of calcium/camodulin protein dependent protein kinase II (CaM kinase II) in the regulation of phosphorylation of CREB after capsaicin injection. We found that capsaicin injection produces a significant upregulation of phosphorylated CREB in the spinal cord of rat. Intrathecal treatment with a CaM kinase II inhibitor, KN-93, significantly blocked the increased phosphorylation of CREB, but did not affect the CREB protein itself. These results suggest that increased phosphorylation of CREB protein may contribute to central sensitization following acute peripheral noxious stimuli, and the effect may be regulated through the activation of CaM kinase cascades.

  17. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  18. Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Olson, N J; Massé, T; Suzuki, T; Chen, J; Alam, D; Kelly, P T

    1995-01-01

    To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element. Images Fig. 1 Fig. 3 PMID:7878035

  19. Actin Dynamics Is Controlled by a Casein Kinase II and Phosphatase 2C Interplay on Toxoplasma gondii Toxofilin

    PubMed Central

    Delorme, Violaine; Cayla, Xavier; Faure, Grazyna; Garcia, Alphonse; Tardieux, Isabelle

    2003-01-01

    Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine53. The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility. PMID:12802063

  20. A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin-dependent kinase II holoenzyme

    PubMed Central

    Chao, Luke H.; Stratton, Margaret M.; Lee, Il-Hyung; Rosenberg, Oren S.; Levitz, Joshua; Mandell, Daniel J.; Kortemme, Tanja; Groves, Jay T.; Schulman, Howard; Kuriyan, John

    2011-01-01

    Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains. PMID:21884935

  1. Chronic antidepressants induce redistribution and differential activation of alphaCaM kinase II between presynaptic compartments.

    PubMed

    Barbiero, Valentina S; Giambelli, Roberto; Musazzi, Laura; Tiraboschi, Ettore; Tardito, Daniela; Perez, Jorge; Drago, Filippo; Racagni, Giorgio; Popoli, Maurizio

    2007-12-01

    Changes in synaptic plasticity are involved in pathophysiology of depression and in the mechanism of antidepressants. Ca(2+)/calmodulin (CaM) kinase II, a protein kinase involved in synaptic plasticity, has been previously shown to be a target of antidepressants. We previously found that antidepressants activate the kinase in hippocampal neuronal cell bodies by increasing phosphorylation at Thr(286), reduce the kinase phosphorylation in synaptic membranes, and in turn its phosphorylation-dependent interaction with syntaxin-1 and the release of glutamate from hippocampal synaptosomes. Here, we investigated the chronic effect of different antidepressants (fluoxetine, desipramine, and reboxetine) on the expression and function of the kinase in distinct subcellular compartments in order to dissect the different kinase pools affected. Acute treatments did not induce any change in the kinase. In total tissue extracts chronic drug treatments induced activation of the kinase; in hippocampus (HC), but not in prefrontal/frontal cortex, this was partially accounted for by increased Thr(286) phosphorylation, suggesting the involvement of different mechanisms of activation. In synaptosomes, all drugs reduced the kinase phosphorylation, particularly in HC where, upon fractionation of the synaptosomal particulate into synaptic vesicles and membranes, we found that the drugs induced a redistribution and differential activation of the kinase between membranes and vesicles. Furthermore, a large decrease in the level and phosphorylation of synapsin I located at synaptic membranes was consistent with the observed decrease of CaM kinase II. Overall, antidepressants induce a complex pattern of modifications in distinct subcellular compartments; at presynaptic level, these changes are in line with a dampening of glutamate release.

  2. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    SciTech Connect

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  3. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

    PubMed

    Nguyen, Le Xuan Truong; Mitchell, Beverly S

    2013-12-17

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

  4. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors.

  5. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  6. Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II

    PubMed Central

    Bhattacharyya, Moitrayee; Stratton, Margaret M; Going, Catherine C; McSpadden, Ethan D; Huang, Yongjian; Susa, Anna C; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L; Barros, Tiago; Schulman, Howard; Williams, Evan R; Kuriyan, John

    2016-01-01

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. DOI: http://dx.doi.org/10.7554/eLife.13405.001 PMID:26949248

  7. Casein kinase II inhibitor enhances production of infectious genotype 1a hepatitis C virus (H77S).

    PubMed

    Kim, Seungtaek; Jin, Bora; Choi, Sung Hoon; Han, Kwang-Hyub; Ahn, Sang Hoon

    2014-01-01

    Genotype 2a JFH1 virus has substantially contributed to the progress of HCV biology by allowing entire viral life cycle of HCV in cell culture. Using this genotype 2a virus, casein kinase II (CKII) was previously identified as a crucial host factor in virus assembly by phosphorylating NS5A. Since most of the prior studies employed genotype 2a JFH1 or JFH1-based intragenotypic chimera, we used genotype 1a H77S to study virus assembly. CKII inhibition by chemical inhibitors enhanced H77S virus production in contrast to that of JFH1 virus, but genetic inhibition of CKII by siRNA did not change H77S virus titer significantly. The different outcomes from these two approaches of CKII inhibition suggested that nonspecific target kinase of CKII inhibitors plays a role in increasing H77S virus production and both viral and host factors were investigated in this study. Our results emphasize substantial differences among the HCV genotypes that should be considered in both basic research and clinical practices.

  8. Activation of Ca2+-calmodulin kinase II induces desensitization by background light in dogfish retinal ‘on’ bipolar cells

    PubMed Central

    Shiells, R A; Falk, G

    2000-01-01

    Retinal ‘on’ bipolar cells possess a metabotropic glutamate receptor (mGluR6) linked to the control of a G-protein and cGMP-activated channels which functions to generate high synaptic amplification of rod signals under dark-adapted conditions. Desensitization of ‘on’ bipolar cells is initiated by a rise in Ca2+ during background light too weak to adapt rod photoreceptors. Desensitization could also be elicited by raising intracellular Ca2+ above 1 μm. In order to investigate the mechanism of desensitization, whole-cell current responses to brief flashes and to steps of light were obtained from voltage-clamped ‘on’ bipolar cells in dark-adapted dogfish retinal slices. The inclusion of Ca2+-calmodulin kinase II (CaMKII) inhibitor peptides in the patch pipette solutions not only blocked desensitization of ‘on’ bipolar cells by dim background light and by 50 μm Ca2+, but also increased their flash sensitivity. The substrate of phosphorylation by CaMKII is the ‘on’ bipolar cell cGMP-activated channels. Desensitization probably results from a reduction in their sensitivity to cGMP and a voltage-dependent decrease in their conductance. A role for protein kinase C (PKC) in this process was excluded since activating PKC independently of Ca2+ with the phorbol ester PMA failed to induce desensitization of ‘on’ bipolar cells. PMID:11034622

  9. Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

    DOE PAGES

    Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.; ...

    2016-03-07

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts themore » hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.« less

  10. A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II.

    PubMed

    Welsby, Philip J; Wang, Hongge; Wolfe, Joshua T; Colbran, Roger J; Johnson, Michael L; Barrett, Paula Q

    2003-11-05

    Low-voltage-activated (LVA) Ca2+ channels are widely distributed throughout the CNS and are important determinants of neuronal excitability, initiating dendritic and somatic Ca2+ spikes that trigger and shape the pattern of action potential firing. Here, we define a molecular mechanism underlying the dynamic regulation of alpha1H channels (Cav3.2), by Ca2+/CaM-dependent protein kinase II (CaMKII). We show that channel regulation is selective for the LVA alpha1H Ca2+ channel subtype, depends on determinants in the alpha1H II-III intracellular loop, and requires the phosphorylation of a serine residue absent from unregulated alpha1G (Cav3.1) channels. These studies identify the alpha1H channel as a new substrate for CaMKII and provide the first molecular mechanism for the direct regulation of T-type Ca2+ channels by a protein kinase. Our data suggest a novel mechanism for modulating the integrative properties of neurons.

  11. Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.; McSpadden, Ethan D.; Huang, Yongjian; Susa, Anna C.; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L.; Barros, Tiago; Schulman, Howard; Williams, Evan R.; Kuriyan, John

    2016-03-07

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.

  12. Multisite phosphorylation of Arabidopsis HFR1 by casein kinase II and a plausible role in regulating its degradation rate.

    PubMed

    Park, Hee-Jin; Ding, Lei; Dai, Mingqiu; Lin, Rongcheng; Wang, Haiyang

    2008-08-22

    Arabidopsis Long Hypocotyl in Far-Red Light 1 (HFR1), a bHLH transcription factor, plays a critical role in promoting seedling photomorphogenesis and in balancing the shade-avoidance response under canopy shade conditions. Previous studies have established that HFR1 protein is degraded in darkness and is stabilized under light conditions to promote light signaling. How light regulates HFR1 stability is not well understood. In this study, we show that Arabidopsis HFR1 can be phosphorylated by recombinant casein kinase II (CKII) and plant extract in vitro and that phosphorylation of HFR1 can be effectively reduced by treatments with two CKII-specific inhibitors, 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and heparin. We demonstrate that HFR1 physically interacts with the CKB1 and CKB2 regulatory subunits of CKII. Mutagenesis studies indicate that HFR1 is phosphorylated at multiple serine (Ser) residues in the N-terminal regulatory domain of HFR1. We also show that phosphorylation of HFR1 is promoted by light and that a predicted CKII site, Ser(122), represents a major phosphorylation site of HFR1 under both dark and light conditions. Comparison of wild-type, phosphorylation-deficient, and phosphorylation-mimic mutant proteins suggests that phosphorylation acts to reduce the degradation rate of HFR1. Together, our results suggest that CKII-mediated phosphorylation represents an important post-translational modification influencing the stability and signaling activity of Arabidopsis HFR1.

  13. Neuronal calcium/calmodulin-dependent protein kinase II mediates nicotine reward in the conditioned place preference test in mice.

    PubMed

    Jackson, Kia J; Muldoon, Pretal P; Walters, Carrie; Damaj, Mohamad Imad

    2016-02-01

    Several recent studies have indicated the involvement of calcium-dependent mechanisms, in particular the abundant calcium-activated kinase, calcium/calmodulin-dependent kinase II (CaMKII), in behaviors associated with nicotine dependence in mice. Behavioral and biochemical studies have shown that CaMKII is involved in acute and chronic nicotine behaviors and nicotine withdrawal; however, evidence of a role for CaMKII in nicotine reward is lacking. Thus, the goal of the current study was to examine the role of CaMKII in nicotine reward. Using pharmacological and genetic tools, we tested nicotine conditioned place preference (CPP) in C57Bl/6 mice after administration of CaMKII antagonists and in α-CaMKII wild-type (+/+) and heterozygote (±) mice. CaMKII antagonists blocked expression of nicotine CPP, and the preference score was significantly reduced in α-CaMKII ± mice compared with their +/+ counterparts. Further, we assessed CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex, and hippocampus after nicotine CPP and found significant increases in CaMKII activity in the mouse VTA and NAc that were blocked by CaMKII antagonists. The findings from this study show that CaMKII mediates nicotine reward and suggest that increases in CaMKII activity in the VTA and NAc are relevant to nicotine reward behaviors.

  14. A novel endogenous PP2C-like phosphatase dephosphorylates casein kinase II-phosphorylated Physarum fragmin.

    PubMed

    Waelkens, E; de Corte, V; Merlevede, W; Vandekerckhove, J; Gettemans, J

    2000-12-20

    Plasmodial fragmin, a Physarum polycephalum F-actin severing and capping protein, is phosphorylated by casein kinase II at Ser(266) (De Corte, V., Gettemans, J., De Ville, Y., Waelkens, E., and Vandekerckchove, J. (1996), Biochemistry 35, 5472-5480). In this study, we report the purification and characterization of the corresponding fragmin phosphatases. One of the enzymes was purified to near homogeneity from a cytosolic extract; it dephosphorylates CKII-phosphorylated fragmin, a peptide encompassing the CKII phosphorylation site of fragmin as well as histone 2A, CKII-phosphorylated casein and the CKII model-peptide substrate: R(3)E(3)S(P)E(3). Its activity was highly stimulated by Mn(2+) and Mg(2+), and based on its lack of sensitivity toward phosphatase effectors we could exclude similarities with PP1, PP2A and PP2B phosphatases. All biochemical properties of the phosphatase point to a PP2C-like enzyme. A second phosphatase dephosphorylating fragmin was identified as a Physarum alkaline phosphatase.

  15. CaM kinase II and phospholamban contribute to caffeine-induced relaxation of murine gastric fundus smooth muscle.

    PubMed

    Kim, Minkyung; Cho, Sang Yun; Han, In Soo; Koh, Sang Don; Perrino, Brian A

    2005-06-01

    Caffeine has been shown to increase the Ca(2+) release frequency (Ca(2+) sparks) from the sarcoplasmic reticulum (SR) through ryanodine-sensitive stores and relax gastric fundus smooth muscle. Increased Ca(2+) store refilling increases the frequency of Ca(2+) release events and store refilling is enhanced by CaM kinase II (CaMKII) phosphorylation of phospholamban (PLB). These findings suggest that transient, localized Ca(2+) release events from the SR may activate CaMKII and contribute to relaxation by enhancing store refilling due to PLB Thr17 phosphorylation. To investigate this possibility, we examined the effects of caffeine on CaMKII, muscle tone, and PLB phosphorylation in murine gastric fundus smooth muscle. Caffeine (1 mM) hyperpolarized and relaxed murine gastric fundus smooth muscle and activated CaMKII. Ryanodine, tetracaine, or cyclopiazonic acid each prevented CaMKII activation and significantly inhibited caffeine-induced relaxation. The large-conductance Ca(2+)-activated K(+) channel blocker iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation. Caffeine-induced CaMKII activation increased PLB Thr17, but not PLB Ser16 phosphorylation. 3-Isobutyl-1-methylxanthine increased PLB Ser16 phosphorylation, but not PLB Thr17 phosphorylation. The CaMKII inhibitor KN-93 inhibited caffeine-induced relaxation and PLB Thr17 phosphorylation. These results show that caffeine-induced CaMKII activation and PLB phosphorylation play a role in the relaxation of gastric fundus smooth muscles.

  16. Tyrosine kinase activity of a Ca{sup 2+}/calmodulin-dependent protein kinase II catalytic fragment

    SciTech Connect

    Sugiyama, Yasunori; Ishida, Atsuhiko; Sueyoshi, Noriyuki; Kameshita, Isamu

    2008-12-12

    A 30-kDa fragment of Ca{sup 2+}/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX{sub 40}-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca{sup 2+}/calmodulin-dependent protein kinase I (CX{sub 40}) were fused to the N-terminal end of 30K-CaMKII. Although CX{sub 40}-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX{sub 40} region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.

  17. Structural Properties of Human CaMKII Ca2+ /Calmodulin-Dependent Protein Kinase II using X-ray Crystallography

    NASA Astrophysics Data System (ADS)

    Cao, Yumeng Melody; McSpadden, Ethan; Kuriyan, John; Department of Molecular; Cell Biology; Department of Chemistry Team

    To this day, human memory storage remains a mystery as we can at most describe the process vaguely on a cellular level. Switch-like properties of Calcium/Calmodulin-Dependent Protein Kinase II make it a leading candidate in understanding the molecular basis of human memory. The protein crystal was placed in the beam of a synchrotron source and the x-ray crystallography data was collected as reflections on a diffraction pattern that undergo Fourier transform to obtain the electron density. We observed two drastic differences from our solved structure at 2.75Å to a similar construct of the mouse CaMKII association domain. Firstly, our structure is a 6-fold symmetric dodecamer, whereas the previously published construct was a 7-fold symmetric tetradecamer. This suggests the association domain of human CaMKII is a dynamic structure that is triggered subunit exchange process. Secondly, in our structure the N-terminal tag is docked as an additional beta-strand on an uncapped beta-sheet present in each association domain protomer. This is concrete evidence of the involvement of the polypeptide docking site in the molecular mechanism underlining subunit exchange. In the future, we would like to selectively inhibit the exchange process while not disrupting the other functionalities of CaMKII.

  18. Developmental iodine deficiency and hypothyroidism reduce phosphorylation of calcium/calmodulin-dependent kinase II in the rat entorhinal cortex.

    PubMed

    Wang, Yi; Hou, Yi; Dong, Jing; Xu, Hongde; Gong, Jian; Chen, Jie

    2010-12-01

    Iodine is essential for the synthesis of triiodothyronine (T₃) and thyroxine (T₄). Iodine deficiency leads to inadequate thyroid hormone. Hypothyroidism induced by iodine deficiency during gestation and postnatal period leads to cognitive deficits in learning and memory. However, the mechanism underlying these deficits is unclear. Calcium-dependent calmodulin kinase II (CaMKII) known as a potential memory molecule regulates important neuronal functions including learning and memory. Recent studies have shown that hypothyroidism alters phosphorylation of CaMKII in hippocampus or even in sympathetic ganglia of rats. Though the entorhinal cortex (EC) is an important functional structure within the neuronal network responsible for learning and memory, little is known about the effect of hypothyroidism on phosphorylation of CaMKII in the EC. Here, we report that iodine deficiency and propylthiouracil treatment through gestation and lactation reduce phosphorylation of CaMKII in the EC of pups. The increase of calcineurin, as well as reduction of neurogranin and calmodulin, may account for the reduced phosphorylation of CaMKII induced by developmental iodine deficiency and hypothyroidism. These findings in the EC may contribute to understanding the mechanisms that underlie impairment of learning and memory induced by developmental iodine deficiency and hypothyroidism.

  19. Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity.

    PubMed

    Kumrungsee, Thanutchaporn; Akiyama, Sayaka; Guo, Jian; Tanaka, Mitsuru; Matsui, Toshiro

    2016-12-15

    Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca(2+)-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1μM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300μM Trp-Val or Trp-Ile. On the basis of Ca(2+)-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca(2+)-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6μM, respectively.

  20. The δ isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload

    PubMed Central

    Backs, Johannes; Backs, Thea; Neef, Stefan; Kreusser, Michael M.; Lehmann, Lorenz H.; Patrick, David M.; Grueter, Chad E.; Qi, Xiaoxia; Richardson, James A.; Hill, Joseph A.; Katus, Hugo A.; Bassel-Duby, Rhonda; Maier, Lars S.; Olson, Eric N.

    2009-01-01

    Acute and chronic injuries to the heart result in perturbation of intracellular calcium signaling, which leads to pathological cardiac hypertrophy and remodeling. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transduction of calcium signals in the heart, but the specific isoforms of CaMKII that mediate pathological cardiac signaling have not been fully defined. To investigate the potential involvement in heart disease of CaMKIIδ, the major CaMKII isoform expressed in the heart, we generated CaMKIIδ-null mice. These mice are viable and display no overt abnormalities in cardiac structure or function in the absence of stress. However, pathological cardiac hypertrophy and remodeling are attenuated in response to pressure overload in these animals. Cardiac extracts from CaMKIIδ-null mice showed diminished kinase activity toward histone deacetylase 4 (HDAC4), a substrate of stress-responsive protein kinases and suppressor of stress-dependent cardiac remodeling. In contrast, phosphorylation of the closely related HDAC5 was unaffected in hearts of CaMKIIδ-null mice, underscoring the specificity of the CaMKIIδ signaling pathway for HDAC4 phosphorylation. We conclude that CaMKIIδ functions as an important transducer of stress stimuli involved in pathological cardiac remodeling in vivo, which is mediated, at least in part, by the phosphorylation of HDAC4. These findings point to CaMKIIδ as a potential therapeutic target for the maintenance of cardiac function in the setting of pressure overload. PMID:19179290

  1. Ocular dominance plasticity is stably maintained in the absence of alpha calcium calmodulin kinase II (alphaCaMKII) autophosphorylation.

    PubMed

    Taha, Sharif A; Stryker, Michael P

    2005-11-08

    The molecule alpha calcium calmodulin kinase II (alphaCaMKII) is known to play a fundamental role in the induction of many forms of synaptic plasticity. A major theory of alphaCaMKII function proposes that autophosphorylation of the molecule mediates not only the induction but also the maintenance of synaptic plasticity. To test this hypothesis, we assessed ocular dominance plasticity in genetically engineered mice that carry a mutation preventing autophosphorylation of alphaCaMKII. These mutant mice are deficient in plasticity after monocular deprivation, but a sufficiently long period of monocular deprivation will induce ocular dominance plasticity. After induction of ocular dominance plasticity, the stability of the induced changes was assayed after binocular deprivation. Plasticity in homozygous mutant animals was as stable as that measured in WT littermates; also, response characteristics did not differ between the two groups. Our results suggest that alphaCaMKII autophosphorylation is required for the induction of ocular dominance plasticity but is not needed for its stable maintenance thereafter.

  2. Intracellular Ca2+ and Ca2+/Calmodulin-Dependent Kinase II Mediate Acute Potentiation of Neurotransmitter Release by Neurotrophin-3

    PubMed Central

    He, Xiang-ping; Yang, Feng; Xie, Zuo-ping; Lu, Bai

    2000-01-01

    Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve–muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca2+ influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca2+ influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca2+. Depletion of intracellular Ca2+ stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca2+/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca2+ release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII. PMID:10811820

  3. Identification of a BET family Bromodomain / Casein Kinase II / TAF-containing complex as a regulator of mitotic condensin function

    PubMed Central

    Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B.; Silva, Andrea C.; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J.; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G.; Greenblatt, Jack F.; Krogan, Nevan J.; Fillingham, Jeffrey S.; Strahl, Brian D.; Bouhassira, Eric E.; Edelmann, Winfried; Keogh, Michael-Christopher

    2014-01-01

    SUMMARY Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell cycle-directed manner to modulate the activity of condensin during chromosome condensation and decondensation. PMID:24565511

  4. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-d-aspartate receptor

    PubMed Central

    Leonard, A. Soren; Lim, Indra A.; Hemsworth, Daniel E.; Horne, Mary C.; Hell, Johannes W.

    1999-01-01

    The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca2+ influx through postsynaptic N-methyl-d-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIα by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca2+ influx but also close to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP. PMID:10077668

  5. Resveratrol Inhibits Neuronal Apoptosis and Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Diabetic Mouse Retina

    PubMed Central

    Kim, Young-Hee; Kim, Yoon-Sook; Kang, Sang-Soo; Cho, Gyeong-Jae; Choi, Wan-Sung

    2010-01-01

    OBJECTIVE This study investigated the effects of resveratrol, a natural polyphenol with neuroprotective properties, on retinal neuronal cell death mediated by diabetes-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). RESEARCH DESIGN AND METHODS Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received buffer. All mice were killed 2 months after the injections, and the extent of neuronal cell death, CaMKII, and phospho-CaMKII protein expression levels and CaMKII kinase activity were examined in the retinas. To assess the role of CaMKII in the death of retinal neurons, a small-interfering RNA (siRNA) or specific inhibitor of CaMKII was injected into the right vitreous humor, and vehicle only was injected into the left vitreous humor, 2 days before death. Resveratrol (20 mg/kg) was administered by oral gavage daily for 4 weeks, beginning 1 month after the fifth injection of either STZ or buffer. RESULTS The death of retinal ganglion cells (RGCs), CaMKII, phospho-CaMKII protein levels, and CaMKII activity were all greatly increased in the retinas of diabetic mice compared with controls, 2 months after induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive signals co-localized with CaMKII- and phospho-CaMKII immunoreactive RGCs. However, in addition to CaMKII knockdown and inhibition by siRNA or a specific inhibitor, respectively, resveratrol provided complete protection from diabetes-induced retinal cell death. CONCLUSIONS In the present study, resveratrol prevented diabetes-induced RGC death via CaMKII downregulation, implying that resveratrol may have potential therapeutic applications for prevention of diabetes-induced visual dysfunction. PMID:20424226

  6. Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II.

    PubMed

    David-Dufilho, Monique; Millanvoye-Van Brussel, Elisabeth; Topal, Gokce; Walch, Laurence; Brunet, Annie; Rendu, Francine

    2005-10-28

    Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

  7. Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation.

    PubMed

    House, Suzanne J; Ginnan, Roman G; Armstrong, Shayn E; Singer, Harold A

    2007-06-01

    There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

  8. CaM kinase II delta2-dependent regulation of vascular smooth muscle cell polarization and migration.

    PubMed

    Mercure, Melissa Z; Ginnan, Roman; Singer, Harold A

    2008-06-01

    Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

  9. Diabetes mellitus affects activity of calcium/calmodulin-dependent protein kinase II alpha in rat trigeminal ganglia.

    PubMed

    Jerić, Milka; Vuica, Ana; Borić, Matija; Puljak, Livia; Jeličić Kadić, Antonia; Grković, Ivica; Filipović, Natalija

    2015-01-01

    The activity of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The aim of this study was to investigate the immunoreactivity of phosphorylated CaMKIIα (pCaMKIIα) in subpopulations of trigeminal ganglion (TG) neurons in rat models of early diabetes type 1 (dm1) and 2 (dm2). DM1 model was induced with intraperitoneally (i.p.) injected streptozotocin (STZ) (55mg/kg). DM2 rats were fed with the high fat diet (HFD) for 2 weeks and then received 35mg/kg of STZ i.p. Two weeks and 2 months after the STZ-diabetes induction, rats were sacrificed and immunohistochemical analysis for detection of pCaMKIIα immunoreactivity and double immunofluorescence labelling with isolectin (IB4) was performed. Increased intensity of pCaMKIIα immunofluorescence, restricted to IB4-negative small-diameter neurons, was seen in TG neurons two months after STZ-DM1 induction. DM1 model, as well as the obesity (control dm2 groups) resulted in neuronal impaired growth while dm2 model led to neuron hypertrophy in TG. Observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. In future, innovative strategies for modulation of CaMKIIα activity in specific subpopulations of neurons could be a novel approach in therapy of diabetic trigeminal neuropathy. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism*

    PubMed Central

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M.

    2016-01-01

    Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex. PMID:27044741

  11. Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism.

    PubMed

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2016-05-06

    Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Curcumin Attenuates Opioid Tolerance and Dependence by Inhibiting Ca2+/Calmodulin-Dependent Protein Kinase II α Activity

    PubMed Central

    Hu, Xiaoyu; Huang, Fang; Szymusiak, Magdalena

    2015-01-01

    Chronic use of opioid analgesics has been hindered by the development of opioid addiction and tolerance. We have reported that curcumin, a natural flavonoid from the rhizome of Curcuma longa, attenuated opioid tolerance, although the underlying mechanism remains unclear. In this study, we tested the hypothesis that curcumin may inhibit Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα), a protein kinase that has been previously proposed to be critical for opioid tolerance and dependence. In this study, we used state-of-the-art polymeric formulation technology to produce poly(lactic-co-glycolic acid) (PLGA)-curcumin nanoparticles (nanocurcumin) to overcome the drug’s poor solubility and bioavailability, which has made it extremely difficult for studying in vivo pharmacological actions of curcumin. We found that PLGA-curcumin nanoparticles reduced the dose requirement by 11- to 33-fold. Pretreatment with PLGA-curcumin (by mouth) prevented the development of opioid tolerance and dependence in a dose-dependent manner, with ED50 values of 3.9 and 3.2 mg/kg, respectively. PLGA-curcumin dose-dependently attenuated already-established opioid tolerance (ED50 = 12.6 mg/kg p.o.) and dependence (ED50 = 3.1 mg/kg p.o.). Curcumin or PLGA-curcumin did not produce antinociception by itself or affect morphine (1–10 mg/kg) antinociception. Moreover, we found that the behavioral effects of curcumin on opioid tolerance and dependence correlated with its inhibition of morphine-induced CaMKIIα activation in the brain. These results suggest that curcumin may attenuate opioid tolerance and dependence by suppressing CaMKIIα activity. PMID:25515789

  13. The Role of Casein Kinase II in Flowering Time Regulation Has Diversified during Evolution1[W][OA

    PubMed Central

    Ogiso, Eri; Takahashi, Yuji; Sasaki, Takuji; Yano, Masahiro; Izawa, Takeshi

    2010-01-01

    Casein kinase II (CK2) is a protein kinase with an evolutionarily conserved function as a circadian clock component in several organisms, including the long-day plant Arabidopsis (Arabidopsis thaliana). The circadian clock component CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a CK2 target in Arabidopsis, where it influences photoperiodic flowering. In rice (Oryza sativa), a short-day plant, Heading date6 (Hd6) encodes a CK2α subunit that delays flowering time under long-day conditions. Here, we demonstrate that control of flowering time in rice by the Hd6 CK2α subunit requires a functional Hd1 gene (an Arabidopsis CONSTANS ortholog) and is independent of the circadian clock mechanism. Our findings from overexpressing the dominant-negative CK2 allele in rice support the independence of CK2 function from the circadian clock. This lack of control of the circadian clock by Hd6 CK2α might be due to the presence of glutamate in OsLHY (a CCA1 ortholog in rice) instead of the serine at the corresponding CK2 target site in CCA1. However, this glutamate is critical for the control of the OsPRR1 gene (a rice ortholog of the Arabidopsis TOC1/PRR1 gene) by OsLHY for regulation of the circadian clock. We also demonstrated that the other conserved CK2 target sites in OsLHY conferred robust rhythmic expression of OsLHY-LUC under diurnal conditions. These findings imply that the role of CK2 in flowering-time regulation in higher plants has diversified during evolution. PMID:20007447

  14. Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

    PubMed

    Diniz, Luan Pereira; Tortelli, Vanessa; Garcia, Matheus Nunes; Araújo, Ana Paula Bérgamo; Melo, Helen M; Silva, Gisele S Seixas da; Felice, Fernanda G De; Alves-Leon, Soniza Vieira; Souza, Jorge Marcondes de; Romão, Luciana Ferreira; Castro, Newton Gonçalves; Gomes, Flávia Carvalho Alcantara

    2014-12-01

    The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-β1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-β pathway. TGF-β1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-β1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-β1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-β1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. © 2014 Wiley Periodicals, Inc.

  15. Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

    PubMed

    Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

    2016-12-16

    In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  17. Linaclotide activates guanylate cyclase-C/cGMP/protein kinase-II-dependent trafficking of CFTR in the intestine.

    PubMed

    Ahsan, Md Kaimul; Tchernychev, Boris; Kessler, Marco M; Solinga, Robert M; Arthur, David; Linde, Cristina I; Silos-Santiago, Inmaculada; Hannig, Gerhard; Ameen, Nadia A

    2017-06-01

    The transmembrane receptor guanylyl cyclase-C (GC-C), expressed on enterocytes along the intestine, is the molecular target of the GC-C agonist peptide linaclotide, an FDA-approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo-2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)-trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase-II (PKG-II) activity assays. Expression and activity of GC-C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo-2BBe cells increased cell surface CFTR levels. Linaclotide-induced activation of the GC-C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASP(ser239) phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide-induced CFTR trafficking to the apical membrane. Inhibition of protein kinase-A (PKA) also attenuated linaclotide-induced CFTR cell surface trafficking, implying cGMP-dependent cross-activation of PKA pathway. Together, these findings support linaclotide-induced activation of the GC-C/cGMP/PKG-II/CFTR pathway as the major pathway of linaclotide-mediated intestinal fluid secretion, and that linaclotide-dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on

  18. Spontaneous Ca Waves in Ventricular Myocytes from Failing Hearts Depend on Ca2+-calmodulin-dependent Protein Kinase II

    PubMed Central

    Curran, Jerry; Brown, Kathy Hayes; Santiago, Demetrio J.; Pogwizd, Steve; Bers, Donald M.; Shannon, Thomas R.

    2010-01-01

    Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca]SRT) versus control. At similar [Ca]SRT (121 µM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 & 1.29±0.39 SCaWs cell−1, respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca]SRT. Inhibiting CaMKII reversed this effect (14%). At similar [Ca]SRT (71 µM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5±0.5; 1.6±0.7 vs. 0.36±0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis. PMID:20353795

  19. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize.

    PubMed

    Lu Y-T; Feldman, L J; Hidaka, H

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

  20. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Hidaka, H.

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

  1. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Hidaka, H.

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

  2. Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

    PubMed

    Lou, Jie; Zhao, Dan; Zhang, Ling-Ling; Song, Shu-Ying; Li, Yan-Chao; Sun, Fei; Ding, Xiao-Qing; Yu, Chang-Jiang; Li, Yuan-Yuan; Liu, Mei-Tong; Dong, Chang-Jiang; Ji, Yong; Li, Hongliang; Chu, Wenfeng; Zhang, Zhi-Ren

    2016-09-01

    The role of type III transforming growth factor-β receptor (TβRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TβRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TβRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TβRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TβRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TβRIII in vitro. Cardiac-specific transgenic expression of TβRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TβRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TβRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TβRIII in vivo. Our data suggest that TβRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of β-arrestin2 with both TβRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TβRIII expression results in cardiac hypertrophy through β-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-β and β-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac

  3. Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a G(o)/G(q) signaling network.

    PubMed Central

    Robatzek, M; Thomas, J H

    2000-01-01

    Caenorhabditis elegans locomotion is a complex behavior generated by a defined set of motor neurons and interneurons. Genetic analysis shows that UNC-43, the C. elegans Ca(2+)/calmodulin protein kinase II (CaMKII), controls locomotion rate. Elevated UNC-43 activity, from a gain-of-function mutation, causes severely lethargic locomotion, presumably by inappropriate phosphorylation of targets. In a genetic screen for suppressors of this phenotype, we identified multiple alleles of four genes in a G(o)/G(q) G-protein signaling network, which has been shown to regulate synaptic activity via diacylglycerol. Mutations in goa-1, dgk-1, eat-16, or eat-11 strongly or completely suppressed unc-43(gf) lethargy, but affected other mutants with reduced locomotion only weakly. We conclude that CaMKII and G(o)/G(q) pathways act in concert to regulate synaptic activity, perhaps through a direct interaction between CaMKII and G(o). PMID:11063685

  4. A Casein Kinase II Phosphorylation Site in AtYY1 Affects Its Activity, Stability, and Function in the ABA Response

    PubMed Central

    Wu, Xiu-Yun; Li, Tian

    2017-01-01

    The phosphorylation and dephosphorylation of proteins are crucial in the regulation of protein activity and stability in various signaling pathways. In this study, we identified an ABA repressor, Arabidopsis Ying Yang 1 (AtYY1) as a potential target of casein kinase II (CKII). AtYY1 physically interacts with two regulatory subunits of CKII, CKB3, and CKB4. Moreover, AtYY1 can be phosphorylated by CKII in vitro, and the S284 site is the major CKII phosphorylation site. Further analyses indicated that S284 phosphorylation can enhance the transcriptional activity and protein stability of AtYY1 and hence strengthen the effect of AtYY1 as a negative regulator in the ABA response. Our study provides novel insights into the regulatory mechanism of AtYY1 mediated by CKII phosphorylation. PMID:28348572

  5. Digoxin and ouabain induce P-glycoprotein by activating calmodulin kinase II and hypoxia-inducible factor-1alpha in human colon cancer cells

    SciTech Connect

    Riganti, Chiara

    2009-11-01

    Digoxin and ouabain are cardioactive glycosides, which inhibit the Na{sup +}/K{sup +}-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca{sup ++}]{sub i}). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca{sup ++}]{sub i} and this event was dependent on the calcium influx via the Na{sup +}/Ca{sup ++} exchanger. The increased [Ca{sup ++}]{sub i} enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na{sup +}/Ca{sup ++} exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

  6. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling

    PubMed Central

    Saddouk, Fatima Z.; Sun, Li-Yan; Liu, Yong Feng; Jiang, Miao; Singer, Diane V.; Backs, Johannes; Van Riper, Dee; Ginnan, Roman; Schwarz, John J.; Singer, Harold A.

    2016-01-01

    Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions.—Saddouk, F. Z., Sun, L.-Y., Liu, Y. F., Jiang, M., Singer, D. V., Backs, J., Van Riper, D., Ginnan, R., Schwarz, J. J., Singer, H. A. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling. PMID:26567004

  7. Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells.

    PubMed

    Borodinsky, Laura N; Coso, Omar A; Fiszman, Mónica L

    2002-03-01

    In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

  8. Current theories of neuronal information processing performed by Ca2+/calmodulin-dependent protein kinase II with support and insights from computer modelling and simulation.

    PubMed

    Coomber, C

    1998-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is concentrated in brain, and is particularly enriched in synaptic structures where it comprises 20-50% of all proteins. The abundant nature of CaMKII and its ability to phosphorylate a wide range of substrate proteins, including itself, earmarks it as a protein kinase that may have a vital role in neuronal information processing and memory. A computer model of CaMKII is investigated that incorporates recent findings about the geometrical arrangement of subunits, the mechanism of Ca(2+)-dependent subunit activation, and Ca(2+)-independent autophosphorylation. The model is framed as a system of nonlinear differential equations. It is demonstrated numerically that (1) CaMKII is tuned to be activated by stimulation protocols associated with the induction of long-term potentiation; (2) the observed slow dissociation of trapped Ca2+/calmodulin may require the autonomy site to be protected from dephosphorylation; and (3) Ca(2+)-independent kinase activity is expressed in a manner akin to a graded switch. The model validates current theories concerning how CaMKII may be a Ca2+ pulse frequency detector, a molecular switch, or a mediator of the threshold for long-term synaptic plasticity.

  9. Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

    PubMed

    Maubach, Gunter; Sokolova, Olga; Wolfien, Markus; Rothkötter, Hermann-Josef; Naumann, Michael

    2013-09-15

    Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

  10. Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+ /calmodulin-dependent protein kinase II

    PubMed Central

    Yao, Junlan; Davies, Lucinda A.; Howard, Jason D.; Adney, Scott K.; Welsby, Philip J.; Howell, Nancy; Carey, Robert M.; Colbran, Roger J.; Barrett, Paula Q.

    2006-01-01

    Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II–elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with α1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression. PMID:16917542

  11. Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    Yao, Junlan; Davies, Lucinda A; Howard, Jason D; Adney, Scott K; Welsby, Philip J; Howell, Nancy; Carey, Robert M; Colbran, Roger J; Barrett, Paula Q

    2006-09-01

    Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II-elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with alpha1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.

  12. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells

    PubMed Central

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  13. Genetic analysis of cell morphogenesis in fission yeast--a role for casein kinase II in the establishment of polarized growth.

    PubMed Central

    Snell, V; Nurse, P

    1994-01-01

    We have initiated a study to identify genes regulating cell morphogenesis in the fission yeast Schizosaccharomyces pombe. Five genes have been identified, orb1-orb5, whose mutation gives rise to spherical cells, indicative of an inability to polarize growth. Two further genes have been identified, tea1 and ban1, whose mutant alleles have disturbed patterns of tip growth, leading to T-shaped and curved cells. In fission yeast, sites of cell wall deposition are defined by actin localization, with actin distributions and therefore growth patterns undergoing cell cycle stage-specific reorganization. Studies of double mutants constructed between orb5-19 and various cdc mutants blocked before and after cell division show that orb5 is required for the re-establishment of polar growth following cytokinesis. This indicates that the mutant allele orb5-19 is defective in the reinitiation of polarized growth, even though actin reorganization to the cell tips occurs normally. orb5 encodes a fission yeast homologue of casein kinase II alpha. We propose that this kinase plays a role in the translation of cell polarity into polarized growth, but not in the establishment of polarity itself. Images PMID:8187760

  14. Ca2+/calmodulin protein kinase II and memory: learning-related changes in a localized region of the domestic chick brain.

    PubMed

    Solomonia, Revaz O; Kotorashvili, Adam; Kiguradze, Tamar; McCabe, Brian J; Horn, Gabriel

    2005-12-01

    The role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the recognition memory of visual imprinting was investigated. Domestic chicks were exposed to a training stimulus and learning strength measured. Trained chicks, together with untrained chicks, were killed either 1 h or 24 h after training. The intermediate and medial hyperstriatum ventrale/mesopallium (IMHV/IMM), a forebrain memory storage site, was removed together with a control brain region, the posterior pole of the neostriatum/nidopallium (PPN). Amounts of membrane total alphaCaMKII (tCaMKII) and Thr286-autophosphorylated alphaCaMKII (apCAMKII) were measured. For the IMHV/IMM 1 h group, apCaMKII amount and apCAMKII/tCaMKII increased as chicks learned. The magnitude of the molecular changes were positively correlated with learning strength. No learning-related effects were observed in PPN, or in either region at 24 h. These results suggest that CaMKII is involved in the formation of memory but not in its maintenance.

  15. Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

    PubMed Central

    Alessi, Amelia F.; Khivansara, Vishal; Han, Ting; Freeberg, Mallory A.; Moresco, James J.; Tu, Patricia G.; Montoye, Eric; Yates, John R.; Karp, Xantha; Kim, John K.

    2015-01-01

    MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway. PMID:26669440

  16. Casein kinase II is required for proper cell division and acts as a negative regulator of centrosome duplication in Caenorhabditis elegans embryos

    PubMed Central

    Medley, Jeffrey C.; Kabara, Megan M.; Stubenvoll, Michael D.; DeMeyer, Lauren E.

    2017-01-01

    ABSTRACT Centrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein kinase II (CK2) in early Caenorhabditis elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner. PMID:27881437

  17. Ca²⁺/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

    PubMed

    Taillebois, Emiliane; Heuland, Emilie; Bourdin, Céline M; Griveau, Audrey; Quinchard, Sophie; Tricoire-Leignel, Helene; Legros, Christian; Thany, Steeve H

    2013-07-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²⁺ signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

  18. The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

    PubMed Central

    Li, Changlin; Cai, Xiangyu; Sun, Haili; Bai, Ting; Zheng, Xilong; Zhou, Xing Wang; Chen, Xiongwen; Gill, Donald L.; Li, Jing; Tang, Xiang D.

    2011-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are 3 δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target. PMID:21554860

  19. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    PubMed

    Haolong, Cong; Du, Ning; Hongchao, Tian; Yang, Yang; Wei, Zhang; Hua, Zhang; Wenliang, Zhang; Lei, Song; Po, Tien

    2013-01-01

    Enterovirus 71 (EV71) is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II) which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  20. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling.

    PubMed

    Saddouk, Fatima Z; Sun, Li-Yan; Liu, Yong Feng; Jiang, Miao; Singer, Diane V; Backs, Johannes; Van Riper, Dee; Ginnan, Roman; Schwarz, John J; Singer, Harold A

    2016-03-01

    Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions. © FASEB.

  1. Curcumin Inhibits Neuronal Loss in the Retina and Elevates Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Diabetic Rats

    PubMed Central

    Wang, Peipei; Zhu, Yanxia; Chen, Zhen; Shi, Tianyan; Lei, Wensheng

    2015-01-01

    Abstract Purpose: To determine whether curcumin offers neuroprotection to minimize the apoptosis of neural cells in the retina of diabetic rats. Methods: Streptozotocin (STZ)-induced diabetic rats and control rats were used in this study. A subgroup of STZ-induced diabetic rats were treated with curcumin for 12 weeks. Retinal histology, apoptosis of neural cells in the retina, electroretinograms, and retinal glutamate content were evaluated after 12 weeks. Retinal levels of Ca2+/calmodulin-dependent protein kinase II (CaMKII), phospho-CaMKII (p-CaMKII), and cleaved caspase-3 were determined by Western blot analysis. Results: The amplitudes a-wave, b-wave, and oscillatory potential were reduced by diabetes, but curcumin treatment suppressed this reduction of amplitudes. Curcumin also prevented cell loss from the outer nuclear, inner nuclear, and ganglion cell layers. Apoptosis of retinal neurons was detected in diabetic rats. The concentration of glutamate in the retina was higher in diabetic rats, but was significantly reduced in the curcumin-treated group. Furthermore, p-CaMKII and cleaved caspase-3 expression were upregulated in the diabetic retina, but reduced in curcumin-treated rats. Conclusions: Curcumin attenuated diabetes-induced apoptosis in retinal neurons by reducing the glutamate level and downregulating CaMKII. Thus, curcumin might be used to prevent neuronal damage in the retina of patients with diabetes mellitus. PMID:26207889

  2. Dopamine D4 receptor transmission in the prefrontal cortex controls the salience of emotional memory via modulation of calcium calmodulin-dependent kinase II.

    PubMed

    Lauzon, Nicole M; Ahmad, Tasha; Laviolette, Steven R

    2012-11-01

    Dopamine (DA) signaling in the medial prefrontal cortex (mPFC) plays a critical role in the processing of emotional information and memory encoding. Activation of DA D4 receptors within the prelimbic (PLC) division of the mPFC bidirectionally modulates emotional memory by strongly potentiating the salience of normally nonsalient emotional memories but blocking the acquisition of suprathreshold emotionally salient fear memories. Previous in vitro studies have shown that activation of cortical DA D4 receptors can bidirectionally modulate levels of α-calcium calmodulin-dependent kinase II (α-CaMKII), a molecule essential for learning and memory. Using an olfactory fear conditioning procedure in rats combined with microinfusions into the mPFC, we examined the potential role of D4 receptor-mediated control of emotional memory salience through signaling via CaMKII, cAMP/protein kinase A (PKA), and protein phosphatase-1 (PP1) signaling. We report that CaMKII blockade prevents the ability of intra-mPFC DA D4 receptor activation to potentiate the salience of subthreshold fear memory. In contrast, blockade of either cAMP/PKA or PP1 signaling pathways rescued the blockade of suprathreshold fear memory via intra-mPFC D4 receptor activation. Our results demonstrate that modulation of emotional memory salience via intra-mPFC DA D4 receptor transmission depends upon downstream signaling via CaMKII, cAMP/PKA, and PP1 substrates.

  3. Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

    PubMed

    Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

    2016-11-01

    Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

  4. Wenxin-Keli Regulates the Calcium/Calmodulin-Dependent Protein Kinase II Signal Transduction Pathway and Inhibits Cardiac Arrhythmia in Rats with Myocardial Infarction

    PubMed Central

    Xing, Yanwei; Gao, Yonghong; Chen, Jianxin; Zhu, Haiyan; Wu, Aiming; Yang, Qing; Teng, Fei; Zhang, Dong-mei; Xing, Yanhui; Gao, Kuo; He, Qingyong; Zhang, Zhenpeng; Wang, Jie; Shang, Hongcai

    2013-01-01

    Wenxin-Keli (WXKL) is a Chinese herbal compound reported to be of benefit in the treatment of cardiac arrhythmia, cardiac inflammation, and heart failure. Amiodarone is a noncompetitive inhibitor of the α- and β-adrenergic receptors and prevents calcium influx in the slow-response cells of the sinoatrial and atrioventricular nodes. Overexpression of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in transgenic mice results in heart failure and arrhythmias. We hypothesised that administration of WXKL and amiodarone can reduce the incidence of arrhythmias by regulating CaMKII signal transduction. A total of 100 healthy Sprague Dawley rats were used in the study. The rats were randomly divided into four groups (a sham group, a myocardial infarction (MI) group, a WXKL-treated group, and an amiodarone-treated group). A myocardial infarction model was established in these rats by ligating the left anterior descending coronary artery for 4 weeks. Western blotting was used to assess CaMKII, p-CaMKII (Thr-286), PLB, p-PLB (Thr-17), RYR2, and FK binding protein 12.6 (FKBP12.6) levels. The Ca2+ content in the sarcoplasmic reticulum (SR) and the calcium transient amplitude were studied by confocal imaging using the fluorescent indicator Fura-4. In conclusion, WXKL may inhibit heart failure and cardiac arrhythmias by regulating the CaMKII signal transduction pathway similar to amiodarone. PMID:23781262

  5. Wenxin-Keli Regulates the Calcium/Calmodulin-Dependent Protein Kinase II Signal Transduction Pathway and Inhibits Cardiac Arrhythmia in Rats with Myocardial Infarction.

    PubMed

    Xing, Yanwei; Gao, Yonghong; Chen, Jianxin; Zhu, Haiyan; Wu, Aiming; Yang, Qing; Teng, Fei; Zhang, Dong-Mei; Xing, Yanhui; Gao, Kuo; He, Qingyong; Zhang, Zhenpeng; Wang, Jie; Shang, Hongcai

    2013-01-01

    Wenxin-Keli (WXKL) is a Chinese herbal compound reported to be of benefit in the treatment of cardiac arrhythmia, cardiac inflammation, and heart failure. Amiodarone is a noncompetitive inhibitor of the α - and β -adrenergic receptors and prevents calcium influx in the slow-response cells of the sinoatrial and atrioventricular nodes. Overexpression of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in transgenic mice results in heart failure and arrhythmias. We hypothesised that administration of WXKL and amiodarone can reduce the incidence of arrhythmias by regulating CaMKII signal transduction. A total of 100 healthy Sprague Dawley rats were used in the study. The rats were randomly divided into four groups (a sham group, a myocardial infarction (MI) group, a WXKL-treated group, and an amiodarone-treated group). A myocardial infarction model was established in these rats by ligating the left anterior descending coronary artery for 4 weeks. Western blotting was used to assess CaMKII, p-CaMKII (Thr-286), PLB, p-PLB (Thr-17), RYR2, and FK binding protein 12.6 (FKBP12.6) levels. The Ca(2+) content in the sarcoplasmic reticulum (SR) and the calcium transient amplitude were studied by confocal imaging using the fluorescent indicator Fura-4. In conclusion, WXKL may inhibit heart failure and cardiac arrhythmias by regulating the CaMKII signal transduction pathway similar to amiodarone.

  6. Intra-nucleus accumbens administration of the calcium/calmodulin-dependent protein kinase II inhibitor AIP induced antinociception in rats with mononeuropathy.

    PubMed

    Bian, Hui; Yu, Long-Chuan

    2015-07-10

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine- dependent protein kinase, which has been implicated in pain modulation at different levels of the central nervous system. The present study was performed in rats with mononeuropathy induced by left common sciatic nerve ligation. Unilateral sciatic nerve loose ligation produced decreases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation. Intra-nucleus accumbens (NAc) injection of 3 μg, 6 μg and 12 μg of myristoylated autocamtide-2-inhibitory peptide (AIP), the CaMKII inhibitor, dose-dependently increased the HWL to noxious thermal and mechanical stimulation in rats with mononeuropathy. Furthermore, intra-NAc administration of morphine, the HWL to noxious thermal and mechanical stimulation increased markedly, and there were no significant differences between morphine group and AIP group. Taken together, the results showed that intra-NAc injection of AIP induced significant antinociceptive effects in rats with mononeuropathy, indicating that CaMKII may play an important role in the transmission and/or modulation of nociceptive information in the NAc in rats with mononeuropathy.

  7. Casein kinase II is required for proper cell division and acts as a negative regulator of centrosome duplication in Caenorhabditis elegans embryos.

    PubMed

    Medley, Jeffrey C; Kabara, Megan M; Stubenvoll, Michael D; DeMeyer, Lauren E; Song, Mi Hye

    2017-01-15

    Centrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein kinase II (CK2) in early Caenorhabditis elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner. © 2017. Published by The Company of Biologists Ltd.

  8. Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca sup 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Gandy, S.; Czernik, A.J.; Greengard, P. )

    1988-08-01

    The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggest a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP. Ca{sup 2+}/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. Using rat cerebral cortex synaptosomes prelabeled with {sup 32}P{sub i}, a {sup 32}P-labeled phosphoprotein of {approx}135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.

  9. 2,5-hexanedione (HD) treatment alters calmodulin, Ca{sup 2+}/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

    SciTech Connect

    Wang Qingshan Hou Liyan; Zhang Cuili; Zhao Xiulan; Yu Sufang; Xie, Ke-Qin

    2008-10-01

    Calcium-dependent mechanisms, particularly those mediated by Ca{sup 2+}/calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50 mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC and SN were significantly increased, which indicated elevation of Ca{sup 2+} concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs.

  10. Atlas of transgenic Tet-Off Ca2+/calmodulin-dependent protein kinase II and prion protein promoter activity in the mouse brain.

    PubMed

    Odeh, Francis; Leergaard, Trygve B; Boy, Jana; Schmidt, Thorsten; Riess, Olaf; Bjaalie, Jan G

    2011-02-14

    Conditional transgenic mouse models are important tools for investigations of neurodegenerative diseases and evaluation of potential therapeutic interventions. A popular conditional transgenic system is the binary tetracycline-responsive gene (Tet-Off) system, in which the expression of the gene of interest depends on a tetracycline-regulatable transactivator (tTA) under the control of a specific promoter construct. The most frequently used Tet-Off promoter mouse lines are the Ca(2+)/calmodulin-dependent protein kinase II (CamKII) and prion protein (PrP) promoter lines, respectively. To target the regulated gene of interest to relevant brain regions, a priori knowledge about the spatial distribution of the regulated gene expression in the brain is important. Such distribution patterns can be investigated using double transgenic mice in which the promoter construct regulates a LacZ reporter gene encoding the marker β-galactosidase which can be histologically detected using its substrate X-gal. We have previously published an atlas showing the brain-wide expression mediated by the Tet-Off PrP promoter mouse line, but the distribution of activity in the Tet-Off CamKII promoter mouse line is less well known. To compare promoter activity distributions in these two Tet-Off mouse lines, we have developed an online digital atlas tailored for side-by-side comparison of histological section images. The atlas provides a comprehensive list of brain regions containing X-gal labeling and an interactive dual image viewer tool for panning and zooming of corresponding section images. Comparison of spatial expression patterns between the two lines show considerable regional and cellular differences, relevant in context of generation and analysis of inducible models based on these two tetracycline responsive promoter mouse lines. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. The inhibitor of calcium/calmodulin-dependent protein kinase II KN93 attenuates bone cancer pain via inhibition of KIF17/NR2B trafficking in mice.

    PubMed

    Liu, Yue; Liang, Ying; Hou, Bailing; Liu, Ming; Yang, Xuli; Liu, Chenglong; Zhang, Juan; Zhang, Wei; Ma, Zhengliang; Gu, Xiaoping

    2014-09-01

    The N-methyl-d-aspartate receptor (NMDAR) containing subunit 2B (NR2B) is critical for the regulation of nociception in bone cancer pain, although the precise molecular mechanisms remain unclear. KIF17, a kinesin motor, plays a key role in the dendritic transport of NR2B. The up-regulation of NR2B and KIF17 transcription results from an increase in phosphorylated cAMP-response element-binding protein (CREB), which is activated by calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we hypothesized that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. The expression of spinal t-CaMKII, p-CaMKII, NR2B and KIF17 after inoculation was also evaluated. These results showed that inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant up-regulation of p-CaMKII, NR2B and KIF17 expression after inoculation. Intrathecal administration of KN93, a CaMKII inhibitor, down-regulated these three proteins and attenuated bone cancer pain in a dose- and time-dependent manner. These findings indicated that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain, and inhibition of CaMKII may be a useful alternative or adjunct therapy for relieving cancer pain.

  12. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  13. Possible interaction of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II on reversal of spatial memory impairment induced by morphine.

    PubMed

    Farahmandfar, Maryam; Kadivar, Mehdi; Naghdi, Nasser

    2015-03-15

    The opioid system plays an important role in learning and memory by modulation of different molecules in the brain. The aim of the present study was to investigate the role of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II (CaMKII) on the morphine-induced modulation of spatial memory consolidation in male rats. Spatial memory was assessed in Morris water maze task by a single training session of eight trials followed by a probe trial and visible test 24h later. Our data indicated that post-training administration of L-arginine, a nitric oxide precursor (6 and 9 µg/rat, intra-CA1) significantly decreased amnesia induced by morphine (10 mg/kg) in spatial memory consolidation. A reversal effect of L-arginine on morphine-induced amnesia prevented by KN-93 (N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl) phenyl]-N-[2-hydroxyethyl] methoxybenzenesulfnamide), CaMKII inhibitor, (10 nmol/0.5 µl/site). In addition, post-training injection of L-NAME, (NG-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor (10 and 15 µg/rat) or KN-93 (10 nmol/0.5 µl/site) with lower dose of morphine (2.5 mg/kg), which did not induce amnesia by itself, caused inhibition of memory consolidation. We also showed that co-administration of L-arginine (9 µg/rat) and morphine (10 mg/kg) significantly increased CaMKII activity in the rat hippocampus. On the other hand, administration of L-NAME (10 µg/rat) led to a decrease in the haippocampal activity of CaMKII in morphine-treated (2.5mg/kg) animals. These results indicate that acute single exposure to morphine can modulate consolidation of spatial memory, which may be mediated by a hippocampal nitrergic system and CaMKII activity.

  14. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  15. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  16. L-type calcium channels and calcium/calmodulin-dependent kinase II differentially mediate behaviors associated with nicotine withdrawal in mice.

    PubMed

    Jackson, K J; Damaj, M I

    2009-07-01

    Smoking is a widespread health problem. Because the nicotine withdrawal syndrome is a major contributor to continued smoking and relapse, it is important to understand the molecular and behavioral mechanisms of nicotine withdrawal to generate more effective smoking cessation therapies. Studies suggest a role for calcium-dependent mechanisms, such as L-type calcium channels and calcium/calmodulin-dependent protein kinase II (CaMKII), in the effects of nicotine dependence; however, the role of these mechanisms in nicotine-mediated behaviors is unclear. Thus, the goal of this study was to elucidate the role of L-type calcium channels and CaMKII in nicotine withdrawal behaviors. Using both pharmacological and genetic methods, our results show that L-type calcium channels are involved in physical, but not affective, nicotine withdrawal behaviors. Although our data do provide evidence of a role for CaMKII in nicotine withdrawal behaviors, our pharmacological and genetic assessments yielded different results concerning the specific role of the kinase. Pharmacological data suggest that CaMKII is involved in somatic signs and affective nicotine withdrawal, and activity level is decreased after nicotine withdrawal, whereas the genetic assessments yielded results suggesting that CaMKII is involved only in the anxiety-related response, yet the kinase activity may be increased after nicotine withdrawal; thus, future studies are necessary to clarify the precise behavioral specifics of the relevance of CaMKII in nicotine withdrawal behaviors. Overall, our data show that L-type calcium channels and CaMKII are relevant in nicotine withdrawal and differentially mediate nicotine withdrawal behaviors.

  17. Phosphorylation of calcium/calmodulin-dependent protein kinase II in the rat dorsal medial prefrontal cortex is associated with alcohol-induced cognitive inflexibility.

    PubMed

    Natividad, Luis A; Steinman, Michael Q; Laredo, Sarah A; Irimia, Cristina; Polis, Ilham Y; Lintz, Robert; Buczynski, Matthew W; Martin-Fardon, Rémi; Roberto, Marisa; Parsons, Loren H

    2017-09-22

    Repeated cycles of alcohol [ethanol (EtOH)] intoxication and withdrawal dysregulate excitatory glutamatergic systems in the brain and induce neuroadaptations in the medial prefrontal cortex (mPFC) that contribute to cognitive dysfunction. The mPFC is composed of subdivisions that are functionally distinct, with dorsal regions facilitating drug-cue associations and ventral regions modulating new learning in the absence of drug. A key modulator of glutamatergic activity is the holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) that phosphorylates ionotropic glutamate receptors. Here, we examined the hypothesis that abstinence from chronic intermittent EtOH (CIE) exposure dysregulates CaMKII activity in the mPFC to impair cognitive flexibility. We used an operant model of strategy set shifting in male Long-Evans rats demonstrating reduced susceptibility to trial omissions during performance in a visual cue-guided task versus albino strains. Relative to naïve controls, rats experiencing approximately 10 days of abstinence from CIE vapor exposure demonstrated impaired performance during a procedural shift from visual cue to spatial location discrimination. Phosphorylation of CaMKII subtype α was upregulated in the dorsal, but not ventral mPFC of CIE-exposed rats, and was positively correlated with perseverative-like responding during the set shift. The findings suggest that abstinence from CIE exposure induces an undercurrent of kinase activity (e.g. CaMKII), which may promote aberrant glutamatergic responses in select regions of the mPFC. Given the role of the mPFC in modulating executive control of behavior, we propose that increased CaMKII subtype α activity reflects a dysregulated 'top-down' circuit that interferes with adaptive behavioral performance under changing environmental demands. © 2017 Society for the Study of Addiction.

  18. Regulation of the multifunctional Ca2+/calmodulin-dependent protein kinase II by the PP2C phosphatase PPM1F in fibroblasts.

    PubMed

    Harvey, Bohdan P; Banga, Satnam S; Ozer, Harvey L

    2004-06-04

    The regulation of the multifunctional calcium/calmodulin dependent protein kinase II (CaMKII) by serine/threonine protein phosphatases has been extensively studied in neuronal cells; however, this regulation has not been investigated previously in fibroblasts. We cloned a cDNA from SV40-transformed human fibroblasts that shares 80% homology to a rat calcium/calmodulin-dependent protein kinase phosphatase that encodes a PPM1F protein. By using extracts from transfected cells, PPM1F, but not a mutant (R326A) in the conserved catalytic domain, was found to dephosphorylate in vitro a peptide corresponding to the auto-inhibitory region of CaMKII. Further analyses demonstrated that PPM1F specifically dephosphorylates the phospho-Thr-286 in autophosphorylated CaMKII substrate and thus deactivates the CaMKII in vitro. Coimmunoprecipitation of CaMKII with PPM1F indicates that the two proteins can interact intracellularly. Binding of PPM1F to CaMKII involves multiple regions and is not dependent on intact phosphatase activity. Furthermore, overexpression of PPM1F in fibroblasts caused a reduction in the CaMKII-specific phosphorylation of the known substrate vimentin(Ser-82) following induction of the endogenous CaM kinase. These results identify PPM1F as a CaM kinase phosphatase within fibroblasts, although it may have additional functions intracellularly since it has been presented elsewhere as POPX2 and hFEM-2. We conclude that PPM1F, possibly together with the other previously described protein phosphatases PP1 and PP2A, can regulate the activity of CaMKII. Moreover, because PPM1F dephosphorylates the critical autophosphorylation site of CaMKII, we propose that this phosphatase plays a key role in the regulation of the kinase intracellularly.

  19. Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis.

    PubMed

    Tsaadon, Lina; Kaplan-Kraicer, Ruth; Shalgi, Ruth

    2008-05-01

    Sperm-egg fusion induces cortical granules exocytosis (CGE), a process that ensures the block to polyspermy. CGE can be induced independently by either a rise in intracellular calcium concentration or protein kinase C (PKC) activation. We have previously shown that myristoylated alanine-rich C kinase substrate (MARCKS) cross-links filamentous actin (F-actin) and regulates its reorganization. This activity is reduced either by PKC-induced MARCKS phosphorylation (PKC pathway) or by its direct binding to calmodulin (CaM; CaM pathway), both inducing MARCKS translocation, F-actin reorganization, and CGE. Currently, we examine the involvement of Ca(2)(+)/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca(2)(+)/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active alphaCaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt alphaCaMKII. Microinjecting eggs with mutant-unphosphorylatable MARCKS reduced the intensity of 12-O-tetradecanoylphorbol 13-acetate or ionomycin-induced CGE by 50%, indicating that phosphorylation of MARCKS by novel and/or conventional PKCs (n/cPKCs) is a pivotal event associated with CGE. Moreover, we were able to demonstrate cPKCs involvement in ionomycin-induced MARCKS translocation and CGE. These results led us to propose that MARCKS, rather than CaMKII, as a key mediator of CGE.

  20. Cardiac CaM Kinase II Genes δ and γ Contribute to Adverse Remodeling but Redundantly Inhibit Calcineurin-Induced Myocardial Hypertrophy

    PubMed Central

    Kreusser, Michael M.; Lehmann, Lorenz H.; Keranov, Stanislav; Hoting, Marc-Oscar; Oehl, Ulrike; Kohlhaas, Michael; Reil, Jan-Christian; Neumann, Kay; Schneider, Michael D.; Hill, Joseph A.; Dobrev, Dobromir; Maack, Christoph; Maier, Lars S.; Gröne, Hermann-Josef; Katus, Hugo A.; Olson, Eric N.; Backs, Johannes

    2014-01-01

    Background Ca2+-dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear. Methods and Results We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca2+ handling nor functional and structural changes. On pathological pressure overload and β-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling. Conclusions We established a mouse model in which CaMKII’s activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure. PMID:25124496

  1. Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

    PubMed

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

    2014-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.

  2. Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase II in spinal cord injury rats

    PubMed Central

    Min, You-jiang; Ding, Li-li-qiang; Cheng, Li-hong; Xiao, Wei-ping; He, Xing-wei; Zhang, Hui; Min, Zhi-yun; Pei, Jia

    2017-01-01

    Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase (ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan (GV3), Dazhui (GV14), Zusanli (ST36) and Ciliao (BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the mRNA and protein expression of Rho-A and Rho-associated kinase II (ROCKII) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKII. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKII. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of RhoA and ROCKII. There was no synergistic effect of electroacupuncture combined with monosialoganglioside. PMID:28400811

  3. Calcium/Calmodulin-dependent Protein Kinase II is a Ubiquitous Molecule in Human Long-term Memory Synaptic Plasticity: A Systematic Review

    PubMed Central

    Ataei, Negar; Sabzghabaee, Ali Mohammad; Movahedian, Ahmad

    2015-01-01

    Background: Long-term memory is based on synaptic plasticity, a series of biochemical mechanisms include changes in structure and proteins of brain's neurons. In this article, we systematically reviewed the studies that indicate calcium/calmodulin kinase II (CaMKII) is a ubiquitous molecule among different enzymes involved in human long-term memory and the main downstream signaling pathway of long-term memory. Methods: All of the observational, case–control and review studies were considered and evaluated by the search engines PubMed, Cochrane Central Register of Controlled Trials and ScienceDirect Scopus between 1990 and February 2015. We did not carry out meta-analysis. Results: At the first search, it was fined 1015 articles which included “synaptic plasticity” OR “neuronal plasticity” OR “synaptic density” AND memory AND “molecular mechanism” AND “calcium/calmodulin-dependent protein kinase II” OR CaMKII as the keywords. A total of 335 articles were duplicates in the databases and eliminated. A total of 680 title articles were evaluated. Finally, 40 articles were selected as reference. Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory. PMID:26445635

  4. Involvement of Amygdalar Protein Kinase A, but not Calcium/Calmodulin-Dependent Protein Kinase II, in the Reconsolidation of Cocaine-Related Contextual Memories in Rats

    PubMed Central

    Arguello, Amy A.; Hodges, Matthew A.; Wells, Audrey M.; Lara, Honorio; Xie, Xiaohu; Fuchs, Rita A.

    2013-01-01

    Rationale Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response-cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon. Objectives The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response-cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior. Methods Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context. Results Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion. Conclusions PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental

  5. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

    PubMed Central

    Rihs, H P; Jans, D A; Fan, H; Peters, R

    1991-01-01

    We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport. Images PMID:1848177

  6. Calcium-calmodulin-dependent kinase II modulates Kv4.2 channel expression and upregulates neuronal A-type potassium currents.

    PubMed

    Varga, Andrew W; Yuan, Li-Lian; Anderson, Anne E; Schrader, Laura A; Wu, Gang-Yi; Gatchel, Jennifer R; Johnston, Daniel; Sweatt, J David

    2004-04-07

    Calcium-calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K(+) channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K(+) current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K(+) channels.

  7. Age-dependent changes in autophosphorylation of alpha calcium/calmodulin dependent kinase II in hippocampus and amygdala after contextual fear conditioning.

    PubMed

    Fang, Ton; Kasbi, Kamillia; Rothe, Stephanie; Aziz, Wajeeha; Giese, K Peter

    2017-09-01

    The hippocampus and amygdala are essential brain regions responsible for contextual fear conditioning (CFC). The autophosphorylation of alpha calcium-calmodulin kinase II (αCaMKII) at threonine-286 (T286) is a critical step implicated in long-term potentiation (LTP), learning and memory. However, the changes in αCaMKII levels with aging and training in associated brain regions are not fully understood. Here, we studied how aging and training affect the levels of phosphorylated (T286) and proportion of phosphorylated:total αCaMKII in the hippocampus and amygdala. Young and aged mice, naïve (untrained) and trained in CFC, were analysed by immunohistochemistry for the levels of total and phosphorylated αCaMKII in the hippocampus and amygdala. We found that two hours after CFC training, young mice exhibited a higher level of phosphorylated and increased ratio of phosphorylated:total αCaMKII in hippocampal CA3 stratum radiatum. Furthermore, aged untrained mice showed a higher ratio of phosphorylated:total αCaMKII in the CA3 region of the hippocampus when compared to the young untrained group. No effect of training or aging were seen in the central, lateral and basolateral amygdala regions, for both phosphorylated and ratio of phosphorylated:total αCaMKII. These results show that aging impairs the training-induced upregulation of autophosphorylated (T286) αCaMKII in the CA3 stratum radiatum of the hippocampus. This indicates that distinct age-related mechanisms underlie CFC that may rely more heavily on NMDA receptor-dependent plasticity in young age. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.

    PubMed

    Marganski, William A; Gangopadhyay, Samudra S; Je, Hyun-Dong; Gallant, Cynthia; Morgan, Kathleen G

    2005-09-16

    Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.

  9. Gating of long-term depression by Ca2+/calmodulin-dependent protein kinase II through enhanced cGMP signalling in cerebellar Purkinje cells

    PubMed Central

    Kawaguchi, Shin-ya; Hirano, Tomoo

    2013-01-01

    Long-term depression (LTD) at parallel fibre synapses on a cerebellar Purkinje cell has been regarded as a cellular basis for motor learning. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the LTD induction as an important Ca2+-sensing molecule, the underlying signalling mechanism remains unclear. Here, we attempted to explore the potential signalling pathway underlying the CaMKII involvement in LTD using a systems biology approach, combined with validation by electrophysiological and FRET imaging experiments on a rat cultured Purkinje cell. Model simulation predicted the following cascade as a candidate mechanism for the CaMKII contribution to LTD: CaMKII negatively regulates phosphodiesterase 1 (PDE1), subsequently facilitates the cGMP/protein kinase G (PKG) signalling pathway and down-regulates protein phosphatase 2A (PP-2A), thus supporting the LTD-inducing positive feedback loop consisting of mutual activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This model suggestion was corroborated by whole-cell patch clamp recording experiments. In addition, FRET measurement of intracellular cGMP concentration revealed that CaMKII activation causes sustained increase of cGMP, supporting the signalling mechanism of LTD induction by CaMKII. Furthermore, we found that activation of the cGMP/PKG pathway by nitric oxide (NO) can support LTD induction without activation of CaMKII. Thus, this study clarified interaction between NO and Ca2+/CaMKII, two important factors required for LTD. PMID:23297306

  10. Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases.

    PubMed

    Walkup, Ward G; Washburn, Lorraine; Sweredoski, Michael J; Carlisle, Holly J; Graham, Robert L; Hess, Sonja; Kennedy, Mary B

    2015-02-20

    synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases*

    PubMed Central

    Walkup, Ward G.; Washburn, Lorraine; Sweredoski, Michael J.; Carlisle, Holly J.; Graham, Robert L.; Hess, Sonja; Kennedy, Mary B.

    2015-01-01

    synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons. PMID:25533468

  12. Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

    PubMed Central

    Banerjee, Chaitali; Khatri, Preeti; Raman, Rajagopal; Bhatia, Himanshi; Datta, Malabika; Mazumder, Shibnath

    2014-01-01

    The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM. PMID:24763432

  13. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo.

    PubMed

    Jackson, K J; Walters, C L; Damaj, M I

    2009-08-01

    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

  14. Ca(2+)/Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy.

    PubMed

    Duran, Javier; Lagos, Daniel; Pavez, Mario; Troncoso, Mayarling F; Ramos, Sebastián; Barrientos, Genaro; Ibarra, Cristian; Lavandero, Sergio; Estrada, Manuel

    2017-01-01

    Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR)-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and myocyte-enhancer factor 2 (MEF2) play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX) rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg⋅kg(-1)⋅week(-1)) for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN) phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286) and phospholambam (PLN) (Thr17) in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that testosterone

  15. Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    SciTech Connect

    Gangopadhyay, Jaya Pal; Ikemoto, Noriaki

    2010-05-28

    We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca{sup 2+} leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca{sup 2+}/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca{sup 2+} transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca{sup 2+} events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional

  16. Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways.

    PubMed

    Luo, Zhirong; Kohli, Meetu R; Yu, Qing; Kim, Syngcuk; Qu, Tiejun; He, Wen-xi

    2014-07-01

    Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. Biodentine is a bioactive and biocompatible material capable

  17. Multivalent interactions of calcium/calmodulin-dependent protein kinase II with the postsynaptic density proteins NR2B, densin-180, and alpha-actinin-2.

    PubMed

    Robison, A J; Bass, Martha A; Jiao, Yuxia; MacMillan, Leigh B; Carmody, Leigh C; Bartlett, Ryan K; Colbran, Roger J

    2005-10-21

    Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.

  18. Membrane actions of 1α,25(OH)2D3 are mediated by Ca(2+)/calmodulin-dependent protein kinase II in bone and cartilage cells.

    PubMed

    Doroudi, Maryam; Plaisance, Marc C; Boyan, Barbara D; Schwartz, Zvi

    2015-01-01

    1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model.

    PubMed

    Lopes, Luciana B; Brophy, Colleen M; Flynn, Charles R; Yi, Zhengping; Bowen, Benjamin P; Smoke, Christopher; Seal, Brandon; Panitch, Alyssa; Komalavilas, Padmini

    2010-12-01

    The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening. The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting. Pretreatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%. Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the

  20. A Novel Cell Permeant Peptide Inhibitor of MAPKAP Kinase II Inhibits Intimal Hyperplasia in a Human Saphenous Vein Organ Culture Model

    PubMed Central

    Lopes, Luciana B.; Brophy, Colleen M.; Flynn, Charles R.; Yi, Zhengping; Bowen, Benjamin P.; Smoke, Christopher; Seal, Brandon; Panitch, Alyssa; Komalavilas, Padmini

    2010-01-01

    Objective The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates HSP27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening. Design of study The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture (SILAC), in combination with liquid chromatography mass spectrometry was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and CTGF expression was evaluated in human saphenous vein (HSV) rings maintained with 30% FBS for 14 days by light microscopy and immunoblotting. Results Pre-treatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36±9% and 33±10% respectively) compared to control (not pre-treated) cells. MK2i also inhibited A7R5 migration, and downregulated the TGF-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40±17%), CTGF expression (17±5%) and intimal thickness (48.2±10.5%) compared to untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48±14%. Conclusion Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure. PMID:20864298

  1. Ca2+/Calmodulin-dependent Protein Kinase II Inhibitors Disrupt AKAP79-dependent PKC Signaling to GluA1 AMPA Receptors*

    PubMed Central

    Brooks, Ian M.; Tavalin, Steven J.

    2011-01-01

    GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31–52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31–52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31–52) in the absence (apoCaM) but not the presence of Ca2+. In contrast, apoCaM association to AKAP79(31–52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31–52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII. PMID:21156788

  2. Mammalian development in space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  3. Mammalian development in space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  4. Mammalian development in space.

    PubMed

    Ronca, April E

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  5. Mammalian touch catches up

    PubMed Central

    Walsh, Carolyn M.; Bautista, Diana M.; Lumpkin, Ellen A.

    2015-01-01

    An assortment of touch receptors innervate the skin and encode different tactile features of the environment. Compared with invertebrate touch and other sensory systems, our understanding of the molecular and cellular underpinnings of mammalian touch lags behind. Two recent breakthroughs have accelerated progress. First, an arsenal of cell-type-specific molecular markers allowed the functional and anatomical properties of sensory neurons to be matched, thereby unraveling a cellular code for touch. Such markers have also revealed key roles of non-neuronal cell types, such as Merkel cells and keratinocytes, in touch reception. Second, the discovery of Piezo genes as a new family of mechanically activated channels has fueled the discovery of molecular mechanisms that mediate and mechanotransduction in mammalian touch receptors. PMID:26100741

  6. Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

    USGS Publications Warehouse

    Park, J.-W.; Moon, C.H.; Harmache, A.; Wargo, A.R.; Purcell, M.K.; Bremont, M.; Kurath, G.

    2011-01-01

    casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

  7. Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

    PubMed

    Park, J W; Moon, C H; Harmache, A; Wargo, A R; Purcell, M K; Bremont, M; Kurath, G

    2011-02-01

    casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

  8. Mammalian glycosylation in immunity.

    PubMed

    Marth, Jamey D; Grewal, Prabhjit K

    2008-11-01

    Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.

  9. Mammalian sperm morphometry.

    PubMed Central

    Gage, M J

    1998-01-01

    Understanding the adaptive significance of sperm form and function has been a challenge to biologists because sperm are highly specialized cells operating at a microscopic level in a complex environment. A fruitful course of investigation has been to use the comparative approach. This comparative study attempts to address some fundamental questions of the evolution of mammalian sperm morphometry. Data on sperm morphometry for 445 mammalian species were collated from published sources. I use contemporary phylogenetic analysis to control for the inherent non-independence of species and explore relationships between the morphometric dimensions of the three essential spermatozoal components: head, mid-piece and flagellum. Energy for flagellar action is metabolized by the mitochondrial-dense mid-piece and these combine to propel the sperm head, carrying the male haplotype, to the ovum. I therefore search for evolutionary associations between sperm morphometry and body mass, karyotype and the duration of oestrus. In contrast to previous findings, there is no inverse correlation between body weight and sperm length. Sperm mid-piece and flagellum lengths are positively associated with both head length and area, and the slopes of these relationships are discussed. Flagellum length is positively associated with mid-piece length but, in contrast to previous research and after phylogenetic control, I find no relationship between flagellum length and the volume of the mitochondrial sheath. Sperm head dimensions are not related to either genome mass or chromosome number, and there are no relationships between sperm morphometry and the duration of oestrus. PMID:9474794

  10. Mammalian Molecular Clocks

    PubMed Central

    Kwon, Ilmin; Choe, Han Kyoung; Son, Gi Hoon

    2011-01-01

    As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock. PMID:22110358

  11. The mammalian blastocyst.

    PubMed

    Frankenberg, Stephen R; de Barros, Flavia R O; Rossant, Janet; Renfree, Marilyn B

    2016-01-01

    The blastocyst is a mammalian invention that carries the embryo from cleavage to gastrulation. For such a simple structure, it exhibits remarkable diversity in its mode of formation, morphology, longevity, and intimacy with the uterine endometrium. This review explores this diversity in the light of the evolution of viviparity, comparing the three main groups of mammals: monotremes, marsupials, and eutherians. The principal drivers in blastocyst evolution were loss of yolk coupled with evolution of the placenta. An important outcome of blastocyst development is differentiation of two extraembryonic lineages (trophoblast and hypoblast) that contribute to the placenta. While in many species trophoblast segregation is often coupled with blastocyst formation, in marsupials and at least some Afrotherians, these events do not coincide. Thus, many questions regarding the conservation of molecular mechanisms controlling these events are of great interest but currently unresolved. For further resources related to this article, please visit the WIREs website.

  12. Mammalian phospholipase C.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2013-01-01

    Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale.

  13. New Mammalian Expression Systems.

    PubMed

    Zhu, Jie; Hatton, Diane

    2017-06-06

    There are an increasing number of recombinant antibodies and proteins in preclinical and clinical development for therapeutic applications. Mammalian expression systems are key to enabling the production of these molecules, and Chinese hamster ovary (CHO) cell platforms continue to be central to delivery of the stable cell lines required for large-scale production. Increasing pressure on timelines and efficiency, further innovation of molecular formats and the shift to new production systems are driving developments of these CHO cell line platforms. The availability of genome and transcriptome data coupled with advancing gene editing tools are increasing the ability to design and engineer CHO cell lines to meet these challenges. This chapter aims to give an overview of the developments in CHO expression systems and some of the associated technologies over the past few years.

  14. Tumor necrosis factor-alpha enhances neutrophil adhesiveness: induction of vascular cell adhesion molecule-1 via activation of Akt and CaM kinase II and modifications of histone acetyltransferase and histone deacetylase 4 in human tracheal smooth muscle cells.

    PubMed

    Lee, Chiang-Wen; Lin, Chih-Chung; Luo, Shue-Fen; Lee, Hui-Chun; Lee, I-Ta; Aird, William C; Hwang, Tsong-Long; Yang, Chuen-Mao

    2008-05-01

    Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) involves adhesions between both circulating and resident leukocytes and the human tracheal smooth muscle cells (HTSMCs) during airway inflammatory reaction. We have demonstrated previously that tumor necrosis factor (TNF)-alpha-induced VCAM-1 expression is regulated by mitogen-activated protein kinases, nuclear factor-kappaB, and p300 activation in HTSMCs. In addition to this pathway, phosphorylation of Akt and CaM kinase II has been implicated in histone acetyltransferase and histone deacetylase 4 (HDAC4) activation. Here, we investigated whether these different mechanisms participated in TNF-alpha-induced VCAM-1 expression and enhanced neutrophil adhesion. TNF-alpha significantly increased HTSMC-neutrophil adhesions, and this effect was associated with increased expression of VCAM-1 on the HTSMCs and was blocked by the selective inhibitors of Src [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], epidermal growth factor receptor [EGFR; 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline, (AG1478)], phosphatidylinositol 3-kinase (PI3K) [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride(LY294002) and wortmannin],calcium[1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; BAPTA-AM], phosphatidylinositol-phospholipase C (PLC) [1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], protein kinase C (PKC) [12-(2-cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), rottlerin, and 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro 31-8220)], CaM (calmidazolium chloride), CaM kinase II [(8R(*),9S(*),11S(*))-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926) and 1-[N,O-bis(5-isoquinolinesulfonyl

  15. Administration of a Ca-super(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor prevents the learning deficit observed in spinal rats after noncontingent shock administration.

    PubMed

    Baumbauer, Kyle M; Young, Erin E; Hoy, Kevin C; Abood, Athena; Joynes, Robin L

    2007-06-01

    Research has shown that spinal rats given shock to the hind leg when it is in an extended position (contingent shock) will learn to maintain a flexion response. However, subjects that experience shock irrespective of leg position (noncontingent shock) do not exhibit this learning. The current studies examined the role of Ca-super(2+)/calmodulin-dependent protein kinase II (CaMKII) in this learning deficit. Subjects were given intrathecal injections of CaMKII inhibitor solution or artificial cerebrospinal fluid (aCSF) 15 min prior to and immediately or 4 hr following noncontingent shock training. Results demonstrate that the CaMKII inhibitor successfully reversed the learning deficit when injected prior to and immediately following training. These results indicate the importance of CaMKII in the learning deficit present in spinal animals trained with noncontingent shock. Copyright (c) 2007 APA, all rights reserved.

  16. Adrenomedullin in mammalian embryogenesis.

    PubMed

    Garayoa, Mercedes; Bodegas, Elena; Cuttitta, Frank; Montuenga, Luis M

    2002-04-01

    Here are summarized data supporting that adrenomedullin (AM) is a multifunctional factor involved in the complex regulatory mechanisms of mammalian development. During rodent embryogenesis, AM is first expressed in the heart, followed by a broader but also defined spatio-temporal pattern of expression in vascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs. AM pattern of expression is suggestive of its involvement in the control of embryonic invasion, proliferation, and differentiation processes, probably through autocrine or paracrine modes of action. AM levels in fetoplacental tissues, uterus, maternal and umbilical plasma are highly increased during normal gestation. These findings in addition to other physiological and gene targeting studies support the importance of AM as a vasorelaxant factor implicated in the regulation of maternal vascular adaptation to pregnancy, as well as of fetal and fetoplacental circulations. AM is also present in amniotic fluid and milk, which is suggestive of additional functions in the maturation and immunological protection of the fetus. Altered expression of AM has been found in some gestational pathologies, although it is not yet clear whether this corresponds to causative or compensatory mechanisms. Future studies in regard to the distribution and expression levels of the molecules known to function as AM receptors, together with data on the action of complement factor H (an AM binding protein), may help to better define the roles of AM during embryonic development. Copyright 2002 Wiley-Liss, Inc.

  17. The Mammalian Septin Interactome

    PubMed Central

    Neubauer, Katharina; Zieger, Barbara

    2017-01-01

    Septins are GTP-binding and membrane-interacting proteins with a highly conserved domain structure involved in various cellular processes, including cytoskeleton organization, cytokinesis, and membrane dynamics. To date, 13 different septin genes have been identified in mammals (SEPT1 to SEPT12 and SEPT14), which can be classified into four distinct subgroups based on the sequence homology of their domain structure (SEPT2, SEPT3, SEPT6, and SEPT7 subgroup). The family members of these subgroups have a strong affinity for other septins and form apolar tri-, hexa-, or octameric complexes consisting of multiple septin polypeptides. The first characterized core complex is the hetero-trimer SEPT2-6-7. Within these complexes single septins can be exchanged in a subgroup-specific manner. Hexamers contain SEPT2 and SEPT6 subgroup members and SEPT7 in two copies each whereas the octamers additionally comprise two SEPT9 subgroup septins. The various isoforms seem to determine the function and regulation of the septin complex. Septins self-assemble into higher-order structures, including filaments and rings in orders, which are typical for different cell types. Misregulation of septins leads to human diseases such as neurodegenerative and bleeding disorders. In non-dividing cells such as neuronal tissue and platelets septins have been associated with exocytosis. However, many mechanistic details and roles attributed to septins are poorly understood. We describe here some important mammalian septin interactions with a special focus on the clinically relevant septin interactions. PMID:28224124

  18. Mammalian clock output mechanisms.

    PubMed

    Kalsbeek, Andries; Yi, Chun-Xia; Cailotto, Cathy; la Fleur, Susanne E; Fliers, Eric; Buijs, Ruud M

    2011-06-30

    In mammals many behaviours (e.g. sleep-wake, feeding) as well as physiological (e.g. body temperature, blood pressure) and endocrine (e.g. plasma corticosterone concentration) events display a 24 h rhythmicity. These 24 h rhythms are induced by a timing system that is composed of central and peripheral clocks. The highly co-ordinated output of the hypothalamic biological clock not only controls the daily rhythm in sleep-wake (or feeding-fasting) behaviour, but also exerts a direct control over many aspects of hormone release and energy metabolism. First, we present the anatomical connections used by the mammalian biological clock to enforce its endogenous rhythmicity on the rest of the body, especially the neuro-endocrine and energy homoeostatic systems. Subsequently, we review a number of physiological experiments investigating the functional significance of this neuro-anatomical substrate. Together, this overview of experimental data reveals a highly specialized organization of connections between the hypothalamic pacemaker and neuro-endocrine system as well as the pre-sympathetic and pre-parasympathetic branches of the autonomic nervous system.

  19. The Mammalian Septin Interactome.

    PubMed

    Neubauer, Katharina; Zieger, Barbara

    2017-01-01

    Septins are GTP-binding and membrane-interacting proteins with a highly conserved domain structure involved in various cellular processes, including cytoskeleton organization, cytokinesis, and membrane dynamics. To date, 13 different septin genes have been identified in mammals (SEPT1 to SEPT12 and SEPT14), which can be classified into four distinct subgroups based on the sequence homology of their domain structure (SEPT2, SEPT3, SEPT6, and SEPT7 subgroup). The family members of these subgroups have a strong affinity for other septins and form apolar tri-, hexa-, or octameric complexes consisting of multiple septin polypeptides. The first characterized core complex is the hetero-trimer SEPT2-6-7. Within these complexes single septins can be exchanged in a subgroup-specific manner. Hexamers contain SEPT2 and SEPT6 subgroup members and SEPT7 in two copies each whereas the octamers additionally comprise two SEPT9 subgroup septins. The various isoforms seem to determine the function and regulation of the septin complex. Septins self-assemble into higher-order structures, including filaments and rings in orders, which are typical for different cell types. Misregulation of septins leads to human diseases such as neurodegenerative and bleeding disorders. In non-dividing cells such as neuronal tissue and platelets septins have been associated with exocytosis. However, many mechanistic details and roles attributed to septins are poorly understood. We describe here some important mammalian septin interactions with a special focus on the clinically relevant septin interactions.

  20. A Rosetta stone of mammalian genetics.

    PubMed

    Nadeau, J H; Grant, P L; Mankala, S; Reiner, A H; Richardson, J E; Eppig, J T

    1995-01-26

    The Mammalian Comparative Database provides genetic maps of mammalian species. Comparative maps are valuable aids for predicting linkages, developing animal models and studying genome organization and evolution.

  1. Stem Cells in Mammalian Gonads.

    PubMed

    Wu, Ji; Ding, Xinbao; Wang, Jian

    Stem cells have great value in clinical application because of their ability to self-renew and their potential to differentiate into many different cell types. Mammalian gonads, including testes for males and ovaries for females, are composed of germline and somatic cells. In male mammals, spermatogonial stem cells maintain spermatogenesis which occurs continuously in adult testis. Likewise, a growing body of evidence demonstrated that female germline stem cells could be found in mammalian ovaries. Meanwhile, prior studies have shown that somatic stem cells exist in both testes and ovaries. In this chapter, we focus on mammalian gonad stem cells and discuss their characteristics as well as differentiation potentials.

  2. Mammalian Interphase Cdks

    PubMed Central

    2012-01-01

    Cyclin-dependent kinases (Cdks) drive cell cycle progression in all eukaryotes. Yeasts have a single major Cdk that mediates distinct cell cycle transitions via association with different cyclins. The closest homolog in mammals, Cdk1, drives mitosis. Mammals have additional Cdks—Cdk2, Cdk4, and Cdk6—that represent the major Cdks activated during interphase (iCdks). A large body of evidence has accrued that suggests that activation of iCdks dictates progression though interphase. In apparent contradiction, deficiency in each individual iCdk, respectively, in knockout mice proved to be compatible with live birth and in some instances fertility. Moreover, murine embryos could be derived with Cdk1 as the only functional Cdk. Thus, none of the iCdks is strictly essential for mammalian cell cycle progression, raising the possibility that Cdk1 is the dominant regulator in interphase. However, an absence of iCdks has been accompanied by major shifts in cyclin association to Cdk1, suggesting gain in function. After considerable tweaking, a chemical genetic approach has recently been able to examine the impact of acute inhibition of Cdk2 activity without marked distortion of cyclin/Cdk complex formation. The results suggest that, when expressed at its normal levels, Cdk2 performs essential roles in driving human cells into S phase and maintaining genomic stability. These new findings appear to have restored order to the cell cycle field, bringing it full circle to the view that iCdks indeed play important roles. They also underscore the caveat in knockdown and knockout approaches that protein underexpression can significantly perturb a protein interaction network. We discuss the implications of the new synthesis for future cell cycle studies and anti–Cdk-based therapy of cancer and other diseases. PMID:23634250

  3. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  4. Maturation of the mammalian secretome

    PubMed Central

    Simpson, Jeremy C; Mateos, Alvaro; Pepperkok, Rainer

    2007-01-01

    A recent use of quantitative proteomics to determine the constituents of the endoplasmic reticulum and Golgi complex is discussed in the light of other available methodologies for cataloging the proteins associated with the mammalian secretory pathway. PMID:17472737

  5. A TRPC5-regulated calcium signaling pathway controls dendrite patterning in the mammalian brain.

    PubMed

    Puram, Sidharth V; Riccio, Antonio; Koirala, Samir; Ikeuchi, Yoshiho; Kim, Albert H; Corfas, Gabriel; Bonni, Azad

    2011-12-15

    Transient receptor potential (TRP) channels have been implicated as sensors of diverse stimuli in mature neurons. However, developmental roles for TRP channels in the establishment of neuronal connectivity remain largely unexplored. Here, we identify an essential function for TRPC5, a member of the canonical TRP subfamily, in the regulation of dendrite patterning in the mammalian brain. Strikingly, TRPC5 knockout mice harbor long, highly branched granule neuron dendrites with impaired dendritic claw differentiation in the cerebellar cortex. In vivo RNAi analyses suggest that TRPC5 regulates dendrite morphogenesis in the cerebellar cortex in a cell-autonomous manner. Correlating with impaired dendrite patterning in the cerebellar cortex, behavioral analyses reveal that TRPC5 knockout mice have deficits in gait and motor coordination. Finally, we uncover the molecular basis of TRPC5's function in dendrite patterning. We identify the major protein kinase calcium/calmodulin-dependent kinase II β (CaMKIIβ) as a critical effector of TRPC5 function in neurons. Remarkably, TRPC5 forms a complex specifically with CaMKIIβ, but not the closely related kinase CaMKIIα, and thereby induces the CaMKIIβ-dependent phosphorylation of the ubiquitin ligase Cdc20-APC at the centrosome. Accordingly, centrosomal CaMKIIβ signaling mediates the ability of TRPC5 to regulate dendrite morphogenesis in neurons. Our findings define a novel function for TRPC5 that couples calcium signaling to a ubiquitin ligase pathway at the centrosome and thereby orchestrates dendrite patterning and connectivity in the brain.

  6. Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

    PubMed Central

    O’Keefe, Rachel A.; Blice-Baum, Anna; Gong, Xuan; Karaca, Esra

    2016-01-01

    Abstract The fragile X mental retardation protein (FMRP) is an mRNA-binding regulator of protein translation that associates with 4-6% of brain transcripts and is central to neurodevelopment. Autism risk genes’ transcripts are overrepresented among FMRP-binding mRNAs, and FMRP loss-of-function mutations are responsible for fragile X syndrome, the most common cause of monogenetic autism. It is thought that FMRP-dependent translational repression is governed by the phosphorylation of serine residue 499 (S499). However, recent evidence suggests that S499 phosphorylation is not modulated by metabotropic glutamate receptor class I (mGluR-I) or protein phosphatase 2A (PP2A), two molecules shown to regulate FMRP translational repression. Moreover, the mammalian FMRP S499 kinase remains unknown. We found that casein kinase II (CK2) phosphorylates murine FMRP S499. Further, we show that phosphorylation of FMRP S499 permits phosphorylation of additional, nearby residues. Evidence suggests that these nearby residues are modulated by mGluR-I and PP2A pathways. These data support an alternative phosphodynamic model of FMRP that is harmonious with prior studies and serves as a framework for further investigation. PMID:27957526

  7. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

    PubMed

    Murthy, Karnam S; Zhou, Huiping; Grider, John R; Brautigan, David L; Eto, Masumi; Makhlouf, Gabriel M

    2003-08-15

    Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities

  8. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

    PubMed Central

    Murthy, Karnam S; Zhou, Huiping; Grider, John R; Brautigan, David L; Eto, Masumi; Makhlouf, Gabriel M

    2003-01-01

    Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities

  9. Sirtuins: Guardians of Mammalian Healthspan

    PubMed Central

    Giblin, William; Skinner, Mary E.; Lombard, David B.

    2014-01-01

    The first link between sirtuins and longevity was made 15 years ago in yeast. These initial studies sparked efforts by many laboratories working in diverse model organisms to elucidate the relationships between sirtuins, lifespan, and age-associated dysfunction. Here we discuss the current understanding of how sirtuins relate to aging. We focus primarily on mammalian sirtuins SIRT1, SIRT3, and SIRT6, the three sirtuins for which the most relevant data are available. Strikingly, a large body of evidence now indicates that these and other mammalian sirtuins suppress a variety of age-related pathologies and promote healthspan. Moreover, increased expression of SIRT1 or SIRT6 extends mouse lifespan. Overall, these data point to important roles for sirtuins in promoting mammalian health, and perhaps in modulating the aging process. PMID:24877878

  10. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  11. Activation of Casein Kinase II and Inhibition of Phosphatase and Tensin Homologue Deleted on Chromosome 10 Phosphatase by Nerve Growth Factor/p75NTR Inhibit Glycogen Synthase Kinase-3β and Stimulate Axonal Growth

    PubMed Central

    Arevalo, María-Angeles

    2006-01-01

    Axonal elongation and guidance are controlled by extracellular factors such as the neurotrophins. Indeed, nerve growth factor (NGF) seems to promote axon growth through binding to its p75NTR receptor and inactivating RhoA. Furthermore, the local inhibition of glycogen synthase kinase (GSK)-3β by NGF also favors microtubule polymerization and axon extension. Inactivation of GSK-3β may be due to the NGF/TrkA-mediated activation of phosphatidylinositol-3 kinase (PI-3 kinase), which increases the levels of phosphatydilinositol 3-phosphate [PI(3)P]. However, we show here that NGF may inactivate GSK-3β through an alternative mechanism. In cultured hippocampal neurons, the capacity of NGF to promote axon elongation is mostly mediated by p75NTR, and the activation of this pathway leads to the inactivation of GSK-3β. However, the signaling pathway triggered by NGF/p75NTR acts through casein kinase II (CK2). NGF/p75NTR-activated CK2 phosphorylates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), thus rendering this phosphatase inactive. Like activation of the PI-3 kinase, PTEN inactivation allows PI(3)P levels to increase, thus favoring GSK-3β inactivation and axon outgrowth. This newly disclosed mechanism may help to extend the repertoire of pharmacological agents that activate CK2 or that inhibit PTEN to stimulate axon regeneration after trauma or disease. PMID:16723502

  12. Mammalian sex hormones in plants.

    PubMed

    Janeczko, Anna; Skoczowski, Andrzej

    2005-01-01

    The occurrence of mammalian sex hormones and their physiological role in plants is reviewed. These hormones, such as 17beta-estradiol, androsterone, testosterone or progesterone, were present in 60-80% of the plant species investigated. Enzymes responsible for their biosynthesis and conversion were also found in plants. Treatment of the plants with sex hormones or their precursors influenced plant development: cell divisions, root and shoot growth, embryo growth, flowering, pollen tube growth and callus proliferation. The regulatory abilities of mammalian sex hormones in plants makes possible their use in practice, especially in plant in vitro culture.

  13. [Comparative embryology and mammalian cloning].

    PubMed

    Sakharova, N Iu; Chaĭlakhian, L M

    2010-01-01

    A hypothesis has been advanced that logically combines "contradictory" facts concerning the early mammalian development and shows a natural relationship between the embryos developing from a fertilized ovum and from cells of the inner cell mass of blastocyst. When studying the theoretical questions of cloning, it is necessary to take into consideration the peculiarities of prenatal mammalian ontogenesis, which make themselves evident upon comparison with other animals. The absence of yolk in the mammalian ovum defines sharp differences in the early development between mammals and other Amniota. The whole asynchronic cleavage results in the formation of the morula followed by the blastocyst, which hatches from zona pellucida and is implanted into the uterus tissue. This fact allows us to consider the blastocyst as a mammalian larva, which is fed thanks to maternal organism. It is known that, in the body of a larva (blastocyst), a new embryo develops from some somatic cells. This process is known as a polyembryony, which is typical for the development of some parasitic insects. The polyembryony in turn is a variant of somatic embryogenesis, which is a form of asexual reproduction. Thus, two different embryos, "conceptus" and "embryo proper", have different origin: the first forms by the sexual way and the second, by the asexual. The investigation of the mechanisms of somatic embryogenesis in mammals will help us to find conditions necessary for the full reprograming of donor somatic nuclei and provide the successful development of reconstructed embryos.

  14. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages.

  15. Upregulation of p21 activates the intrinsic apoptotic pathway in β-cells.

    PubMed

    Hernandez, Angelina M; Colvin, E Scott; Chen, Yi-Chun; Geiss, Steven L; Eller, Lindsay E; Fueger, Patrick T

    2013-06-15

    Diabetes manifests from a loss in functional β-cell mass, which is regulated by a dynamic balance of various cellular processes, including β-cell growth, proliferation, and death as well as secretory function. The cell cycle machinery comprised of cyclins, kinases, and inhibitors regulates proliferation. However, their involvement during β-cell stress during the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two pharmacological inducers of β-cell stress, dexamethasone and thapsigargin. We hypothesized that β-cell stress upregulates p21 to activate the apoptotic pathway and suppress cell survival signaling. To this end, p21 was adenovirally overexpressed in pancreatic rat islets and 832/13 cells. As expected, p21 overexpression resulted in decreased [(3)H]thymidine incorporation. Flow cytometry analysis in p21-transduced 832/13 cells verified lower replication, as indicated by a decreased cell population in the S phase and a block in G2/M transition. The sub-G0 cell population was higher with p21 overexpression and was attributable to apoptosis, as demonstrated by increased annexin-positive stained cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the antiapoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the proapoptotic proteins Bax and Bak. Therefore, an intact intrinsic apoptotic pathway is central for p21-mediated cell death. In summary, our findings indicate that β-cell apoptosis can be triggered by p21 during stress and is thus a potential target to inhibit for protection of functional β-cell mass.

  16. ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration

    PubMed Central

    Yuan, Liangping; Santi, Mariarita; Rushing, Elisabeth J.; Cornelison, Robert

    2010-01-01

    We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity. PMID:20526801

  17. Disruption of p21-activated kinase 1 gene diminishes atherosclerosis in apolipoprotein E-deficient mice

    PubMed Central

    Singh, Nikhlesh K.; Kotla, Sivareddy; Dyukova, Elena; Traylor Jr., James G.; Orr, A. Wayne; Chernoff, Jonathan; Marion, Tony N.; Rao, Gadiparthi N.

    2015-01-01

    Pak1 plays an important role in various cellular processes, including cell motility, polarity, survival and proliferation. To date, its role in atherogenesis has not been explored. Here we report the effect of Pak1 on atherogenesis using atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice as a model. Disruption of Pak1 in ApoE−/− mice results in reduced plaque burden, significantly attenuates circulating IL-6 and MCP-1 levels, limits the expression of adhesion molecules and diminishes the macrophage content in the aortic root of ApoE−/− mice. We also observed reduced oxidized LDL uptake and increased cholesterol efflux by macrophages and smooth muscle cells of ApoE−/−:Pak1−/− mice as compared with ApoE−/− mice. In addition, we detect increased Pak1 phosphorylation in human atherosclerotic arteries, suggesting its role in human atherogenesis. Altogether, these results identify Pak1 as an important factor in the initiation and progression of atherogenesis. PMID:26104863

  18. Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

    DTIC Science & Technology

    2010-01-01

    PBS and lysed in a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P - 40 , and 0.25% sodium deoxycholate, 1 mM Na3VO4, and 1 mM NaF... P - 40 , 10 mM NaCl, 50 µg/ml propidium iodide and 70 Kunitz units/ml RNase A. FACS analysis was performed using CELLQuest™ software (Becton...and differences were considered statistically significant at P < 0.05. Results Chernoff, Jonathan 40 Establishing Merlin-deficient cells that

  19. Tamoxifen Dependent Interaction Between in Estrogen Receptor and a Novel p21 Activated Kinase

    DTIC Science & Technology

    2004-06-01

    Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine- proline-tyrosine, TPY ) in the activation...similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6...the other catalytic domain and blocks its function. Binding of GTP-Cdc42 or -Rac causes the AID to dissociate from the catalytic domain and activates

  20. Mesalamine modulates intercellular adhesion through inhibition of p-21 activated kinase-1

    PubMed Central

    Khare, Vineeta; Lyakhovich, Alex; Dammann, Kyle; Lang, Michaela; Borgmann, Melanie; Tichy, Boris; Pospisilova, Sarka; Luciani, Gloria; Campregher, Christoph; Evstatiev, Rayko; Pflueger, Maren; Hundsberger, Harald; Gasche, Christoph

    2013-01-01

    Mesalamine (5-ASA) is widely used for the treatment of ulcerative colitis, a remitting condition characterized by chronic inflammation of the colon. Knowledge about the molecular and cellular targets of 5-ASA is limited and a clear understanding of its activity in intestinal homeostasis and interference with neoplastic progression is lacking. We sought to identify molecular pathways interfered by 5-ASA, using CRC cell lines with different genetic background. Microarray was performed for gene expression profile of 5-ASA-treated and untreated cells (HCT116 and HT29). Filtering and analysis of data identified three oncogenic pathways interfered by 5-ASA: MAPK/ERK pathway, cell adhesion and β-catenin/Wnt signaling. PAK1 emerged as a consensus target of 5-ASA, orchestrating these pathways. We further investigated the effect of 5-ASA on cell adhesion. 5-ASA increased cell adhesion which was measured by cell adhesion assay and transcellular-resistance measurement. Moreover, 5-ASA treatment restored membranous expression of adhesion molecules E-cadherin and β-catenin. Role of PAK1 as a mediator of mesalamine activity was validated in vitro and in vivo. Inhibition of PAK1 by RNA interference also increased cell adhesion. PAK1 expression was elevated in APCmin polyps and 5-ASA treatment reduced its expression. Our data demonstrates novel pharmacological mechanism of mesalamine in modulation of cell adhesion and role of PAK1 in APCmin polyposis. We propose that inhibition of PAK1 expression by 5-ASA can impede with neoplastic progression in colorectal carcinogenesis. The mechanism of PAK1 inhibition and induction of membranous translocation of adhesion proteins by 5-ASA might be independent of its known anti-inflammatory action. PMID:23146664

  1. p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics

    PubMed Central

    Allen, Jayme D.; Jaffer, Zahara M.; Park, Su-Jung; Burgin, Sarah; Hofmann, Clemens; Sells, Mary Ann; Chen, Shi; Derr-Yellin, Ethel; Michels, Elizabeth G.; McDaniel, Andrew; Bessler, Waylan K.; Ingram, David A.; Atkinson, Simon J.; Travers, Jeffrey B.

    2009-01-01

    Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcϵRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, Wsh/Wsh mast cell–deficient mice locally reconstituted with Pak1−/− bone marrow–derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1−/− BMMCs exhibited a reduction in FcϵRI-induced degranulation. Further, Pak1−/− BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcϵRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases. PMID:19124833

  2. Interaction Between a Novel p21 Activated Kinase (PAK6) and Androgen Receptor in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    cancer. The cell cycle signaling regulated by the mitogen activated protein/extracellular-signal-regulated kinase (MAPK/ERK) have been linked to tumor...biological roles of PAK6 in prostate cancer cells , and to examine the expression of PAK6 in prostate tissues. We anticipate that by completing the...of prostate cells (Balk, 2002; Gelmann, 2002). Androgen ablation is an effective treatment for the majority of advanced prostate cancer patients

  3. Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

    DTIC Science & Technology

    2009-01-01

    SchΔ(39-121) Pak1+/+ mice developed NF2-related pathologies (schwannomatosis, nerve sheath tumors, sarcomas ), whereas 2/34 P0- SchΔ(39-121) Pak1...Pak3 and Pak5 HEK293 cells were transfected with 0.5 mg Pak3 or myc-Pak5 DNA using lipo - some-mediated transfection (Lipofectamine 2000, Invitrogen

  4. p21-activated kinase 1 restricts tonic endocannabinoid signaling in the hippocampus

    PubMed Central

    Xia, Shuting; Zhou, Zikai; Leung, Celeste; Zhu, Yuehua; Pan, Xingxiu; Qi, Junxia; Morena, Maria; Hill, Matthew N; Xie, Wei; Jia, Zhengping

    2016-01-01

    PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not 2-arachidonoylglycerol (2-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-2 (COX-2), and COX-2 inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-2, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies. DOI: http://dx.doi.org/10.7554/eLife.14653.001 PMID:27296803

  5. Calcium/calmodulin‐dependent kinase II and nitric oxide synthase 1‐dependent modulation of ryanodine receptors during β‐adrenergic stimulation is restricted to the dyadic cleft

    PubMed Central

    Dries, Eef; Santiago, Demetrio J.; Johnson, Daniel M.; Gilbert, Guillaume; Holemans, Patricia; Korte, Sanne M.; Roderick, H. Llewelyn

    2016-01-01

    Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation.Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII.Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs.In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent.The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. Abstract In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However

  6. Activation of ROS/NF-{kappa}B and Ca{sup 2+}/CaM kinase II are necessary for VCAM-1 induction in IL-1{beta}-treated human tracheal smooth muscle cells

    SciTech Connect

    Luo, S.-F.; Chang, C.-C.; Lee, I-T.; Lee, C.-W.; Lin, W.-N.; Lin, C.-C.; Yang, C.-M.

    2009-05-15

    Histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in the expression of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes have been shown to induce VCAM-1 expression. Here, we investigated the mechanisms underlying IL-1{beta}-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs). Our results showed that IL-1{beta} enhanced HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by pretreatment with selective inhibitors of PKC{alpha} (Goe6976), c-Src (PP1), NADPH oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (garcinol), NF-{kappa}B (Bay11-7082), HDAC (trichostatin A), and ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88, PKC{alpha}, Src, p47{sup phox}, p300, and HDAC4. Moreover, IL-1{beta} stimulated NF-{kappa}B and CaMKII phosphorylation through MyD88-dependent PI-PLC/PKC{alpha}/c-Src/ROS and PI-PLC/Ca{sup 2+}/CaM pathways, respectively. Activation of NF-{kappa}B and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylases. These findings suggested that IL-1{beta} induced VCAM-1 expression via these multiple signaling pathways in HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

  7. Ca2+–calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

    PubMed Central

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-01-01

    Abstract Recent studies have demonstrated that changes in the activity of calcium–calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation–contraction (E–C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM–GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+–CaMKII–DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII. PMID:21486818

  8. Moderate Alcohol Drinking and the Amygdala Proteome: Identification and Validation of Calcium/Calmodulin Dependent Kinase II and AMPA Receptor Activity as Novel Molecular Mechanisms of the Positive Reinforcing Effects of Alcohol

    PubMed Central

    Salling, Michael C.; Faccidomo, Sara P.; Li, Chia; Psilos, Kelly; Galunas, Christina; Spanos, Marina; Agoglia, Abigail E.; Kash, Thomas L.; Hodge, Clyde W.

    2015-01-01

    BACKGROUND Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood. METHODS Here, we conducted a high-through put screen of the amygdala proteome in mice after moderate alcohol drinking (n = 12/group) followed by behavioral studies (n = 6–8/group) to uncover novel molecular mechanisms of the positive reinforcing properties of alcohol that strongly influence the development of addiction. RESULTS Two-dimensional difference in-gel electrophoresis with matrix assisted laser desorption ionization tandem time-of-flight identified 29 differentially expressed proteins in the amygdala of nondependent C57BL/6J mice following 24 days of alcohol drinking. Alcohol-sensitive proteins included calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and a network of functionally linked proteins that regulate neural plasticity and glutamate-mediated synaptic activity. Accordingly, alcohol drinking increased α-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) in central amygdala (CeA) and phosphorylation of AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in CeA and lateral amygdala. Further, CaMKIIα-Thr286 and GluA1-Ser831 phosphorylation was increased in CeA and lateral amygdala of mice that lever-pressed for alcohol versus the nondrug reinforcer sucrose. Mechanistic studies showed that targeted pharmacologic inhibition of amygdala CaMKII or AMPAR activity specifically inhibited the positive reinforcing properties of alcohol but not sucrose. CONCLUSIONS Moderate alcohol drinking increases the activity and function of plasticity-linked protein networks in the amygdala that regulate the positive reinforcing effects of the drug. Given the prominence of positive reinforcement in the etiology of addiction, we propose that alcohol-induced adaptations in CaMKIIα and AMPAR signaling in the amygdala may serve as a molecular gateway from use to abuse. PMID:25579851

  9. Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

    PubMed

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function.

  10. Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice.

    PubMed

    Jackson, Kia J; Imad Damaj, M

    2013-02-15

    Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the β2-selective antagonist dihydro-β-erythroidine (DHβE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20min prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHβE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through β2-containing nicotinic receptors.

  11. Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

    PubMed

    Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

    2016-07-15

    In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. Low Dose Ultraviolet B Irradiation Increases Hyaluronan Synthesis in Epidermal Keratinocytes via Sequential Induction of Hyaluronan Synthases Has1–3 Mediated by p38 and Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Signaling*

    PubMed Central

    Rauhala, Leena; Hämäläinen, Lasse; Salonen, Pauliina; Bart, Geneviève; Tammi, Markku; Pasonen-Seppänen, Sanna; Tammi, Raija

    2013-01-01

    Hyaluronan, a major epidermal extracellular matrix component, responds strongly to different kinds of injuries. This also occurs by UV radiation, but the mechanisms involved are poorly understood. The effects of a single ultraviolet B (UVB) exposure on hyaluronan content and molecular mass, and expression of genes involved in hyaluronan metabolism were defined in monolayer and differentiated, organotypic three-dimensional cultures of rat epidermal keratinocytes. The signals regulating the response were characterized using specific inhibitors and Western blotting. In monolayer cultures, UVB increased hyaluronan synthase Has1 mRNA already 4 h postexposure, with a return to control level by 24 h. In contrast, Has2 and Has3 were persistently elevated from 8 h onward. Silencing of Has2 and especially Has3 decreased the UVB-induced accumulation of hyaluronan. p38 and Ca2+/calmodulin-dependent protein kinase II pathways were found to be involved in the UVB-induced up-regulation of Has2 and Has3 expression, respectively, and their inhibition reduced hyaluronan deposition. However, the expressions of the hyaluronan-degrading enzymes Hyal1 and Hyal2 and the hyaluronan receptor Cd44 were also up-regulated by UVB. In organotypic cultures, UVB treatment also resulted in increased expression of both Has and Hyal genes and shifted hyaluronan toward a smaller size range. Histochemical stainings indicated localized losses of hyaluronan in the epidermis. The data show that exposure of keratinocytes to acute, low dose UVB increases hyaluronan synthesis via up-regulation of Has2 and Has3. The simultaneously enhanced catabolism of hyaluronan demonstrates the complexity of the UVB-induced changes. Nevertheless, enhanced hyaluronan metabolism is an important part of the adaptation of keratinocytes to radiation injury. PMID:23645665

  13. Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

    PubMed

    Yilmaz, Mehtap; Gangopadhyay, Samudra S; Leavis, Paul; Grabarek, Zenon; Morgan, Kathleen G

    2013-02-07

    CaMKII (Ca²⁺/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²⁶, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, we generated a phosphospecific Ser²⁶ antibody and demonstrated an increase in Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, we mutated Ser²⁶ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²⁸⁷ autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²⁶ of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most probably by blocking ATP binding. We propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity.

  14. Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin.

    PubMed

    Robison, A J; Bartlett, Ryan K; Bass, Martha A; Colbran, Roger J

    2005-11-25

    Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.

  15. Activation of ROS/NF-kappaB and Ca2+/CaM kinase II are necessary for VCAM-1 induction in IL-1beta-treated human tracheal smooth muscle cells.

    PubMed

    Luo, Shue-Fen; Chang, Chia-Chi; Lee, I-Ta; Lee, Chiang-Wen; Lin, Wei-Ning; Lin, Chih-Chung; Yang, Chuen-Mao

    2009-05-15

    Histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in the expression of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes have been shown to induce VCAM-1 expression. Here, we investigated the mechanisms underlying IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs). Our results showed that IL-1beta enhanced HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by pretreatment with selective inhibitors of PKCalpha (Gö6976), c-Src (PP1), NADPH oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (garcinol), NF-kappaB (Bay11-7082), HDAC (trichostatin A), and ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88, PKCalpha, Src, p47(phox), p300, and HDAC4. Moreover, IL-1beta stimulated NF-kappaB and CaMKII phosphorylation through MyD88-dependent PI-PLC/PKCalpha/c-Src/ROS and PI-PLC/Ca2+/CaM pathways, respectively. Activation of NF-kappaB and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylases. These findings suggested that IL-1beta induced VCAM-1 expression via these multiple signaling pathways in HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.

  16. ET-1-induced growth promoting responses involving ERK1/2 and PKB signaling and Egr-1 expression are mediated by Ca2+/CaM-dependent protein kinase-II in vascular smooth muscle cells.

    PubMed

    Bouallegue, Ali; Simo Cheyou, Estelle R; Anand-Srivastava, Madhu B; Srivastava, Ashok K

    2013-12-01

    Endothelin-1 (ET-1), a potent vasoactive peptide with a pathogenic role in vascular diseases, has been shown to induce the activation of ERK1/2, PKB and the expression of a transcriptional regulator, the early growth response 1 (Egr-1), key mediators of hypertrophic and proliferative responses in vascular smooth muscle cells (VSMC). We have demonstrated earlier that ET-1 requires H2O2 generation to activate these signaling pathways and Ca2+, calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII), play a critical role to trigger H2O2-induced effects in VSMC. However, an involvement of CaMKII in mediating ET-1-induced responses in VSMC remains unknown. Therefore, by utilizing pharmacological inhibitors of CaM, CaMKII, a CaMKII inhibitor peptide and CaMKII knockdown techniques, we have investigated the contribution of CaM and CaMKII in ET-1-induced ERK1/2 and PKB signaling, Egr-1 expression and hypertrophic and proliferative responses in VSMC. W-7 and calmidazolium, antagonists of CaM, as well as KN-93, an inhibitor of CaMKII activity, attenuated ET-1-induced ERK1/2 and PKB phosphorylation. In addition, transfection of VSMC with a CaMKII inhibitory peptide suppressed ET-1-evoked ERK1/2 and PKB phosphorylation. Similarly, siRNA-mediated CaMKII silencing reduced ET-1-produced ERK1/2 and PKB phosphorylation. CaM and CaMKII blockade also significantly lowered the ET-1-induced protein and DNA synthesis as well as Egr-1 expression. These findings demonstrate that CaMKII plays a critical role in ET-1-induced growth promoting signaling pathways as well as hypertrophic and proliferative responses in VSMC. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel alpha(1C)-subunit gene (Cacna1c) by DREAM translocation.

    PubMed

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-06-01

    Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

  18. The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.

    PubMed

    Dhavan, Rani; Greer, Paul L; Morabito, Maria A; Orlando, Lianna R; Tsai, Li-Huei

    2002-09-15

    Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

  19. The fine-structural distribution of G-protein receptor kinase 3, beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl(-)-cotransporter in rodent olfactory epithelia.

    PubMed

    Menco, Bert Ph M

    2005-03-01

    The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na+, K(+)-2Cl(-)-cotransporter NKCC1 regulates Cl(-)-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to beta-arrestin-2, five to CaMKII (one to both the alpha and beta form, and two each specific to CaMKII alpha and beta), two to PDE1C2, and three to Cl(-)-cotransporters. Only antibodies to Cl(-)-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, beta- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.

  20. Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice.

    PubMed

    Moriguchi, Shigeki; Yamamoto, Yui; Ikuno, Tatsuya; Fukunaga, Kohji

    2011-06-01

    Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  1. Mechanisms of mammalian iron homeostasis

    PubMed Central

    Pantopoulos, Kostas; Porwal, Suheel Kumar; Tartakoff, Alan; Devireddy, L.

    2012-01-01

    Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in acquisition or distribution of the metal causes anemia; whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanisms to regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways, as well as in mechanisms underlying intracellular iron trafficking, an important but less-studied area of mammalian iron homeostasis. PMID:22703180

  2. Architecture of the Mammalian Golgi

    PubMed Central

    Klumperman, Judith

    2011-01-01

    Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described. PMID:21502307

  3. Bioenergetics of Mammalian Sperm Capacitation

    PubMed Central

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods. PMID:24791005

  4. Ceramide signaling in mammalian epidermis

    PubMed Central

    Uchida, Yoshikazu

    2013-01-01

    Ceramide, the backbone structure of all sphingolipids, as well as a minor component of cellular membranes, has a unique role in the skin, by forming the epidermal permeability barrier at the extracellular domains of the outermost layer of skin, the stratum corneum, which is required for terrestrial mammalian survival. In contrast to the role of ceramide in forming the permeability barrier, the signaling roles of ceramide and its metabolites have not yet been recognized. Ceramide and/or its metabolites regulate proliferation, differentiation, and apoptosis in epidermal keratinocytes. Recent studies have further demonstrated that a ceramide metabolite, sphingosine-1-phosphate, modulates innate immune function. Ceramide already has been applied to therapeutic approaches for treatment of eczema associated with attenuated epidermal permeability barrier function. Pharmacological modulation of ceramide and its metabolites signaling can also be applied to cutaneous disease prevention and therapy. The author here describes the signaling roles of ceramide and its metabolites in mammalian cells and tissues, including epidermis. PMID:24055887

  5. Mammalian Polyamine Metabolism and Function

    PubMed Central

    Pegg, Anthony E.

    2009-01-01

    Summary Polyamines are ubiquitous small basic molecules that play multiple essential roles in mammalian physiology. Their cellular content is highly regulated and there is convincing evidence that altered metabolism is involvement in many disease states. Drugs altering polyamine levels may therefore have a variety of important targets. This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism. PMID:19603518

  6. Evaluating and treating mammalian bites.

    PubMed

    Rasmussen, Donna; Landon, Alexandra; Powell, Jennifer; Brown, Gina R

    2017-03-01

    Mammalian bites, typically from dogs, cats, or humans, are a common presentation in EDs and family practice settings, and patients present with varying degrees of complexity. Injuries can range from local to systemic, including aggressive bacterial infections and permanent limb impairment. Using a systematic approach to initial wound assessment, followed by appropriate diagnostic testing and treatment, is critical to improved long-term patient outcomes.

  7. GLUTs and mammalian sperm metabolism.

    PubMed

    Bucci, Diego; Rodriguez-Gil, Juan Enrique; Vallorani, Claudia; Spinaci, Marcella; Galeati, Giovanna; Tamanini, Carlo

    2011-01-01

    Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

  8. Scalable architecture in mammalian brains.

    PubMed

    Clark, D A; Mitra, P P; Wang, S S

    2001-05-10

    Comparison of mammalian brain parts has often focused on differences in absolute size, revealing only a general tendency for all parts to grow together. Attempts to find size-independent effects using body weight as a reference variable obscure size relationships owing to independent variation of body size and give phylogenies of questionable significance. Here we use the brain itself as a size reference to define the cerebrotype, a species-by-species measure of brain composition. With this measure, across many mammalian taxa the cerebellum occupies a constant fraction of the total brain volume (0.13 +/- 0.02), arguing against the hypothesis that the cerebellum acts as a computational engine principally serving the neocortex. Mammalian taxa can be well separated by cerebrotype, thus allowing the use of quantitative neuroanatomical data to test evolutionary relationships. Primate cerebrotypes have progressively shifted and neocortical volume fractions have become successively larger in lemurs and lorises, New World monkeys, Old World monkeys, and hominoids, lending support to the idea that primate brain architecture has been driven by directed selection pressure. At the same time, absolute brain size can vary over 100-fold within a taxon, while maintaining a relatively uniform cerebrotype. Brains therefore constitute a scalable architecture.

  9. Mammalian Alphaherpesvirus miRNAs

    PubMed Central

    Jurak, Igor; Griffiths, Anthony; Coen, Donald M.

    2012-01-01

    Mammalian alphaherpesviruses are major causes of human and veterinary disease. During productive infection, these viruses exhibit complex and robust patterns of gene expression. These viruses also form latent infections in neurons of sensory ganglia in which productive cycle gene expression is highly repressed. Both modes of infection provide advantageous opportunities for regulation by microRNAs. Thus far, published data regarding microRNAs are available for six mammalian alphaherpesviruses. No microRNAs have yet been detected from varicella zoster virus. The five other viruses -- herpes simplex viruses-1 and -2, herpes B virus, bovine herpesvirus-1, and pseudorabies virus -- representing both genera of mammalian alphaherpesviruses have been shown to express microRNAs. In this article, we discuss these microRNAs in terms of where they are encoded in the viral genome relative to other viral transcripts; whether they are expressed during productive or latent infection; their potential targets; what little is known about their actual targets and functions during viral infection; and what little is known about the interactions of these viruses with the host microRNA machinery. PMID:21736960

  10. Functional characterization of mammalian Wntless homolog in mammalian system.

    PubMed

    Wang, Li-Ting; Wang, Shih-Jong; Hsu, Shih-Hsien

    2012-07-01

    Wntless (GPR177) protein is a newly identified regulator of Wnt signals in Drosophila, but its cellular function in mammals is still unclear. In this study, we explored the expression pattern and potential cellular function of Wntless in mammalian cells. Wntless mRNA was expressed in many mouse tissues, including the spleen, lung, kidney, thymus, and stomach, and lower levels of expression were detected in the mouse brain and testis. Expression of Wntless protein analyzed by Western blot and immunohistochemical staining was only detected in the submucosa, muscle, ganglia, and nerve cells of murine large intestines. Both immunofluorescence staining and subcellular fraction extraction analysis revealed that endogenous Wntless protein was expressed predominantly in the cytoplasmic organelles with a morphologically dot-shaped distribution. Furthermore, overexpression of Wntless could be corrected by and may activate the nuclear factor-κB (NF-κB) signaling pathway in cancer (HeLa) cells. These results suggest that Wntless plays a role in signaling regulation during the formation of cancer in addition to its role as a retromer protein in mammalian systems.

  11. Evolutionary paths to mammalian cochleae.

    PubMed

    Manley, Geoffrey A

    2012-12-01

    Evolution of the cochlea and high-frequency hearing (>20 kHz; ultrasonic to humans) in mammals has been a subject of research for many years. Recent advances in paleontological techniques, especially the use of micro-CT scans, now provide important new insights that are here reviewed. True mammals arose more than 200 million years (Ma) ago. Of these, three lineages survived into recent geological times. These animals uniquely developed three middle ear ossicles, but these ossicles were not initially freely suspended as in modern mammals. The earliest mammalian cochleae were only about 2 mm long and contained a lagena macula. In the multituberculate and monotreme mammalian lineages, the cochlea remained relatively short and did not coil, even in modern representatives. In the lineage leading to modern therians (placental and marsupial mammals), cochlear coiling did develop, but only after a period of at least 60 Ma. Even Late Jurassic mammals show only a 270 ° cochlear coil and a cochlear canal length of merely 3 mm. Comparisons of modern organisms, mammalian ancestors, and the state of the middle ear strongly suggest that high-frequency hearing (>20 kHz) was not realized until the early Cretaceous (~125 Ma). At that time, therian mammals arose and possessed a fully coiled cochlea. The evolution of modern features of the middle ear and cochlea in the many later lineages of therians was, however, a mosaic and different features arose at different times. In parallel with cochlear structural evolution, prestins in therian mammals evolved into effective components of a new motor system. Ultrasonic hearing developed quite late-the earliest bat cochleae (~60 Ma) did not show features characteristic of those of modern bats that are sensitive to high ultrasonic frequencies.

  12. Mammalian cloning: advances and limitations.

    PubMed

    Solter, D

    2000-12-01

    For many years, researchers cloning mammals experienced little success, but recent advances have led to the successful cloning of several mammalian species. However, cloning by the transfer of nuclei from adult cells is still a hit-and-miss procedure, and it is not clear what technical and biological factors underlie this. Our understanding of the molecular basis of reprogramming remains extremely limited and affects experimental approaches towards increasing the success rate of cloning. Given the future practical benefits that cloning can offer, the time has come to address what should be done to resolve this problem.

  13. 1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle.

    PubMed Central

    Brozinick, J T; Reynolds, T H; Dean, D; Cartee, G; Cushman, S W

    1999-01-01

    Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia

  14. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    SciTech Connect

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  15. Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells.

    PubMed

    Heise, Nicole; Palme, Daniela; Misovic, Milan; Koka, Saisudha; Rudner, Justine; Lang, Florian; Salih, Helmut R; Huber, Stephan M; Henke, Guido

    2010-01-01

    Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

  16. Rapid 1α,25(OH)₂D ₃ membrane-mediated activation of Ca²⁺/calmodulin-dependent protein kinase II in growth plate chondrocytes requires Pdia3, PLAA and caveolae.

    PubMed

    Doroudi, Maryam; Boyan, Barbara D; Schwartz, Zvi

    2014-08-01

    1α,25-Dihydroxy vitamin D3 [1α,25(OH)2D3] regulates growth zone chondrocytes (GC) via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. 1α,25(OH)2D3 initiates its membrane effects via its specific membrane-associated receptor (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating PLA2, resulting in prostaglandin E2 (PGE2) release and protein kinase C activation. Recently, we reported that 1α,25(OH)2D3 rapidly activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in GC cells. However, the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3-dependent rapid activation of CaMKII are not clear. The aim of the present study was to evaluate the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3 membrane-stimulated CaMKII activation. Pre-treating chondrocytes from the growth zone of the rat costochondral cartilage with antibodies against PLAA or Pdia3 blocked activation of CaMKII by 1α,25(OH)2D3. PLAA peptide rapidly activated CaMKII in GC cells. Caveolae disruption abolished CaMKII activation in response to 1α,25(OH)2D3 or PLAA peptide treatment. Immunoprecipitation studies showed increased CaM binding to PLAA in response to 1α,25(OH)2D3. The results indicated that Pdia3, PLAA and caveolae are required for rapid 1α,25(OH)2D3 membrane-mediated activation of CaMKII. 1α,25(OH)2D3 signaling via Pdia3 receptor triggered the interaction between PLAA and CaM suggesting that CaM may play a major role linking PLAA to CaMKII in membrane-mediated actions of 1α,25(OH)2D3.

  17. Ceramide signaling in mammalian epidermis.

    PubMed

    Uchida, Yoshikazu

    2014-03-01

    Ceramide, the backbone structure of all sphingolipids, as well as a minor component of cellular membranes, has a unique role in the skin, by forming the epidermal permeability barrier at the extracellular domains of the outermost layer of the skin, the stratum corneum, which is required for terrestrial mammalian survival. In contrast to the role of ceramide in forming the permeability barrier, the signaling roles of ceramide and its metabolites have not yet been recognized. Ceramide and/or its metabolites regulate proliferation, differentiation, and apoptosis in epidermal keratinocytes. Recent studies have further demonstrated that a ceramide metabolite, sphingosine-1-phosphate, modulates innate immune function. Ceramide has already been applied to therapeutic approaches for treatment of eczema associated with attenuated epidermal permeability barrier function. Pharmacological modulation of ceramide and its metabolites' signaling can also be applied to cutaneous disease prevention and therapy. The author here describes the signaling roles of ceramide and its metabolites in mammalian cells and tissues, including the epidermis. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

  18. Modeling the mammalian sleep cycle.

    PubMed

    Weber, Franz

    2017-08-24

    During sleep, the mammalian brain transitions through repeated cycles of non-rapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep. The physiological implementation of this slow ultradian brain rhythm is largely unknown. Two differing dynamical mechanisms have been proposed to underlie the NREM-REM cycle. The first model type relies on reciprocal interactions between inhibitory and excitatory neural populations resulting in stable limit cycle oscillations. Recent experimental findings instead favor a model, in which mutually inhibitory interactions between REM sleep-promoting (REM-on) and REM sleep-suppressing (REM-off) neural populations stabilize the brain state. Slow modulations in the neural excitability, that are hypothesized to reflect the homeostatic need for REM sleep, abruptly switch the brain in and out of REM sleep. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Polarity in Mammalian Epithelial Morphogenesis

    PubMed Central

    Roignot, Julie; Peng, Xiao; Mostov, Keith

    2013-01-01

    Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires the intervention of several fundamental cell processes, whose integration in space and time is only starting to be elucidated. To understand what governs the building of epithelial tissues during development, it is essential to consider the polarization process in the context of the whole tissue. To this end, the development of three-dimensional organotypic cell culture models has brought new insights into the mechanisms underlying the establishment and maintenance of higher-order epithelial tissue architecture, and in the dynamic remodeling of cell polarity that often occurs during development of epithelial organs. Here we discuss some important aspects of mammalian epithelial morphogenesis, from the establishment of cell polarity to epithelial tissue generation. PMID:23378592

  20. Producing Newborn Synchronous Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R.; Helmstetter, Charles E.; Thornton, Maureen

    2008-01-01

    A method and bioreactor for the continuous production of synchronous (same age) population of mammalian cells have been invented. The invention involves the attachment and growth of cells on an adhesive-coated porous membrane immersed in a perfused liquid culture medium in a microgravity analog bioreactor. When cells attach to the surface divide, newborn cells are released into the flowing culture medium. The released cells, consisting of a uniform population of synchronous cells are then collected from the effluent culture medium. This invention could be of interest to researchers investigating the effects of the geneotoxic effects of the space environment (microgravity, radiation, chemicals, gases) and to pharmaceutical and biotechnology companies involved in research on aging and cancer, and in new drug development and testing.

  1. Mammalian glutaminase isozymes in brain.

    PubMed

    Márquez, Javier; Cardona, Carolina; Campos-Sandoval, José A; Peñalver, Ana; Tosina, Marta; Matés, José M; Martín-Rufián, Mercedes

    2013-06-01

    Glutamine/glutamate homeostasis must be exquisitely regulated in mammalian brain and glutaminase (GA, E.C. 3.5.1.2) is one of the main enzymes involved. The products of GA reaction, glutamate and ammonia, are essential metabolites for energy and biosynthetic purposes but they are also hazardous compounds at concentrations beyond their normal physiological thresholds. The classical pattern of GA expression in mammals has been recently challenged by the discovery of novel transcript variants and protein isoforms. Furthermore, the interactome of brain GA is also starting to be uncovered adding a new level of regulatory complexity. GA may traffic in brain and unexpected locations, like cytosol and nucleus, have been found for GA isoforms. Finally, the expression of GA in glial cells has been reported and its potential implications in ammonia homeostasis are discussed.

  2. Interaction theory of mammalian mitochondria.

    PubMed

    Nakada, K; Inoue, K; Hayashi, J

    2001-11-09

    We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit.

  3. Determinants of Mammalian Nucleolar Architecture

    PubMed Central

    Farley, Katherine I.; Surovtseva, Yulia; Merkel, Janie; Baserga, Susan J.

    2015-01-01

    The nucleolus is responsible for the production of ribosomes, essential machines which synthesize all proteins needed by the cell. The structure of human nucleoli is highly dynamic and is directly related to its functions in ribosome biogenesis. Despite the importance of this organelle, the intricate relationship between nucleolar structure and function remains largely unexplored. How do cells control nucleolar formation and function? What are the minimal requirements for making a functional nucleolus? Here we review what is currently known regarding mammalian nucleolar formation at nucleolar organizer regions (NORs), which can be studied by observing the dissolution and reformation of the nucleolus during each cell division. Additionally, the nucleolus can be examined by analyzing how alterations in nucleolar function manifest in differences in nucleolar architecture. Furthermore, changes in nucleolar structure and function are correlated with cancer, highlighting the importance of studying the determinants of nucleolar formation. PMID:25670395

  4. Pharmacology of mammalian olfactory receptors.

    PubMed

    Smith, Richard S; Peterlin, Zita; Araneda, Ricardo C

    2013-01-01

    Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000-different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand-receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the "odor space" of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.

  5. Body Size in Mammalian Paleobiology

    NASA Astrophysics Data System (ADS)

    Damuth, John; MacFadden, Bruce J.

    1990-11-01

    This valuable collection of essays presents and evaluates techniques of body-mass estimation and reviews current and potential applications of body-size estimates in paleobiology. Papers discuss explicitly the errors and biases of various regression techniques and predictor variables, and the identification of functionally similar groups of species for improving the accuracy of estimates. At the same time other chapters review and discuss the physiological, ecological, and behavioral correlates of body size in extant mammals; the significance of body-mass distributions in mammalian faunas; and the ecology and evolution of body size in particular paleofaunas. Coverage is particularly detailed for carnivores, primates, and ungulates, but information is also presented on marsupials, rodents, and proboscideans.

  6. Mammalian skin evolution: a reevaluation.

    PubMed

    Maderson, P F A

    2003-06-01

    A 1972 model for the evolutionary origin of hair suggested a primary mechanoreceptor role improving behavioral thermoregulation contributed to the success of late Paleozoic mammal-like reptiles. An insulatory role appeared secondarily subsequent to protohair multiplication. That model is updated in light of new data on (a) palaeoecology of mammalian ancestors; (b) involvement of HRPs in keratinization; (c) lipogenic lamellar bodies that form the barrier to cutaneous water loss; and (d) growth factors involved in hair follicle embryogenesis and turnover. It is now proposed that multiplication of sensory protohairs caused by mutations in patterning genes initially protected the delicate barrier tissues and eventually produced the minimal morphology necessary for an insulatory pelage. The latter permitted Mesozoic mammals to occupy the nocturnal niche 'in the shadow of dinosaurs'. When the giant reptiles became extinct, mammals underwent rapid radiation and reemerged as the dominant terrestrial vertebrates.

  7. The mammalian Cretaceous cochlear revolution.

    PubMed

    Manley, Geoffrey A

    2016-12-19

    The hearing organs of amniote vertebrates show large differences in their size and structure between the species' groups. In spite of this, their performance in terms of hearing sensitivity and the frequency selectivity of auditory-nerve units shows unexpectedly small differences. The only substantial difference is that therian, defined as live-bearing, mammalian groups are able to hear ultrasonic frequencies (above 15-20 kHz), whereas in contrast monotreme (egg laying) mammals and all non-mammalian amniotes cannot. This review compares the structure and physiology of the cochleae of the main groups and asks the question as to why the many structural differences seen in therian mammals arose, yet did not result in greater differences in physiology. The likely answers to this question are found in the history of the mammals during the Cretaceous period that ended 65 million years ago. During that period, the therian cochlea lost its lagenar macula, leading to a fall in endolymph calcium levels. This likely resulted in a small revolution and an auditory crisis that was compensated for by a subsequent series of structural and physiological adaptations. The end result was a system of equivalent performance to that independently evolved in other amniotes but with the additional - and of course "unforeseen" - advantage that ultrasonic-frequency responses became an available option. That option was not always availed of, but in most groups of therian mammals it did evolve and is used for communication and orientation based on improved sound localization, with micro-bats and toothed whales relying on it for prey capture.

  8. Genome regulation in mammalian cells.

    PubMed

    Puck, T T; Krystosek, A; Chan, D C

    1990-05-01

    A theory is presented proposing that genetic regulation in mammalian cells is at least a two-tiered effect; that one level of regulation involves the transition between gene exposure and sequestration; that normal differentiation requires a different spectrum of genes to be exposed in each separate state of differentiation; that the fiber systems of the cell cytoskeleton and the nuclear matrix together control the degree of gene exposure; that specific phosphorylation of these elements causes them to assume a different organizational network and to impose a different pattern of sequestration and exposure on the elements of the genome; that the varied gene phosphorylation mechanisms in the cell are integrated in this function; that attachment of this network system to specific parts of the chromosomes brings about sequestration or exposure of the genes in their neighborhood in a fashion similar to that observed when microtubule elements attach through the kinetochore to the centromeric DNA; that one function of repetitive sequences is to serve as elements for the final attachment of this fibrous network to the specific chromosomal loci; and that at least an important part of the calcium manifestation as a metabolic trigger of different differentiation states involves its acting as a binding agent to centers of electronegativity, in particular proteins and especially phosphorylated groups, so as to change the conformation of the fiber network that ultimately controls gene exposure in the mammalian cell. It would appear essential to determine what abnormal gene exposures and sequestrations are characteristic of each type of cancer; which agonists, if any, will bring about reverse transformation; and whether these considerations can be used in therapy.

  9. Mammalian Mitochondrial ncRNA Database.

    PubMed

    Anandakumar, Shanmugam; Vijayakumar, Saravanan; Arumugam, Nagarajan; Gromiha, M Michael

    2015-01-01

    Mammalian Mitochondrial ncRNA is a web-based database, which provides specific information on non-coding RNA in mammals. This database includes easy searching, comparing with BLAST and retrieving information on predicted structure and its function about mammalian ncRNAs. The database is available for free at http://www.iitm.ac.in/bioinfo/mmndb/.

  10. Recent advances in mammalian protein production

    PubMed Central

    Bandaranayake, Ashok D.; Almo, Steven C.

    2014-01-01

    Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones. PMID:24316512

  11. Photodynamic inactivation of mammalian viruses and bacteriophages.

    PubMed

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  12. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  13. Autophagosome formation in mammalian cells.

    PubMed

    Burman, Chloe; Ktistakis, Nicholas T

    2010-12-01

    Autophagy is a fundamental intracellular trafficking pathway conserved from yeast to mammals. It is generally thought to play a pro-survival role, and it can be up regulated in response to both external and intracellular factors, including amino acid starvation, growth factor withdrawal, low cellular energy levels, endoplasmic reticulum (ER) stress, hypoxia, oxidative stress, pathogen infection, and organelle damage. During autophagy initiation a portion of the cytosol is surrounded by a flat membrane sheet known as the isolation membrane or phagophore. The isolation membrane then elongates and seals itself to form an autophagosome. The autophagosome fuses with normal endocytic traffic to mature into a late autophagosome, before fusing with lysosomes. The molecular machinery that enables formation of an autophagosome in response to the various autophagy stimuli is almost completely identified in yeast and-thanks to the observed conservation-is also being rapidly elucidated in higher eukaryotes including mammals. What are less clear and currently under intense investigation are the mechanism by which these various autophagy components co-ordinate in order to generate autophagosomes. In this review, we will discuss briefly the fundamental importance of autophagy in various pathophysiological states and we will then review in detail the various players in early autophagy. Our main thesis will be that a conserved group of heteromeric protein complexes and a relatively simple signalling lipid are responsible for the formation of autophagosomes in mammalian cells.

  14. Mitochondrial dysfunction in mammalian ageing.

    PubMed

    Terzioglu, Mügen; Larsson, Nils-Göran

    2007-01-01

    Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. Increasing age in mammals correlates with increased levels of mitochondrial DNA (mtDNA) mutations and deteriorating respiratory chain function. Mosaic respiratory chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. Experimental evidence in the mouse has linked increased levels of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, greying of the hair, weight reduction and decreased fertility. It has been known for a long time that respiratory chain-deficient cells are more prone to undergo apoptosis and increased cell loss is therefore likely of importance in age-associated mitochondrial dysfunction. There is a tendency to automatically link mitochondrial dysfunction to increased production of reactive oxygen species (ROS). However, the experimental support for this concept is rather weak. Mouse models with respiratory chain deficiency induced by tissue-specific mtDNA depletion or by massive increase of point mutations in mtDNA have very minor or no increase of oxidative stress. Future studies are needed to address the relative importance of mitochondrial dysfunction and ROS in mammalian ageing.

  15. Structure of the mammalian kinetochore.

    PubMed

    Ris, H; Witt, P L

    1981-01-01

    The structure of the mammalian trilaminar kinetochore was investigated using stereo electron microscopy of chromosomes in hypotonic solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed wihout pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore out layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observation of thick sections in stereo which made it possible to identify microtubules ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer.

  16. Nuclear Organization of Mammalian Genomes

    PubMed Central

    Sadoni, Nicolas; Langer, Sabine; Fauth, Christine; Bernardi, Giorgio; Cremer, Thomas; Turner, Bryan M.; Zink, Daniele

    1999-01-01

    We investigated the nuclear higher order compartmentalization of chromatin according to its replication timing (Ferreira et al. 1997) and the relations of this compartmentalization to chromosome structure and the spatial organization of transcription. Our aim was to provide a comprehensive and integrated view on the relations between chromosome structure and functional nuclear architecture. Using different mammalian cell types, we show that distinct higher order compartments whose DNA displays a specific replication timing are stably maintained during all interphase stages. The organizational principle is clonally inherited. We directly demonstrate the presence of polar chromosome territories that align to build up higher order compartments, as previously suggested (Ferreira et al. 1997). Polar chromosome territories display a specific orientation of early and late replicating subregions that correspond to R- or G/C-bands of mitotic chromosomes. Higher order compartments containing G/C-bands replicating during the second half of the S phase display no transcriptional activity detectable by BrUTP pulse labeling and show no evidence of transcriptional competence. Transcriptionally competent and active chromatin is confined to a coherent compartment within the nuclear interior that comprises early replicating R-band sequences. As a whole, the data provide an integrated view on chromosome structure, nuclear higher order compartmentalization, and their relation to the spatial organization of functional nuclear processes. PMID:10491386

  17. Technology of mammalian cell encapsulation.

    PubMed

    Uludag, H; De Vos, P; Tresco, P A

    2000-08-20

    Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates a cell mass from an outside environment and aims to maintain normal cellular physiology within a desired permeability barrier. Numerous encapsulation techniques have been developed over the years. These techniques are generally classified as microencapsulation (involving small spherical vehicles and conformally coated tissues) and macroencapsulation (involving larger flat-sheet and hollow-fiber membranes). This review is intended to summarize techniques of cell encapsulation as well as methods for evaluating the performance of encapsulated cells. The techniques reviewed include microencapsulation with polyelectrolyte complexation emphasizing alginate-polylysine capsules, thermoreversible gelation with agarose as a prototype system, interfacial precipitation and interfacial polymerization, as well as the technology of flat sheet and hollow fiber-based macroencapsulation. Four aspects of encapsulated cells that are critical for the success of the technology, namely the capsule permeability, mechanical properties, immune protection and biocompatibility, have been singled out and methods to evaluate these properties were summarized. Finally, speculations regarding future directions of cell encapsulation research and device development are included from the authors' perspective.

  18. Mammalian cell cultivation in space

    NASA Astrophysics Data System (ADS)

    Gmünder, Felix K.; Suter, Robert N.; Kiess, M.; Urfer, R.; Nordau, C.-G.; Cogoli, A.

    Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 μl. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.

  19. [Chemical defense of plant to mammalian herbivore].

    PubMed

    Li, J; Liu, J

    2001-06-01

    The research progress in the chemical defense of plant to mammalian herbivore was reviewed in this paper. The plant secondary compounds mainly are phenolics, terpenoids and nitrogen-containing compounds. The defense efficiency of plant to mammalian herbivores is different with the types and content of secondary compounds in plant. Secondary compounds inhibited the foraging of mammalian herbivores by affecting the intake, digestion, metabolites and reproduction of animal. It is the main trends to study the mode of coevolution of plant and animals mediated by plant secondary compounds.

  20. Ghrelin Receptors in Non-Mammalian Vertebrates

    PubMed Central

    Kaiya, Hiroyuki; Kangawa, Kenji; Miyazato, Mikiya

    2012-01-01

    The growth hormone secretagogue-receptor (GHS-R) was discovered in humans and pigs in 1996. The endogenous ligand, ghrelin, was discovered 3 years later, in 1999, and our understanding of the physiological significance of the ghrelin system in vertebrates has grown steadily since then. Although the ghrelin system in non-mammalian vertebrates is a subject of great interest, protein sequence data for the receptor in non-mammalian vertebrates has been limited until recently, and related biological information has not been well organized. In this review, we summarize current information related to the ghrelin receptor in non-mammalian vertebrates. PMID:23882259

  1. High resolution thermal denaturation of mammalian DNAs.

    PubMed Central

    Guttmann, T; Vítek, A; Pivec, L

    1977-01-01

    High resolution melting profiles of different mammalian DNAs are presented. Melting curves of various mammalian DNAs were compared with respect to the degree of asymmetry, first moment, transition breath and Tmi of individual subtransitions. Quantitative comparison of the shape of all melting curves was made. Correlation between phylogenetical relations among mammals and shape of the melting profiles of their DNAs was demonstrated. The difference between multi-component heterogeneity of mammalian DNAs found by optical melting analysis and sedimentation in CsCl-netropsin density gradient is also discussed. PMID:840642

  2. Enzymology of Mammalian DNA Methyltransferases.

    PubMed

    Jurkowska, Renata Z; Jeltsch, Albert

    2016-01-01

    DNA methylation is currently one of the hottest topics in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA nucleotide methyltransferases (DNMTs), principles of their regulation in cells have only begun to be uncovered. In mammals, DNA methylation is introduced by the DNMT1, DNMT3A, and DNMT3B enzymes, which are all large multi-domain proteins. These enzymes contain a catalytic C-terminal domain with a characteristic cytosine-C5 methyltransferase fold and an N-terminal part with different domains that interacts with other proteins and chromatin and is involved in targeting and regulation of the DNMTs. The subnuclear localization of the DNMT enzymes plays an important role in their biological function: DNMT1 is localized to replicating DNA via interaction with PCNA and UHRF1. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD and PWWP domains. Recently, a novel regulatory mechanism has been discovered in DNMTs, as latest structural and functional data demonstrated that the catalytic activities of all three enzymes are under tight allosteric control of their N-terminal domains having autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of autoinhibitory domains by protein factors, noncoding RNAs, or by posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, including their specificity and processivity, and afterward we focus on the regulation of their activity and targeting via allosteric processes, protein interactors, and posttranslational modifications.

  3. Chemosignals, Hormones and Mammalian Reproduction

    PubMed Central

    Petrulis, Aras

    2013-01-01

    Many mammalian species use chemosignals to coordinate reproduction by altering the physiology and behavior of both sexes. Chemosignals prime reproductive physiology so that individuals become sexually mature and active at times when mating is most probable and suppress it when it is not. Once in reproductive condition, odors produced and deposited by both males and females are used to find and select individuals for mating. The production, dissemination and appropriate responses to these cues are modulated heavily by organizational and activational effects of gonadal sex steroids and thereby intrinsically link chemical communication to the broader reproductive context. Many compounds have been identified as “pheromones” but very few have met the expectations of that term: a unitary, species-typical substance that is both necessary and sufficient for an experience-independent behavioral or physiological response. In contrast, most responses to chemosignals are dependent or heavily modulated by experience, either in adulthood or during development. Mechanistically, chemosignals are perceived by both main and accessory (vomeronasal) olfactory systems with the importance of each system tied strongly to the nature of the stimulus rather than to the response. In the central nervous system, the vast majority of responses to chemosignals are mediated by cortical and medial amygdala connections with hypothalamic and other forebrain structures. Despite the importance of chemosignals in mammals, many details of chemical communication differ even among closely related species and defy clear categorization. Although generating much research and public interest, strong evidence for the existence of a robust chemical communication among humans is lacking. PMID:23545474

  4. Chemosignals, hormones and mammalian reproduction.

    PubMed

    Petrulis, Aras

    2013-05-01

    Many mammalian species use chemosignals to coordinate reproduction by altering the physiology and behavior of both sexes. Chemosignals prime reproductive physiology so that individuals become sexually mature and active at times when mating is most probable and suppress it when it is not. Once in reproductive condition, odors produced and deposited by both males and females are used to find and select individuals for mating. The production, dissemination and appropriate responses to these cues are modulated heavily by organizational and activational effects of gonadal sex steroids and thereby intrinsically link chemical communication to the broader reproductive context. Many compounds have been identified as "pheromones" but very few have met the expectations of that term: a unitary, species-typical substance that is both necessary and sufficient for an experience-independent behavioral or physiological response. In contrast, most responses to chemosignals are dependent or heavily modulated by experience, either in adulthood or during development. Mechanistically, chemosignals are perceived by both main and accessory (vomeronasal) olfactory systems with the importance of each system tied strongly to the nature of the stimulus rather than to the response. In the central nervous system, the vast majority of responses to chemosignals are mediated by cortical and medial amygdala connections with hypothalamic and other forebrain structures. Despite the importance of chemosignals in mammals, many details of chemical communication differ even among closely related species and defy clear categorization. Although generating much research and public interest, strong evidence for the existence of a robust chemical communication among humans is lacking. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Bats and Rodents Shape Mammalian Retroviral Phylogeny.

    PubMed

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-11-09

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general.

  6. Mammalian synthetic biology: emerging medical applications.

    PubMed

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M; Krams, Rob

    2015-05-06

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON-OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes.

  7. Mammalian Response to Cenozoic Climatic Change

    NASA Astrophysics Data System (ADS)

    Blois, Jessica L.; Hadly, Elizabeth A.

    2009-05-01

    Multiple episodes of rapid and gradual climatic changes influenced the evolution and ecology of mammalian species and communities throughout the Cenozoic. Climatic change influenced the abundance, genetic diversity, morphology, and geographic ranges of individual species. Within communities these responses interacted to catalyze immigration, speciation, and extinction. Combined they affected long-term patterns of community stability, functional turnover, biotic turnover, and diversity. Although the relative influence of climate on particular evolutionary processes is oft debated, an understanding of processes at the root of biotic change yields important insights into the complexity of mammalian response. Ultimately, all responses trace to events experienced by populations. However, many such processes emerge as patterns above the species level, where shared life history traits and evolutionary history allow us to generalize about mammalian response to climatic change. These generalizations provide the greatest power to understand and predict mammalian responses to current and future global change.

  8. Mammalian synthetic biology: emerging medical applications

    PubMed Central

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

    2015-01-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

  9. Bats and Rodents Shape Mammalian Retroviral Phylogeny

    PubMed Central

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  10. Circadian Plasticity of Mammalian Inhibitory Interneurons

    PubMed Central

    2017-01-01

    Inhibitory interneurons participate in all neuronal circuits in the mammalian brain, including the circadian clock system, and are indispensable for their effective function. Although the clock neurons have different molecular and electrical properties, their main function is the generation of circadian oscillations. Here we review the circadian plasticity of GABAergic interneurons in several areas of the mammalian brain, suprachiasmatic nucleus, neocortex, hippocampus, olfactory bulb, cerebellum, striatum, and in the retina. PMID:28367335

  11. Pathways of mammalian replication fork restart.

    PubMed

    Petermann, Eva; Helleday, Thomas

    2010-10-01

    Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation.

  12. Simplified Bioreactor For Growing Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F.

    1995-01-01

    Improved bioreactor for growing mammalian cell cultures developed. Designed to support growth of dense volumes of mammalian cells by providing ample, well-distributed flows of nutrient solution with minimal turbulence. Cells relatively delicate and, unlike bacteria, cannot withstand shear forces present in turbulent flows. Bioreactor vessel readily made in larger sizes to accommodate greater cell production quantities. Molding equipment presently used makes cylinders up to 30 centimeters long. Alternative sintered plastic techniques used to vary pore size and quantity, as necessary.

  13. Hacking the genetic code of mammalian cells.

    PubMed

    Schwarzer, Dirk

    2009-07-06

    A genetic shuttle: The highlighted article, which was recently published by Schultz, Geierstanger and co-workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line.

  14. cDNA cloning, expression analysis, and chromosomal localization of a gene with high homology to wheat eIF-(iso)4F and mammalian eIF-4G

    SciTech Connect

    Shaughnessy, J.D. Jr.; Jenkins, N.A.; Copeland, N.G.

    1997-01-15

    A novel mammalian gene, Eif4g2, with a high degree of homology to the p82 subunit of the wheat germ eukaryotic translation initiation factor eIF-(iso)4F and mammalian eIF-4G has been isolated. Zoo blot analysis indicates that Eif4g2 is a single-copy gene that is highly conserved among vertebrates. Northern blot analysis shows that Eif4g2 is ubiquitously expressed at high levels in all human and mouse tissues examined. The 3810-nucleotide Eif4g2 cDNA contains a 907-amino-acid open reading frame that codes for a polypeptide with a predicted molecular mass of 102 kDa. The Eif4g2 polypeptide exhibits an overall similarity to wheat p82 of 52%. A 248-amino-acid segment at the amino-terminal end of both peptides exhibits 63% similarity and contains conserved potential RNA binding domains and a phosphorylation site. The Eif4g2 polypeptide contains multiple potential N-linked glycosylation sites as well as protein kinase C and casein kinase II phosphorylation sites. Southern blot analysis of DNA from interspecific backcross mice shows that Eif4g2 is localized to distal mouse chromosome 7 in a region syntenic with human chromosome 11p15. 25 refs., 5 figs.

  15. Archetype, adaptation and the mammalian heart.

    PubMed

    Meijler, F L; Meijler, T D

    2011-03-01

    Forty years ago, we started our quest for 'The Holy Grail' of understanding ventricular rate control and rhythm in atrial fibrillation (AF). We therefore studied the morphology and function of a wide range of mammalian hearts. From mouse to whale, we found that all hearts show similar structural and functional characteristics. This suggests that the mammalian heart remained well conserved during evolution and in this aspect it differs from other organs and parts of the mammalian body. The archetype of the mammalian heart was apparently so successful that adaptation by natural selection (evolution) caused by varying habitat demands, as occurred in other organs and many other aspects of mammalian anatomy, bypassed the heart. The structure and function of the heart of placental mammals have thus been strikingly conserved throughout evolution. The changes in the mammalian heart that did take place were mostly adjustments (scaling), to compensate for variations in body size and shape. A remarkable scaling effect is, for instance, the difference in atrioventricular (AV) conduction time, which is vital for optimal cardiac function in all mammals, small and large. Scaling of AV conduction takes place in the AV node (AVN), but its substrate is unknown. This sheds new light on the vital role of the AVN in health and disease. The AVN is master and servant of the heart at the same time and is of salient importance for our understanding of supraventricular arrhythmias in humans, especially AF. In Information Technology a software infra-structure called 'enterprise service bus' (ESB) may provide understanding of the mammalian heart's conservation during evolution. The ESB is quite unspecific (and thus general) when compared with the specialised components it has to support. For instance, one of the functions of an ESB is the routing of messages between system nodes. This routing is independent and unaware of the content of the messages. The function of the heart is likewise

  16. Mammalian Cell-Based Sensor System

    NASA Astrophysics Data System (ADS)

    Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

    Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

  17. A Comparative Study of Mammalian Diversification Pattern

    PubMed Central

    Yu, Wenhua; Xu, Junxiao; Wu, Yi; Yang, Guang

    2012-01-01

    Although mammals have long been regarded as a successful radiation, the diversification pattern among the clades is still poorly known. Higher-level phylogenies are conflicting and comprehensive comparative analyses are still lacking. Using a recently published supermatrix encompassing nearly all extant mammalian families and a novel comparative likelihood approach (MEDUSA), the diversification pattern of mammalian groups was examined. Both order- and family-level phylogenetic analyses revealed the rapid radiation of Boreoeutheria and Euaustralidelphia in the early mammalian history. The observation of a diversification burst within Boreoeutheria at approximately 100 My supports the Long Fuse model in elucidating placental diversification progress, and the rapid radiation of Euaustralidelphia suggests an important role of biogeographic dispersal events in triggering early Australian marsupial rapid radiation. Diversification analyses based on family-level diversity tree revealed seven additional clades with exceptional diversification rate shifts, six of which represent accelerations in net diversification rate as compared to the background pattern. The shifts gave origin to the clades Muridae+Cricetidae, Bovidae+Moschidae+Cervidae, Simiiformes, Echimyidae, Odontoceti (excluding Physeteridae+Kogiidae+Platanistidae), Macropodidae, and Vespertilionidae. Moderate to high extinction rates from background and boreoeutherian diversification patterns indicate the important role of turnovers in shaping the heterogeneous taxonomic richness observed among extant mammalian groups. Furthermore, the present results emphasize the key role of extinction on erasing unusual diversification signals, and suggest that further studies are needed to clarify the historical radiation of some mammalian groups for which MEDUSA did not detect exceptional diversification rates. PMID:22457604

  18. Effect of Microgravity on Mammalian Lymphocytes

    NASA Technical Reports Server (NTRS)

    Banerjee, H.; Blackshear, M.; Mahaffey, K.; Knight, C.; Khan, A. A.; Delucas, L.

    2004-01-01

    The effect of microgravity on mammalian system is an important and interesting topic for scientific investigation, since NASA s objective is to send manned flights to planets like Mars and eventual human colonization.The Astronauts will be exposed to microgravity environment for a long duration of time during these flights.Our objective of research is to conduct in vitro studies for the effect of microgravity on mammalian immune system.We did our preliminary investigations by exposing mammalian lymphocytes to a microgravity simulator cell bioreactor designed by NASA and manufactured at Synthecon Inc (USA).Our initial results showed no significant change in cytokine expression in these cells for a time period of forty eight hours exposure.Our future experiments will involve exposure for a longer period of time.

  19. Effect of Microgravity on Mammalian Lymphocytes

    NASA Technical Reports Server (NTRS)

    Banerjee, H.; Blackshear, M.; Mahaffey, K.; Khan, A. A.; Delucas, L.

    2004-01-01

    The effect of microgravity on mammalian system is an important and interesting topic for scientific investigation, since NASA s objective is to send manned flights to planets like Mars and eventual human colonization. The Astronauts will be exposed to microgravity environment for a long duration of time during these flights. Our objective of research is to conduct in vitro studies for the effect of microgravity on mammalian immune system and nervous system. We did our preliminary investigations by exposing mammalian lymphocytes and astrocyte cells to a microgravity simulator cell bioreactor designed by NASA and manufactured at Synthecon, Inc. (USA).Our initial results showed no significant change in cytokine expression in these cells up to a time period of 120 hours exposure. Our future experiments will involve exposure for a longer period of time.

  20. Involvement of opsins in mammalian sperm thermotaxis

    PubMed Central

    Pérez-Cerezales, Serafín; Boryshpolets, Sergii; Afanzar, Oshri; Brandis, Alexander; Nevo, Reinat; Kiss, Vladimir; Eisenbach, Michael

    2015-01-01

    A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of <0.0006 °C as they swim their body-length distance. The identity of the sensing system that confers this exceptional sensitivity on spermatozoa is not known. Here we show that the temperature-sensing system of mammalian spermatozoa involves opsins, known to be G-protein-coupled receptors that act as photosensors in vision. We demonstrate by molecular, immunological, and functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways—the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors. PMID:26537127

  1. Mammalian diversity: gametes, embryos and reproduction.

    PubMed

    Behringer, Richard R; Eakin, Guy S; Renfree, Marilyn B

    2006-01-01

    The class Mammalia is composed of approximately 4800 extant species. These mammalian species are divided into three subclasses that include the monotremes, marsupials and eutherians. Monotremes are remarkable because these mammals are born from eggs laid outside of the mother's body. Marsupial mammals have relatively short gestation periods and give birth to highly altricial young that continue a significant amount of 'fetal' development after birth, supported by a highly sophisticated lactation. Less than 10% of mammalian species are monotremes or marsupials, so the great majority of mammals are grouped into the subclass Eutheria, including mouse and human. Mammals exhibit great variety in morphology, physiology and reproduction. In the present article, we highlight some of this remarkable diversity relative to the mouse, one of the most widely used mammalian model organisms, and human. This diversity creates challenges and opportunities for gamete and embryo collection, culture and transfer technologies.

  2. Involvement of opsins in mammalian sperm thermotaxis.

    PubMed

    Pérez-Cerezales, Serafín; Boryshpolets, Sergii; Afanzar, Oshri; Brandis, Alexander; Nevo, Reinat; Kiss, Vladimir; Eisenbach, Michael

    2015-11-05

    A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of <0.0006 °C as they swim their body-length distance. The identity of the sensing system that confers this exceptional sensitivity on spermatozoa is not known. Here we show that the temperature-sensing system of mammalian spermatozoa involves opsins, known to be G-protein-coupled receptors that act as photosensors in vision. We demonstrate by molecular, immunological, and functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways-the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors.

  3. Requirement of mammalian Timeless for circadian rhythmicity.

    PubMed

    Barnes, Jessica W; Tischkau, Shelley A; Barnes, Jeffrey A; Mitchell, Jennifer W; Burgoon, Penny W; Hickok, Jason R; Gillette, Martha U

    2003-10-17

    Despite a central circadian role in Drosophila for the transcriptional regulator Timeless (dTim), the relevance of mammalian Timeless (mTim) remains equivocal. Conditional knockdown of mTim protein expression in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms, and altered levels of known core clock elements. Full-length mTim protein (mTIM-fl) exhibited a 24-hour oscillation, where as a truncated isoform (mTIM-s) was constitutively expressed. mTIM-fl associated with the mammalian clock Period proteins (mPERs) in oscillating SCN cells. These data suggest that mTim is required for rhythmicity and is a functional homolog of dTim on the negative-feedback arm of the mammalian molecular clockwork.

  4. Synthetic therapeutic gene circuits in mammalian cells.

    PubMed

    Ye, Haifeng; Fussenegger, Martin

    2014-08-01

    In the emerging field of synthetic biology, scientists are focusing on designing and creating functional devices, systems, and organisms with novel functions by engineering and assembling standardised biological building blocks. The progress of synthetic biology has significantly advanced the design of functional gene networks that can reprogram metabolic activities in mammalian cells and provide new therapeutic opportunities for future gene- and cell-based therapies. In this review, we describe the most recent advances in synthetic mammalian gene networks designed for biomedical applications, including how these synthetic therapeutic gene circuits can be assembled to control signalling networks and applied to treat metabolic disorders, cancer, and immune diseases. We conclude by discussing the various challenges and future prospects of using synthetic mammalian gene networks for disease therapy. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Synthetic mammalian gene circuits for biomedical applications.

    PubMed

    Ye, Haifeng; Aubel, Dominique; Fussenegger, Martin

    2013-12-01

    Synthetic biology is the science of reassembling cataloged and standardized biological items in a systematic and rational manner to create and engineer functional biological designer devices, systems and organisms with novel and useful, preferably therapeutic functions. Synthetic biology has significantly advanced the design of complex genetic networks that can reprogram metabolic activities in mammalian cells and provide novel therapeutic strategies for future gene-based and cell-based therapies. Synthetic biology-inspired therapeutic strategies provide new opportunities for improving human health in the 21st century. This review covers the most recent synthetic mammalian circuits designed for therapy of diseases such as metabolic disorders, cancer, and immune disorders. We conclude by discussing current challenges and future perspectives for biomedical applications of synthetic mammalian gene networks.

  6. Better Smelling Through Genetics: Mammalian Odor Perception

    PubMed Central

    Keller, Andreas; Vosshall, Leslie B.

    2008-01-01

    SUMMARY The increasing availability of genomic and genetic tools to study olfaction—the sense of smell—has brought important new insights into how this chemosensory modality functions in different species. Newly sequenced mammalian genomes—from platypus to dog—have made it possible to infer how smell has evolved to suit the needs of a given species and how variation within a species may affect individual olfactory perception. This review will focus on recent advances in the genetics and genomics of mammalian smell, with a primary focus on rodents and humans. PMID:18938244

  7. A new modification for mammalian messenger RNA.

    PubMed

    Liu, Fange; He, Chuan

    2017-09-01

    The discovery of multiple RNA modifications in the past few years has broadened our views of the structures and potential functions of RNA species, but deciphering which modifications are made where and how remains a challenge. A new study by Xu et al. applies a combination of mass spectrometry, biochemistry, genetics, and cellular biology tools to reveal the two mammalian methyltransferases that are responsible for m(3)C installation in tRNA and a third that mediates the previously unknown installation of m(3)C in mammalian mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Mammalian cell cultures for biologics manufacturing.

    PubMed

    Kantardjieff, Anne; Zhou, Weichang

    2014-01-01

    Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations.

  9. The mammalian blastema: regeneration at our fingertips

    PubMed Central

    Simkin, Jennifer; Sammarco, Mimi C.; Dawson, Lindsay A.; Schanes, Paula P.; Yu, Ling

    2015-01-01

    Abstract In the mouse, digit tip regeneration progresses through a series of discrete stages that include inflammation, histolysis, epidermal closure, blastema formation, and redifferentiation. Recent studies reveal how each regenerative stage influences subsequent stages to establish a blastema that directs the successful regeneration of a complex mammalian structure. The focus of this review is on early events of healing and how an amputation wound transitions into a functional blastema. The stepwise formation of a mammalian blastema is proposed to provide a model for how specific targeted treatments can enhance regenerative performance in humans. PMID:27499871

  10. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

    1993-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  11. Crossroads between Bacterial and Mammalian Glycosyltransferases

    PubMed Central

    Brockhausen, Inka

    2014-01-01

    Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

  12. The cytogenetics of mammalian autosomal rearrangements

    SciTech Connect

    Daniel, A.

    1988-01-01

    Combining data from animal and clinical studies with classical cytogenetic observations, the volume provides information on various aspects of mammalian autosomal rearrangements. Topics range from the reproductive consequences to carriers of autosomal rearrangements to the application of structural rearrangements and DNA probes to gene mapping. In addition, the book presents an overview of new perspectives and future directions for research.

  13. Phospholipid synthesis and transport in mammalian cells.

    PubMed

    Vance, Jean E

    2015-01-01

    Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter-organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria-associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter-organelle phospholipid transport in mammalian cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Architecture of mammalian respiratory complex I.

    PubMed

    Vinothkumar, Kutti R; Zhu, Jiapeng; Hirst, Judy

    2014-11-06

    Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved 'core' subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 'supernumerary' subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.

  15. Architecture of mammalian respiratory complex I

    PubMed Central

    Hirst, Judy

    2014-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The fourteen conserved ‘core’ subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 ‘supernumerary’ subunits are unknown. Here, we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we significantly advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases. PMID:25209663

  16. Medical and experimental mammalian genetics: A perspective

    SciTech Connect

    McKusick, V.A.; Roderick, T.H.; Mori, J.; Paul, N.W.

    1987-01-01

    This book contains 14 papers. Some of the titles are: Structure and Organization of Mammalian Chromosomes: Normal and Abnormal; Globin Gene Structure and the Nature of Mutation; Retroviral DNA Content of the Mouse Genome; Maternal Genes: Mitochondrial Diseases; Human Evolution; and Prospects for Gene Replacement Therapy.

  17. Structure of mammalian respiratory complex I

    PubMed Central

    Hirst, Judy

    2016-01-01

    Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner membrane. Mammalian complex I1 contains 45 subunits, comprising 14 core subunits that house the catalytic machinery and are conserved from bacteria to humans, and a mammalian-specific cohort of 31 supernumerary subunits1,2. Knowledge about the structures and functions of the supernumerary subunits is fragmentary. Here, we describe a 4.2 Å resolution single-particle cryoEM structure of complex I from Bos taurus. We locate and model all 45 subunits to provide the entire structure of the mammalian complex. Furthermore, computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally-dynamic regions and match biochemical descriptions of the ‘active-to-deactive’ enzyme transition that occurs during hypoxia3,4. Thus, our structures provide a foundation for understanding complex I assembly5 and the effects of mutations that cause clinically-relevant complex I dysfunctions6, insights into the structural and functional roles of the supernumerary subunits, and new information on the mechanism and regulation of catalysis. PMID:27509854

  18. A promoter-level mammalian expression atlas

    PubMed Central

    2015-01-01

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. PMID:24670764

  19. Ticks Take Cues from Mammalian Interferon.

    PubMed

    de Silva, Aravinda M

    2016-07-13

    Interferons are considered a first line of immune defense restricted to vertebrates. In this issue of Cell Host & Microbe, Smith et al. (2016) demonstrate that mammalian interferon γ activates an antimicrobial response within ticks feeding on blood. The study suggests that arthropods have a parallel interferon-like defense system.

  20. A promoter-level mammalian expression atlas.

    PubMed

    Forrest, Alistair R R; Kawaji, Hideya; Rehli, Michael; Baillie, J Kenneth; de Hoon, Michiel J L; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V; Lizio, Marina; Itoh, Masayoshi; Andersson, Robin; Mungall, Christopher J; Meehan, Terrence F; Schmeier, Sebastian; Bertin, Nicolas; Jørgensen, Mette; Dimont, Emmanuel; Arner, Erik; Schmidl, Christian; Schaefer, Ulf; Medvedeva, Yulia A; Plessy, Charles; Vitezic, Morana; Severin, Jessica; Semple, Colin A; Ishizu, Yuri; Young, Robert S; Francescatto, Margherita; Alam, Intikhab; Albanese, Davide; Altschuler, Gabriel M; Arakawa, Takahiro; Archer, John A C; Arner, Peter; Babina, Magda; Rennie, Sarah; Balwierz, Piotr J; Beckhouse, Anthony G; Pradhan-Bhatt, Swati; Blake, Judith A; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Burroughs, A Maxwell; Califano, Andrea; Cannistraci, Carlo V; Carbajo, Daniel; Chen, Yun; Chierici, Marco; Ciani, Yari; Clevers, Hans C; Dalla, Emiliano; Davis, Carrie A; Detmar, Michael; Diehl, Alexander D; Dohi, Taeko; Drabløs, Finn; Edge, Albert S B; Edinger, Matthias; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Fagiolini, Michela; Fairbairn, Lynsey; Fang, Hai; Farach-Carson, Mary C; Faulkner, Geoffrey J; Favorov, Alexander V; Fisher, Malcolm E; Frith, Martin C; Fujita, Rie; Fukuda, Shiro; Furlanello, Cesare; Furino, Masaaki; Furusawa, Jun-ichi; Geijtenbeek, Teunis B; Gibson, Andrew P; Gingeras, Thomas; Goldowitz, Daniel; Gough, Julian; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J; Hamaguchi, Masahide; Hara, Mitsuko; Harbers, Matthias; Harshbarger, Jayson; Hasegawa, Akira; Hasegawa, Yuki; Hashimoto, Takehiro; Herlyn, Meenhard; Hitchens, Kelly J; Ho Sui, Shannan J; Hofmann, Oliver M; Hoof, Ilka; Hori, Furni; Huminiecki, Lukasz; Iida, Kei; Ikawa, Tomokatsu; Jankovic, Boris R; Jia, Hui; Joshi, Anagha; Jurman, Giuseppe; Kaczkowski, Bogumil; Kai, Chieko; Kaida, Kaoru; Kaiho, Ai; Kajiyama, Kazuhiro; Kanamori-Katayama, Mutsumi; Kasianov, Artem S; Kasukawa, Takeya; Katayama, Shintaro; Kato, Sachi; Kawaguchi, Shuji; Kawamoto, Hiroshi; Kawamura, Yuki I; Kawashima, Tsugumi; Kempfle, Judith S; Kenna, Tony J; Kere, Juha; Khachigian, Levon M; Kitamura, Toshio; Klinken, S Peter; Knox, Alan J; Kojima, Miki; Kojima, Soichi; Kondo, Naoto; Koseki, Haruhiko; Koyasu, Shigeo; Krampitz, Sarah; Kubosaki, Atsutaka; Kwon, Andrew T; Laros, Jeroen F J; Lee, Weonju; Lennartsson, Andreas; Li, Kang; Lilje, Berit; Lipovich, Leonard; Mackay-Sim, Alan; Manabe, Ri-ichiroh; Mar, Jessica C; Marchand, Benoit; Mathelier, Anthony; Mejhert, Niklas; Meynert, Alison; Mizuno, Yosuke; de Lima Morais, David A; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Motakis, Efthymios; Motohashi, Hozumi; Mummery, Christine L; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nakazato, Kenichi; van Nimwegen, Erik; Ninomiya, Noriko; Nishiyori, Hiromi; Noma, Shohei; Noma, Shohei; Noazaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohimiya, Hiroko; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A; Pain, Arnab; Passier, Robert; Patrikakis, Margaret; Persson, Helena; Piazza, Silvano; Prendergast, James G D; Rackham, Owen J L; Ramilowski, Jordan A; Rashid, Mamoon; Ravasi, Timothy; Rizzu, Patrizia; Roncador, Marco; Roy, Sugata; Rye, Morten B; Saijyo, Eri; Sajantila, Antti; Saka, Akiko; Sakaguchi, Shimon; Sakai, Mizuho; Sato, Hiroki; Savvi, Suzana; Saxena, Alka; Schneider, Claudio; Schultes, Erik A; Schulze-Tanzil, Gundula G; Schwegmann, Anita; Sengstag, Thierry; Sheng, Guojun; Shimoji, Hisashi; Shimoni, Yishai; Shin, Jay W; Simon, Christophe; Sugiyama, Daisuke; Sugiyama, Takaai; Suzuki, Masanori; Suzuki, Naoko; Swoboda, Rolf K; 't Hoen, Peter A C; Tagami, Michihira; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tatum, Zuotian; Thompson, Mark; Toyodo, Hiroo; Toyoda, Tetsuro; Valen, Elvind; van de Wetering, Marc; van den Berg, Linda M; Verado, Roberto; Vijayan, Dipti; Vorontsov, Ilya E; Wasserman, Wyeth W; Watanabe, Shoko; Wells, Christine A; Winteringham, Louise N; Wolvetang, Ernst; Wood, Emily J; Yamaguchi, Yoko; Yamamoto, Masayuki; Yoneda, Misako; Yonekura, Yohei; Yoshida, Shigehiro; Zabierowski, Susan E; Zhang, Peter G; Zhao, Xiaobei; Zucchelli, Silvia; Summers, Kim M; Suzuki, Harukazu; Daub, Carsten O; Kawai, Jun; Heutink, Peter; Hide, Winston; Freeman, Tom C; Lenhard, Boris; Bajic, Vladimir B; Taylor, Martin S; Makeev, Vsevolod J; Sandelin, Albin; Hume, David A; Carninci, Piero; Hayashizaki, Yoshihide

    2014-03-27

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  1. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  2. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  3. Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase

    PubMed Central

    Molosh, Andrei I.; Johnson, Philip L.; Spence, John P.; Arendt, David; Federici, Lauren M.; Bernabe, Cristian; Janasik, Steven P.; Segu, Zaneer M.; Khanna, Rajesh; Goswami, Chirayu; Zhu, Weiguo; Park, Su-Jung; Li, Lang; Mechref, Yehia S.; Clapp, D. Wade; Shekhar, Anantha

    2014-01-01

    Children with Neurofibromatosis type 1 (NF1) are increasingly recognized to have high prevalence of social difficulties and autism spectrum disorders (ASD). We demonstrated selective social learning deficit in mice with deletion of a single Nf1 gene (Nf1+/−), along with greater activation of mitogen activated protein kinase pathway in neurons from amygdala and frontal cortex, structures relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate/GABA neurotransmission; deficits in long-term potentiation; and specific disruptions in expression of two proteins associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (ADAM22) and heat shock protein 70 (HSP70), respectively. All of these amygdala disruptions were normalized by co-deletion of p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide novel insights and therapeutic targets for NF1 and ASD patients. PMID:25242307

  4. Lactate Metabolism is Associated with Mammalian Mitochondria

    PubMed Central

    Chen, Ying-Jr; Mahieu, Nathaniel G.; Huang, Xiaojing; Singh, Manmilan; Crawford, Peter A; Johnson, Stephen L.; Gross, Richard W.; Schaefer, Jacob

    2016-01-01

    It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. Within the fermenting cell itself, however, it is generally assumed that lactate is produced to replenish NAD+ and then is secreted. Here we explored the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. With high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells. PMID:27618187

  5. Mammalian lipoxygenases and their biological relevance

    PubMed Central

    Kuhn, Hartmut; Banthiya, Swathi; van Leyen, Klaus

    2015-01-01

    Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOX oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, lipoxygenases are key players in regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. PMID:25316652

  6. Circadian aspects of mammalian parturition: a review.

    PubMed

    Olcese, James

    2012-02-05

    The identification of circadian clocks in endocrine tissues has added considerable depth and complexity to our understanding of their physiology. A growing body of research reveals circadian clock gene expression in the uterus of non-pregnant and pregnant rodents. This review will focus on the mammalian uterus and its rhythmicity, particularly as it pertains to the circadian timing of parturition. This key event in the reproductive axis shows dramatic species-specific differences in its circadian phase. It is proposed here that these differences in the phasing of mammalian parturition are likely a function of opposite uterine cell responses to humoral cues. The argument will be made that melatonin fulfills many of the criteria to serve as a circadian signal in the initiation of human parturition, including specific actions on uterine smooth muscle cells that are consistent with a role for this hormone in the circadian timing of parturition.

  7. Avian and Mammalian Facilitative Glucose Transporters.

    PubMed

    Byers, Mary Shannon; Howard, Christianna; Wang, Xiaofei

    2017-04-05

    The GLUT members belong to a family of glucose transporter proteins that facilitate glucose transport across the cell membrane. The mammalian GLUT family consists of thirteen members (GLUTs 1-12 and H⁺-myo-inositol transporter (HMIT)). Humans have a recently duplicated GLUT member, GLUT14. Avians express the majority of GLUT members. The arrangement of multiple GLUTs across all somatic tissues signifies the important role of glucose across all organisms. Defects in glucose transport have been linked to metabolic disorders, insulin resistance and diabetes. Despite the essential importance of these transporters, our knowledge regarding GLUT members in avians is fragmented. It is clear that there are no chicken orthologs of mammalian GLUT4 and GLUT7. Our examination of GLUT members in the chicken revealed that some chicken GLUT members do not have corresponding orthologs in mammals. We review the information regarding GLUT orthologs and their function and expression in mammals and birds, with emphasis on chickens and humans.

  8. Potassium transport in the mammalian collecting duct.

    PubMed

    Muto, S

    2001-01-01

    The mammalian collecting duct plays a dominant role in regulating K(+) excretion by the nephron. The collecting duct exhibits axial and intrasegmental cell heterogeneity and is composed of at least two cell types: collecting duct cells (principal cells) and intercalated cells. Under normal circumstances, the collecting duct cell in the cortical collecting duct secretes K(+), whereas under K(+) depletion, the intercalated cell reabsorbs K(+). Assessment of the electrochemical driving forces and of membrane conductances for transcellular and paracellular electrolyte movement, the characterization of several ATPases, patch-clamp investigation, and cloning of the K(+) channel have provided important insights into the role of pumps and channels in those tubule cells that regulate K(+) secretion and reabsorption. This review summarizes K(+) transport properties in the mammalian collecting duct. Special emphasis is given to the mechanisms of how K(+) transport is regulated in the collecting duct.

  9. Mammalian Sperm Motility: Observation and Theory

    NASA Astrophysics Data System (ADS)

    Gaffney, E. A.; Gadêlha, H.; Smith, D. J.; Blake, J. R.; Kirkman-Brown, J. C.

    2011-01-01

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics.

  10. Expanding the genetic code of mammalian cells.

    PubMed

    Italia, James S; Zheng, Yunan; Kelemen, Rachel E; Erickson, Sarah B; Addy, Partha S; Chatterjee, Abhishek

    2017-04-15

    In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  11. Ricin trafficking in plant and mammalian cells.

    PubMed

    Lord, J Michael; Spooner, Robert A

    2011-07-01

    Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans). The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin) and Ricinus communis agglutinin l (RCA). These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.

  12. Synaptic Release at Mammalian Bipolar Cell Terminals

    PubMed Central

    Wan, Qun-Fang; Heidelberger, Ruth

    2011-01-01

    Bipolar cells play a vital role in the transfer of visual information across the vertebrate retina. The synaptic output of these neurons is regulated by factors that are extrinsic and intrinsic. Relatively little is known about the intrinsic factors that regulate neurotransmitter exocytosis. Much of what we know about intrinsic presynaptic mechanisms that regulate glutamate release has come from the study of the unusually large and accessible synaptic terminal of the goldfish rod-dominant bipolar cell, the Mb1 bipolar cell. However, over the past several years, examination of presynaptic mechanisms governing neurotransmitter release has been extended to the mammalian rod bipolar cell. In this review, we discuss the recent advances in our understanding of synaptic vesicle dynamics and neurotransmitter release in rodent rod bipolar cells and consider how these properties help shape the synaptic output of the mammalian retina. PMID:21272392

  13. Basic techniques in mammalian cell tissue culture.

    PubMed

    Phelan, Katy; May, Kristin M

    2015-03-02

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.

  14. Isolation of genomic DNA from mammalian cells.

    PubMed

    Koh, Cheryl M

    2013-01-01

    The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Mammalian Evolution May not Be Strictly Bifurcating

    PubMed Central

    Hallström, Björn M.; Janke, Axel

    2010-01-01

    The massive amount of genomic sequence data that is now available for analyzing evolutionary relationships among 31 placental mammals reduces the stochastic error in phylogenetic analyses to virtually zero. One would expect that this would make it possible to finally resolve controversial branches in the placental mammalian tree. We analyzed a 2,863,797 nucleotide-long alignment (3,364 genes) from 31 placental mammals for reconstructing their evolution. Most placental mammalian relationships were resolved, and a consensus of their evolution is emerging. However, certain branches remain difficult or virtually impossible to resolve. These branches are characterized by short divergence times in the order of 1–4 million years. Computer simulations based on parameters from the real data show that as little as about 12,500 amino acid sites could be sufficient to confidently resolve short branches as old as about 90 million years ago (Ma). Thus, the amount of sequence data should no longer be a limiting factor in resolving the relationships among placental mammals. The timing of the early radiation of placental mammals coincides with a period of climate warming some 100–80 Ma and with continental fragmentation. These global processes may have triggered the rapid diversification of placental mammals. However, the rapid radiations of certain mammalian groups complicate phylogenetic analyses, possibly due to incomplete lineage sorting and introgression. These speciation-related processes led to a mosaic genome and conflicting phylogenetic signals. Split network methods are ideal for visualizing these problematic branches and can therefore depict data conflict and possibly the true evolutionary history better than strictly bifurcating trees. Given the timing of tectonics, of placental mammalian divergences, and the fossil record, a Laurasian rather than Gondwanan origin of placental mammals seems the most parsimonious explanation. PMID:20591845

  16. Structure and function of mammalian cilia.

    PubMed

    Satir, Peter; Christensen, Søren T

    2008-06-01

    In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease.

  17. Comparison of amphibian and mammalian thyroperoxidase ...

    EPA Pesticide Factsheets

    Thyroperoxidase (TPO) catalyzes the production of thyroid hormones in the vertebrate thyroid gland by oxidizing iodide (I- ) to produce iodinated tyrosines on thyroglobulin, and further coupling of specific mono- or di-iodinated tyrosines to generate the triiodo- and tetra-iodothyronine, precursors to thyroid hormone. This enzyme is a target for thyroid disrupting chemicals. TPO-inhibition by xenobiotics is a molecular initiating event that is known to perturb the thyroid axis by preventing synthesis of thyroid hormone. Previous work on TPO-inhibition has been focused on mammalian TPO; specifically, the rat and pig. A primary objective of this experiment was to directly measure TPO activity in a non-mammalian system, in this case a thyroid gland homogenate from Xenopus laevis; as well as compare chemical inhibition from past mammalian studies to the amphibian data generated. Thyroid glands obtained from X. laevis tadpoles at NF stages 58-60, were pooled and homogenized by sonication in phosphate buffer. This homogenate was then used to test 24 chemicals for inhibition of TPO as measured by conversion of Amplex UltraRed (AUR) substrate to its fluorescent product. The test chemicals were selected based upon previous results from rat in vitro TPO assays, and X. laevis in vitro and in vivo studies for thyroid disrupting endpoints, and included both positive and negative chemicals in these assays. An initial screening of the chemicals was done at a single high con

  18. Epigenetic Regulation of the Mammalian Cell

    PubMed Central

    Baverstock, Keith; Rönkkö, Mauno

    2008-01-01

    Background Understanding how mammalian cells are regulated epigenetically to express phenotype is a priority. The cellular phenotypic transition, induced by ionising radiation, from a normal cell to the genomic instability phenotype, where the ability to replicate the genotype accurately is compromised, illustrates important features of epigenetic regulation. Based on this phenomenon and earlier work we propose a model to describe the mammalian cell as a self assembled open system operating in an environment that includes its genotype, neighbouring cells and beyond. Phenotype is represented by high dimensional attractors, evolutionarily conditioned for stability and robustness and contingent on rules of engagement between gene products encoded in the genetic network. Methodology/Findings We describe how this system functions and note the indeterminacy and fluidity of its internal workings which place it in the logical reasoning framework of predicative logic. We find that the hypothesis is supported by evidence from cell and molecular biology. Conclusions Epigenetic regulation and memory are fundamentally physical, as opposed to chemical, processes and the transition to genomic instability is an important feature of mammalian cells with probable fundamental relevance to speciation and carcinogenesis. A source of evolutionarily selectable variation, in terms of the rules of engagement between gene products, is seen as more likely to have greater prominence than genetic variation in an evolutionary context. As this epigenetic variation is based on attractor states phenotypic changes are not gradual; a phenotypic transition can involve the changed contribution of several gene products in a single step. PMID:18523589

  19. Mutation hot spots in mammalian mitochondrial DNA.

    PubMed

    Galtier, Nicolas; Enard, David; Radondy, Yoan; Bazin, Eric; Belkhir, Khalid

    2006-02-01

    Animal mitochondrial DNA is characterized by a remarkably high level of within-species homoplasy, that is, phylogenetic incongruence between sites of the molecule. Several investigators have invoked recombination to explain it, challenging the dogma of maternal, clonal mitochondrial inheritance in animals. Alternatively, a high level of homoplasy could be explained by the existence of mutation hot spots. By using an exhaustive mammalian data set, we test the hot spot hypothesis by comparing patterns of site-specific polymorphism and divergence in several groups of closely related species, including hominids. We detect significant co-occurrence of synonymous polymorphisms among closely related species in various mammalian groups, and a correlation between the site-specific levels of variability within humans (on one hand) and between Hominoidea species (on the other hand), indicating that mutation hot spots actually exist in mammalian mitochondrial coding regions. The whole data, however, cannot be explained by a simple mutation hot spots model. Rather, we show that the site-specific mutation rate quickly varies in time, so that the same sites are not hypermutable in distinct lineages. This study provides a plausible mutation model that potentially accounts for the peculiar distribution of mitochondrial sequence variation in mammals without the need for invoking recombination. It also gives hints about the proximal causes of mitochondrial site-specific hypermutability in humans.

  20. Mammalian Synthetic Biology: Engineering Biological Systems.

    PubMed

    Black, Joshua B; Perez-Pinera, Pablo; Gersbach, Charles A

    2017-06-21

    The programming of new functions into mammalian cells has tremendous application in research and medicine. Continued improvements in the capacity to sequence and synthesize DNA have rapidly increased our understanding of mechanisms of gene function and regulation on a genome-wide scale and have expanded the set of genetic components available for programming cell biology. The invention of new research tools, including targetable DNA-binding systems such as CRISPR/Cas9 and sensor-actuator devices that can recognize and respond to diverse chemical, mechanical, and optical inputs, has enabled precise control of complex cellular behaviors at unprecedented spatial and temporal resolution. These tools have been critical for the expansion of synthetic biology techniques from prokaryotic and lower eukaryotic hosts to mammalian systems. Recent progress in the development of genome and epigenome editing tools and in the engineering of designer cells with programmable genetic circuits is expanding approaches to prevent, diagnose, and treat disease and to establish personalized theranostic strategies for next-generation medicines. This review summarizes the development of these enabling technologies and their application to transforming mammalian synthetic biology into a distinct field in research and medicine.

  1. Aneuploidy in mammalian somatic cells in vivo.

    PubMed

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed.

  2. Mammalian masticatory muscles: homology, nomenclature, and diversification.

    PubMed

    Druzinsky, Robert E; Doherty, Alison H; De Vree, Frits L

    2011-08-01

    There is a deep and rich literature of comparative studies of jaw muscles in mammals but no recent analyses employ modern phylogenetic techniques to better understand evolutionary changes that have occurred in these muscles. In order to fully develop and utilize the Feeding Experiments End-user Database (FEED), we are constructing a comprehensive ontology of mammalian jaw muscles. This process has led to a careful consideration of nomenclature and homologies of the muscles and their constituent parts. Precise determinations of muscle attachments have shown that muscles with similar names are not necessarily homologous. Using new anatomical descriptions derived from the literature, we defined character states for the jaw muscles in diverse mammalian species. We then mapped those characters onto a recent phylogeny of mammals with the aid of the Mesquite software package. Our data further elucidate how muscle groups associated with the feeding apparatus differ and have become highly specialized in certain mammalian orders, such as Rodentia, while remaining conserved in other orders. We believe that careful naming of muscles and statistical analyses of their distributions among mammals, in association with the FEED database, will lead to new, significant insights into the functional, structural, and evolutionary morphology of the jaw muscles.

  3. Some principles of regeneration in mammalian systems.

    PubMed

    Carlson, Bruce M

    2005-11-01

    This article presents some general principles underlying regenerative phenomena in vertebrates, starting with the epimorphic regeneration of the amphibian limb and continuing with tissue and organ regeneration in mammals. Epimorphic regeneration following limb amputation involves wound healing, followed shortly by a phase of dedifferentiation that leads to the formation of a regeneration blastema. Up to the point of blastema formation, dedifferentiation is guided by unique regenerative pathways, but the overall developmental controls underlying limb formation from the blastema generally recapitulate those of embryonic limb development. Damaged mammalian tissues do not form a blastema. At the cellular level, differentiation follows a pattern close to that seen in the embryo, but at the level of the tissue and organ, regeneration is strongly influenced by conditions inherent in the local environment. In some mammalian systems, such as the liver, parenchymal cells contribute progeny to the regenerate. In others, e.g., skeletal muscle and bone, tissue-specific progenitor cells constitute the main source of regenerating cells. The substrate on which regeneration occurs plays a very important role in determining the course of regeneration. Epimorphic regeneration usually produces an exact replica of the structure that was lost, but in mammalian tissue regeneration the form of the regenerate is largely determined by the mechanical environment acting on the regenerating tissue, and it is normally an imperfect replica of the original. In organ hypertophy, such as that occurring after hepatic resection, the remaining liver mass enlarges, but there is no attempt to restore the original form.

  4. Comparison of amphibian and mammalian thyroperoxidase ...

    EPA Pesticide Factsheets

    Thyroperoxidase (TPO) catalyzes the production of thyroid hormones in the vertebrate thyroid gland by oxidizing iodide (I- ) to produce iodinated tyrosines on thyroglobulin, and further coupling of specific mono- or di-iodinated tyrosines to generate the triiodo- and tetra-iodothyronine, precursors to thyroid hormone. This enzyme is a target for thyroid disrupting chemicals. TPO-inhibition by xenobiotics is a molecular initiating event that is known to perturb the thyroid axis by preventing synthesis of thyroid hormone. Previous work on TPO-inhibition has been focused on mammalian TPO; specifically, the rat and pig. A primary objective of this experiment was to directly measure TPO activity in a non-mammalian system, in this case a thyroid gland homogenate from Xenopus laevis; as well as compare chemical inhibition from past mammalian studies to the amphibian data generated. Thyroid glands obtained from X. laevis tadpoles at NF stages 58-60, were pooled and homogenized by sonication in phosphate buffer. This homogenate was then used to test 24 chemicals for inhibition of TPO as measured by conversion of Amplex UltraRed (AUR) substrate to its fluorescent product. The test chemicals were selected based upon previous results from rat in vitro TPO assays, and X. laevis in vitro and in vivo studies for thyroid disrupting endpoints, and included both positive and negative chemicals in these assays. An initial screening of the chemicals was done at a single high con

  5. MAMMALIAN CELLS CONTAIN A SECOND NUCLEOCYTOPLASMIC HEXOSAMINIDASE

    PubMed Central

    Gutternigg, Martin; Rendić, Dubravko; Voglauer, Regina; Iskratsch, Thomas; Wilson, Iain B. H.

    2010-01-01

    Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterised in recombinant form and has an important role in cellular function due to its ability to cleave β-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterise for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability; furthermore, a myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localisation. Transcripts of the corresponding gene are expressed in a number of murine tissues. Based on its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be found from mammalian sources. PMID:19040401

  6. Protein and genome evolution in Mammalian cells for biotechnology applications.

    PubMed

    Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

    2009-06-01

    Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

  7. Mammalian Niche Conservation through Deep Time

    PubMed Central

    DeSantis, Larisa R. G.; Beavins Tracy, Rachel A.; Koontz, Cassandra S.; Roseberry, John C.; Velasco, Matthew C.

    2012-01-01

    Climate change alters species distributions, causing plants and animals to move north or to higher elevations with current warming. Bioclimatic models predict species distributions based on extant realized niches and assume niche conservation. Here, we evaluate if proxies for niches (i.e., range areas) are conserved at the family level through deep time, from the Eocene to the Pleistocene. We analyze the occurrence of all mammalian families in the continental USA, calculating range area, percent range area occupied, range area rank, and range polygon centroids during each epoch. Percent range area occupied significantly increases from the Oligocene to the Miocene and again from the Pliocene to the Pleistocene; however, mammalian families maintain statistical concordance between rank orders across time. Families with greater taxonomic diversity occupy a greater percent of available range area during each epoch and net changes in taxonomic diversity are significantly positively related to changes in percent range area occupied from the Eocene to the Pleistocene. Furthermore, gains and losses in generic and species diversity are remarkably consistent with ∼2.3 species gained per generic increase. Centroids demonstrate southeastern shifts from the Eocene through the Pleistocene that may correspond to major environmental events and/or climate changes during the Cenozoic. These results demonstrate range conservation at the family level and support the idea that niche conservation at higher taxonomic levels operates over deep time and may be controlled by life history traits. Furthermore, families containing megafauna and/or terminal Pleistocene extinction victims do not incur significantly greater declines in range area rank than families containing only smaller taxa and/or only survivors, from the Pliocene to Pleistocene. Collectively, these data evince the resilience of families to climate and/or environmental change in deep time, the absence of terminal Pleistocene

  8. Mammalian niche conservation through deep time.

    PubMed

    DeSantis, Larisa R G; Beavins Tracy, Rachel A; Koontz, Cassandra S; Roseberry, John C; Velasco, Matthew C

    2012-01-01

    Climate change alters species distributions, causing plants and animals to move north or to higher elevations with current warming. Bioclimatic models predict species distributions based on extant realized niches and assume niche conservation. Here, we evaluate if proxies for niches (i.e., range areas) are conserved at the family level through deep time, from the Eocene to the Pleistocene. We analyze the occurrence of all mammalian families in the continental USA, calculating range area, percent range area occupied, range area rank, and range polygon centroids during each epoch. Percent range area occupied significantly increases from the Oligocene to the Miocene and again from the Pliocene to the Pleistocene; however, mammalian families maintain statistical concordance between rank orders across time. Families with greater taxonomic diversity occupy a greater percent of available range area during each epoch and net changes in taxonomic diversity are significantly positively related to changes in percent range area occupied from the Eocene to the Pleistocene. Furthermore, gains and losses in generic and species diversity are remarkably consistent with ~2.3 species gained per generic increase. Centroids demonstrate southeastern shifts from the Eocene through the Pleistocene that may correspond to major environmental events and/or climate changes during the Cenozoic. These results demonstrate range conservation at the family level and support the idea that niche conservation at higher taxonomic levels operates over deep time and may be controlled by life history traits. Furthermore, families containing megafauna and/or terminal Pleistocene extinction victims do not incur significantly greater declines in range area rank than families containing only smaller taxa and/or only survivors, from the Pliocene to Pleistocene. Collectively, these data evince the resilience of families to climate and/or environmental change in deep time, the absence of terminal Pleistocene

  9. Mammalian developmental genetics in the twentieth century.

    PubMed

    Artzt, Karen

    2012-12-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas.

  10. Mammalian Gravity Receptors: Structure and Metabolism

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    1985-01-01

    Calcium metabolism in mammalian gravity receptors is examined. To accomplish this objective it is necessary to study both the mineral deposits of the receptors, the otoconia, and the sensory areas themselves, the saccular and utricular maculas. The main focus was to elucidate the natures of the organic and inorganic phases of the crystalline masses, first in rat otoconia but more recently in otoliths and otoconia of a comparative series of vertebrates. Some of the ultrastructural findings in rat maculas, however, have prompted a more thorough study of the organization of the hair cells and innervation patterns in graviceptors.

  11. Primer removal during mammalian mitochondrial DNA replication.

    PubMed

    Uhler, Jay P; Falkenberg, Maria

    2015-10-01

    The small circular mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause mitochondrial disease in humans. A fundamental step in mitochondrial DNA (mtDNA) replication and maintenance is the removal of the RNA primers needed for replication initiation. The nucleases RNase H1, FEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA replication initiation. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Mammalian Gravity Receptors: Structure and Metabolism

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    1985-01-01

    Calcium metabolism in mammalian gravity receptors is examined. To accomplish this objective it is necessary to study both the mineral deposits of the receptors, the otoconia, and the sensory areas themselves, the saccular and utricular maculas. The main focus was to elucidate the natures of the organic and inorganic phases of the crystalline masses, first in rat otoconia but more recently in otoliths and otoconia of a comparative series of vertebrates. Some of the ultrastructural findings in rat maculas, however, have prompted a more thorough study of the organization of the hair cells and innervation patterns in graviceptors.

  13. Derivation of the mammalian skull vault

    PubMed Central

    MORRISS-KAY, GILLIAN M.

    2001-01-01

    This review describes the evolutionary history of the mammalian skull vault as a basis for understanding its complex structure. Current information on the developmental tissue origins of the skull vault bones (mesoderm and neural crest) is assessed for mammals and other tetrapods. This information is discussed in the context of evolutionary changes in the proportions of the skull vault bones at the sarcopterygian-tetrapod transition. The dual tissue origin of the skull vault is considered in relation to the molecular mechanisms underlying osteogenic cell proliferation and differentiation in the sutural growth centres and in the proportionate contributions of different sutures to skull growth. PMID:11523816

  14. Mammalian odorant receptors: functional evolution and variation

    PubMed Central

    Jiang, Yue; Matsunami, Hiroaki

    2015-01-01

    In mammals, the perception of smell starts with the activation of odorant receptors (ORs) by volatile molecules in the environment. The mammalian OR repertoire has been subject to rapid evolution, and is highly diverse within the human population. Recent advances in the functional expression and ligand identification of ORs allow for functional analysis of OR evolution, and reveal that changes in OR protein sequences translate into high degrees of functional variations. Moreover, in several cases the functional variation of a single OR affects the perception of its cognate odor ligand, providing clues as to how an odor is coded at the receptor level. PMID:25660959

  15. The transcriptional landscape of the mammalian genome.

    PubMed

    Carninci, P; Kasukawa, T; Katayama, S; Gough, J; Frith, M C; Maeda, N; Oyama, R; Ravasi, T; Lenhard, B; Wells, C; Kodzius, R; Shimokawa, K; Bajic, V B; Brenner, S E; Batalov, S; Forrest, A R R; Zavolan, M; Davis, M J; Wilming, L G; Aidinis, V; Allen, J E; Ambesi-Impiombato, A; Apweiler, R; Aturaliya, R N; Bailey, T L; Bansal, M; Baxter, L; Beisel, K W; Bersano, T; Bono, H; Chalk, A M; Chiu, K P; Choudhary, V; Christoffels, A; Clutterbuck, D R; Crowe, M L; Dalla, E; Dalrymple, B P; de Bono, B; Della Gatta, G; di Bernardo, D; Down, T; Engstrom, P; Fagiolini, M; Faulkner, G; Fletcher, C F; Fukushima, T; Furuno, M; Futaki, S; Gariboldi, M; Georgii-Hemming, P; Gingeras, T R; Gojobori, T; Green, R E; Gustincich, S; Harbers, M; Hayashi, Y; Hensch, T K; Hirokawa, N; Hill, D; Huminiecki, L; Iacono, M; Ikeo, K; Iwama, A; Ishikawa, T; Jakt, M; Kanapin, A; Katoh, M; Kawasawa, Y; Kelso, J; Kitamura, H; Kitano, H; Kollias, G; Krishnan, S P T; Kruger, A; Kummerfeld, S K; Kurochkin, I V; Lareau, L F; Lazarevic, D; Lipovich, L; Liu, J; Liuni, S; McWilliam, S; Madan Babu, M; Madera, M; Marchionni, L; Matsuda, H; Matsuzawa, S; Miki, H; Mignone, F; Miyake, S; Morris, K; Mottagui-Tabar, S; Mulder, N; Nakano, N; Nakauchi, H; Ng, P; Nilsson, R; Nishiguchi, S; Nishikawa, S; Nori, F; Ohara, O; Okazaki, Y; Orlando, V; Pang, K C; Pavan, W J; Pavesi, G; Pesole, G; Petrovsky, N; Piazza, S; Reed, J; Reid, J F; Ring, B Z; Ringwald, M; Rost, B; Ruan, Y; Salzberg, S L; Sandelin, A; Schneider, C; Schönbach, C; Sekiguchi, K; Semple, C A M; Seno, S; Sessa, L; Sheng, Y; Shibata, Y; Shimada, H; Shimada, K; Silva, D; Sinclair, B; Sperling, S; Stupka, E; Sugiura, K; Sultana, R; Takenaka, Y; Taki, K; Tammoja, K; Tan, S L; Tang, S; Taylor, M S; Tegner, J; Teichmann, S A; Ueda, H R; van Nimwegen, E; Verardo, R; Wei, C L; Yagi, K; Yamanishi, H; Zabarovsky, E; Zhu, S; Zimmer, A; Hide, W; Bult, C; Grimmond, S M; Teasdale, R D; Liu, E T; Brusic, V; Quackenbush, J; Wahlestedt, C; Mattick, J S; Hume, D A; Kai, C; Sasaki, D; Tomaru, Y; Fukuda, S; Kanamori-Katayama, M; Suzuki, M; Aoki, J; Arakawa, T; Iida, J; Imamura, K; Itoh, M; Kato, T; Kawaji, H; Kawagashira, N; Kawashima, T; Kojima, M; Kondo, S; Konno, H; Nakano, K; Ninomiya, N; Nishio, T; Okada, M; Plessy, C; Shibata, K; Shiraki, T; Suzuki, S; Tagami, M; Waki, K; Watahiki, A; Okamura-Oho, Y; Suzuki, H; Kawai, J; Hayashizaki, Y

    2005-09-02

    This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

  16. Genome Editing Using Mammalian Haploid Cells

    PubMed Central

    Horii, Takuro; Hatada, Izuho

    2015-01-01

    Haploid cells are useful for studying gene functions because disruption of a single allele can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem cells (ESCs) in mice, rats, and monkeys provides a new platform for simple genetic manipulation of the mammalian genome. Use of haploid ESCs enhances the genome-editing potential of the CRISPR/Cas system. For example, CRISPR/Cas was used in haploid ESCs to generate multiple knockouts and large deletions at high efficiency. In addition, genome-wide screening is facilitated by haploid cell lines containing gene knockout libraries. PMID:26437403

  17. The virome in mammalian physiology and disease

    PubMed Central

    Virgin, Herbert W.

    2014-01-01

    The virome contains the most abundant and fastest-mutating genetic elements on Earth. The mammalian virome is constituted of viruses that infect host cells, virus-derived elements in our chromosomes, and viruses that infect the broad array of other types of organisms that inhabit us. Virome interactions with the host cannot be encompassed by a monotheistic view of viruses as pathogens. Instead, the genetic and transcriptional identity of mammals is defined in part by our co-evolved virome, a concept with profound implications for understanding health and disease. PMID:24679532

  18. Mammalian Kidney Development: Principles, Progress, and Projections

    PubMed Central

    Little, Melissa H.; McMahon, Andrew P.

    2012-01-01

    The mammalian kidney is a vital organ with considerable cellular complexity and functional diversity. Kidney development is notable for requiring distinct but coincident tubulogenic processes involving reciprocal inductive signals between mesenchymal and epithelial progenitor compartments. Key molecular pathways mediating these interactions have been identified. Further, advances in the analysis of gene expression and gene activity, coupled with a detailed knowledge of cell origins, are enhancing our understanding of kidney morphogenesis and unraveling the normal processes of postnatal repair and identifying disease-causing mechanisms. This article focuses on recent insights into central regulatory processes governing organ assembly and renal disease, and predicts future directions for the field. PMID:22550230

  19. AS52/GPT Mammalian Mutagenesis Assay

    DTIC Science & Technology

    1996-05-10

    dimethylnitrosamine (DMN) at 50 and 100 f.J.g/rnl was used as a 3 TLS Project Nn. A0ŗ-003: AS52/GPT Mammalian Mutagenesis Assay promutagen that requires metabolic...Chemical Source Lot No. air Air Products N/A calcium chloride Sigma 84F-0723 d imeth y !sulfoxide Fisher 933274 dimethylnitrosamine Sigma 82B0365...methanesulfonate (EMS) at 150 and 300 J.i-g/ml is used as a direct-acting mutagen for the nonactivated portion, and dimethylnitrosamine (DMN) at 150 and 300

  20. Structure and function of mammalian cilia

    PubMed Central

    Satir, Peter

    2008-01-01

    In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease. PMID:18365235

  1. Mammalian cell culture capacity for biopharmaceutical manufacturing.

    PubMed

    Ecker, Dawn M; Ransohoff, Thomas C

    2014-01-01

    : With worldwide sales of biopharmaceuticals increasing each year and continuing growth on the horizon, the manufacture of mammalian biopharmaceuticals has become a major global enterprise. We describe the current and future industry wide supply of manufacturing capacity with regard to capacity type, distribution, and geographic location. Bioreactor capacity and the use of single-use products for biomanufacturing are also profiled. An analysis of the use of this capacity is performed, including a discussion of current trends that will influence capacity growth, availability, and utilization in the coming years.

  2. Mammalian Developmental Genetics in the Twentieth Century

    PubMed Central

    Artzt, Karen

    2012-01-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas. PMID:23212897

  3. Molecular architecture of the mammalian circadian clock.

    PubMed

    Partch, Carrie L; Green, Carla B; Takahashi, Joseph S

    2014-02-01

    Circadian clocks coordinate physiology and behavior with the 24h solar day to provide temporal homeostasis with the external environment. The molecular clocks that drive these intrinsic rhythmic changes are based on interlocked transcription/translation feedback loops that integrate with diverse environmental and metabolic stimuli to generate internal 24h timing. In this review we highlight recent advances in our understanding of the core molecular clock and how it utilizes diverse transcriptional and post-transcriptional mechanisms to impart temporal control onto mammalian physiology. Understanding the way in which biological rhythms are generated throughout the body may provide avenues for temporally directed therapeutics to improve health and prevent disease.

  4. An Adaptive Threshold in Mammalian Neocortical Evolution

    PubMed Central

    Kalinka, Alex T.; Tomancak, Pavel; Huttner, Wieland B.

    2014-01-01

    Expansion of the neocortex is a hallmark of human evolution. However, determining which adaptive mechanisms facilitated its expansion remains an open question. Here we show, using the gyrencephaly index (GI) and other physiological and life-history data for 102 mammalian species, that gyrencephaly is an ancestral mammalian trait. We find that variation in GI does not evolve linearly across species, but that mammals constitute two principal groups above and below a GI threshold value of 1.5, approximately equal to 109 neurons, which may be characterized by distinct constellations of physiological and life-history traits. By integrating data on neurogenic period, neuroepithelial founder pool size, cell-cycle length, progenitor-type abundances, and cortical neuron number into discrete mathematical models, we identify symmetric proliferative divisions of basal progenitors in the subventricular zone of the developing neocortex as evolutionarily necessary for generating a 14-fold increase in daily prenatal neuron production, traversal of the GI threshold, and thus establishment of two principal groups. We conclude that, despite considerable neuroanatomical differences, changes in the length of the neurogenic period alone, rather than any novel neurogenic progenitor lineage, are sufficient to explain differences in neuron number and neocortical size between species within the same principal group. PMID:25405475

  5. Ecological adaptation determines functional mammalian olfactory subgenomes

    PubMed Central

    Hayden, Sara; Bekaert, Michaël; Crider, Tess A.; Mariani, Stefano; Murphy, William J.; Teeling, Emma C.

    2010-01-01

    The ability to smell is governed by the largest gene family in mammalian genomes, the olfactory receptor (OR) genes. Although these genes are well annotated in the finished human and mouse genomes, we still do not understand which receptors bind specific odorants or how they fully function. Previous comparative studies have been taxonomically limited and mostly focused on the percentage of OR pseudogenes within species. No study has investigated the adaptive changes of functional OR gene families across phylogenetically and ecologically diverse mammals. To determine the extent to which OR gene repertoires have been influenced by habitat, sensory specialization, and other ecological traits, to better understand the functional importance of specific OR gene families and thus the odorants they bind, we compared the functional OR gene repertoires from 50 mammalian genomes. We amplified more than 2000 OR genes in aquatic, semi-aquatic, and flying mammals and coupled these data with 48,000 OR genes from mostly terrestrial mammals, extracted from genomic projects. Phylogenomic, Bayesian assignment, and principle component analyses partitioned species by ecotype (aquatic, semi-aquatic, terrestrial, flying) rather than phylogenetic relatedness, and identified OR families important for each habitat. Functional OR gene repertoires were reduced independently in the multiple origins of aquatic mammals and were significantly divergent in bats. We reject recent neutralist views of olfactory subgenome evolution and correlate specific OR gene families with physiological requirements, a preliminary step toward unraveling the relationship between specific odors and respective OR gene families. PMID:19952139

  6. Structure and function in mammalian societies

    PubMed Central

    Clutton-Brock, Tim

    2009-01-01

    Traditional interpretations of the evolution of animal societies have suggested that their structure is a consequence of attempts by individuals to maximize their inclusive fitness within constraints imposed by their social and physical environments. In contrast, some recent re-interpretations have argued that many aspects of social organization should be interpreted as group-level adaptations maintained by selection operating between groups or populations. Here, I review our current understanding of the evolution of mammalian societies, focusing, in particular, on the evolution of reproductive strategies in societies where one dominant female monopolizes reproduction in each group and her offspring are reared by other group members. Recent studies of the life histories of females in these species show that dispersing females often have little chance of establishing new breeding groups and so are likely to maximize their inclusive fitness by helping related dominants to rear their offspring. As in eusocial insects, increasing group size can lead to a progressive divergence in the selection pressures operating on breeders and helpers and to increasing specialization in their behaviour and life histories. As yet, there is little need to invoke group-level adaptations in order to account for the behaviour of individuals or the structure of mammalian groups. PMID:19805430

  7. Epidemiology of mammalian hepatitis E virus infection.

    PubMed

    Kaba, Mamadou; Moal, Valérie; Gérolami, René; Colson, Philippe

    2013-01-01

    Mammalian hepatitis E virus (HEV), the etiological agent of hepatitis E in humans, is a recently discovered infectious agent. It was identified for the first time in 1983 using electron microscopy on a faecal specimen of a person infected with non-A, non-B enterically-transmitted hepatitis. Based on retrospective and prospective studies, HEV was long described as one of the leading causes of acute viral hepatitis in tropical and subtropical countries, whereas in developed countries hepatitis E was considered an imported disease from HEV hyperendemic countries. Data from studies conducted during the past decade have greatly shifted our knowledge on the epidemiology and clinical spectrum of HEV. Recently, it has been shown that contrary to previous beliefs, hepatitis E is also an endemic disease in several developed countries, particularly in Japan and in Europe, as evidenced by reports of high anti-HEV immunoglobulin G prevalence in healthy individuals and an increasing number of non-travel-related acute hepatitis E cases. Moreover, a porcine reservoir and growing evidence of zoonotic transmission have been reported in these countries. This review summarizes the current knowledge on the epidemiology and prevention of transmission of mammalian HEV. Copyright © 2013 S. Karger AG, Basel.

  8. Ecology and evolution of mammalian biodiversity.

    PubMed

    Jones, Kate E; Safi, Kamran

    2011-09-12

    Mammals have incredible biological diversity, showing extreme flexibility in eco-morphology, physiology, life history and behaviour across their evolutionary history. Undoubtedly, mammals play an important role in ecosystems by providing essential services such as regulating insect populations, seed dispersal and pollination and act as indicators of general ecosystem health. However, the macroecological and macroevolutionary processes underpinning past and present biodiversity patterns are only beginning to be explored on a global scale. It is also particularly important, in the face of the global extinction crisis, to understand these processes in order to be able to use this knowledge to prevent future biodiversity loss and loss of ecosystem services. Unfortunately, efforts to understand mammalian biodiversity have been hampered by a lack of data. New data compilations on current species' distributions, ecologies and evolutionary histories now allow an integrated approach to understand this biodiversity. We review and synthesize these new studies, exploring the past and present ecology and evolution of mammalian biodiversity, and use these findings to speculate about the mammals of our future.

  9. Catabolic flexibility of mammalian-associated lactobacilli

    PubMed Central

    2013-01-01

    Metabolic flexibility may be generally defined as “the capacity for the organism to adapt fuel oxidation to fuel availability”. The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus. PMID:23680304

  10. Metabolic flux estimation in mammalian cell cultures.

    PubMed

    Goudar, Chetan T; Biener, Richard K; Piret, James M; Konstantinov, Konstantin B

    2014-01-01

    Metabolic flux analysis with its ability to quantify cellular metabolism is an attractive tool for accelerating cell line selection, medium optimization, and other bioprocess development activities. In the stoichiometric flux estimation approach, unknown fluxes are determined using intracellular metabolite mass balance expressions and measured extracellular rates. The simplicity of the stoichiometric approach extends its application to most cell culture systems, and the steps involved in metabolic flux estimation by the stoichiometric method are presented in detail in this chapter. Specifically, overdetermined systems are analyzed since the extra measurements can be used to check for gross measurement errors and system consistency. Cell-specific rates comprise the input data for flux estimation, and the logistic modeling approach is described for robust-specific rate estimation in batch and fed-batch systems. A simplified network of mammalian cell metabolism is used to illustrate the flux estimation procedure, and the steps leading up the consistency index determination are presented. If gross measurement errors are detected, a technique for determining the source of gross measurement error is also described. A computer program that performs most of the calculation described in this chapter is presented, and references to flux estimation software are provided. The procedure presented in this chapter should enable rapid metabolic flux estimation in any mammalian cell bioreaction network by the stoichiometric approach.

  11. The Mammalian Ovary from Genesis to Revelation

    PubMed Central

    Edson, Mark A.; Nagaraja, Ankur K.; Matzuk, Martin M.

    2009-01-01

    Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago. PMID:19776209

  12. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  13. Ecology and evolution of mammalian biodiversity

    PubMed Central

    Jones, Kate E.; Safi, Kamran

    2011-01-01

    Mammals have incredible biological diversity, showing extreme flexibility in eco-morphology, physiology, life history and behaviour across their evolutionary history. Undoubtedly, mammals play an important role in ecosystems by providing essential services such as regulating insect populations, seed dispersal and pollination and act as indicators of general ecosystem health. However, the macroecological and macroevolutionary processes underpinning past and present biodiversity patterns are only beginning to be explored on a global scale. It is also particularly important, in the face of the global extinction crisis, to understand these processes in order to be able to use this knowledge to prevent future biodiversity loss and loss of ecosystem services. Unfortunately, efforts to understand mammalian biodiversity have been hampered by a lack of data. New data compilations on current species' distributions, ecologies and evolutionary histories now allow an integrated approach to understand this biodiversity. We review and synthesize these new studies, exploring the past and present ecology and evolution of mammalian biodiversity, and use these findings to speculate about the mammals of our future. PMID:21807728

  14. Kinetic Analysis of a Mammalian Phospholipase D

    PubMed Central

    Henage, Lee G.; Exton, John H.; Brown, H. Alex

    2013-01-01

    In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals, including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon phospholipase D to modulate cellular actions, such as cell growth, shape, and secretion. We examined the kinetics of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Cα, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. PKCα activation of PLD1 involves N- and C-terminal PLD domains. Rho GTPases were binding activators, enhancing the catalytic efficiency of a purified PLD1 catalytic domain via effects on Km. Arf-1, a catalytic activator, stimulated PLD1 by enhancing the catalytic constant, kcat. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. Synergy was observed only when PLD1 was simultaneously stimulated by a binding activator and a catalytic activator. Surprisingly, synergistic activation was steeply dependent on phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Together, these findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane. PMID:16339153

  15. The cellular code for mammalian thermosensation

    PubMed Central

    Pogorzala, Leah A.; Mishra, Santosh K.; Hoon, Mark A.

    2013-01-01

    Mammalian somatosenory neurons respond to thermal stimuli allowing animals to reliably discriminate hot from cold and select their preferred environments. We previously generated mice that are completely insensitive to temperatures from noxious cold to painful heat (−5 to 55 °C) by ablating several different classes of nociceptor early in development. Here we have adopted a selective ablation strategy in adult mice to dissect this phenotype and thereby demonstrated that separate populations of molecularly defined neurons respond to hot and cold. TRPV1-expressing neurons are responsible for all behavioral responses to temperatures between 40 and 50°C, while TRPM8-neurons are required for cold aversion. We also show that more extreme cold and heat activate additional populations of nociceptors including cells expressing Mrgprd. Thus, although eliminating Mrgprd-neurons alone does not affect behavioral responses to temperature, when combined with ablation of TRPV1- or TRPM8-cells, it significantly decreases responses to extreme heat and cold respectively. Notably, ablation of TRPM8-neurons distorts responses to preferred temperatures suggesting that the pleasant thermal sensation of warmth may in fact just reflect reduced aversive-input form TRPM8 and TRPV1-neurons. As predicted by this hypothesis, mice lacking both these classes of thermosensor exhibited neither aversive nor attractive responses to temperatures between 10 and 50 °C. Taken together these results provide a simple cellular basis for mammalian thermosensation whereby two molecularly defined classes of sensory neurons detect and encode both attractive and aversive cues. PMID:23536068

  16. Power amplification in the mammalian cochlea.

    PubMed

    Lukashkin, Andrei N; Walling, Mark N; Russell, Ian J

    2007-08-07

    It was first suggested by Gold in 1948 [1] that the exquisite sensitivity and frequency selectivity of the mammalian cochlea is due to an active process referred to as the cochlear amplifier. It is thought that this process works by pumping energy to augment the otherwise damped sound-induced vibrations of the basilar membrane [2-4], a mechanism known as negative damping. The existence of the cochlear amplifier has been inferred from comparing responses of sensitive and compromised cochleae [5] and observations of acoustic emissions [6, 7] and through mathematical modeling [8, 9]. However, power amplification has yet to be demonstrated directly. Here, we prove that energy is indeed produced in the cochlea on a cycle-by-cycle basis. By using laser interferometry [10], we show that the nonlinear component of basilar-membrane responses to sound stimulation leads the forces acting on the membrane. This is possible only in active systems with negative damping [11]. Our finding provides the first direct evidence for power amplification in the mammalian cochlea. The finding also makes redundant current hypotheses of cochlear frequency sharpening and sensitization that are not based on negative damping.

  17. The terminal DNA structure of mammalian chromosomes.

    PubMed Central

    McElligott, R; Wellinger, R J

    1997-01-01

    In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated. PMID:9218811

  18. Genomic imprinting: a mammalian epigenetic discovery model.

    PubMed

    Barlow, Denise P

    2011-01-01

    Genomic imprinting is an epigenetic process leading to parental-specific expression of one to two percent of mammalian genes that offers one of the best model systems for a molecular analysis of epigenetic regulation in development and disease. In the twenty years since the first imprinted gene was identified, this model has had a significant impact on decoding epigenetic information in mammals. So far it has led to the discovery of long-range cis-acting control elements whose epigenetic state regulates small clusters of genes and of unusual macro noncoding RNAs (ncRNAs) that directly repress genes in cis, and critically, it has demonstrated that one biological role of DNA methylation is to allow expression of genes normally repressed by default. This review describes the progress in understanding how imprinted protein-coding genes are silenced; in particular, it focuses on the role of macro ncRNAs that have broad relevance as a potential new layer of regulatory information in the mammalian genome.

  19. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  20. Focusing on RISC assembly in mammalian cells

    SciTech Connect

    Hong Junmei; Wei Na; Chalk, Alistair; Wang Jue; Song, Yutong; Yi Fan; Qiao Renping; Sonnhammer, Erik L.L.; Wahlestedt, Claes; Liang Zicai Du, Quan

    2008-04-11

    RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.

  1. Expression of mammalian membrane proteins in mammalian cells using Semliki Forest virus vectors.

    PubMed

    Lundstrom, Kenneth

    2010-01-01

    One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation and bacterial systems to expression in animal cells. In contrast to soluble proteins, the expression levels have been relatively low due to the following reasons: The topology of membrane proteins requires special, posttranslational processing, folding, and insertion into membranes, which often are mammalian cell specific. Despite these strict demands, functional membrane proteins (G protein-coupled receptors, ion channels, and transporters) have been successfully expressed in bacterial, yeast, and insect cells. A general drawback observed in prokaryotic cells is that accumulation of foreign protein in membranes is toxic and results in growth arrest and therefore low yields of recombinant protein.In this chapter, the focus is on expression of recombinant mammalian membrane proteins in mammalian host cells, particularly applying Semliki Forest virus (SFV) vectors. Replication-deficient SFV vectors are rapidly generated at high titers in BHK-21 (Baby Hamster Kidney) cells, which then are applied for a broad range of mammalian and nonmammalian cells. The SFV system has provided high expression levels of topologically different proteins, especially for membrane proteins. Robust ligand-binding assays and functional coupling to G proteins and electrophysiological recordings have made the SFV system an attractive tool in drug discovery. Furthermore, the high susceptibility of SFV vectors to primary neurons has allowed various applications in neuroscience. Establishment of large-scale production in mammalian adherent and suspension cultures has allowed production of hundreds of milligrams of membrane proteins that has allowed their submission to serious structural biology approaches. In this

  2. Mammalian social odours: attraction and individual recognition

    PubMed Central

    Brennan, Peter A; Kendrick, Keith M

    2006-01-01

    Mammalian social systems rely on signals passed between individuals conveying information including sex, reproductive status, individual identity, ownership, competitive ability and health status. Many of these signals take the form of complex mixtures of molecules sensed by chemosensory systems and have important influences on a variety of behaviours that are vital for reproductive success, such as parent–offspring attachment, mate choice and territorial marking. This article aims to review the nature of these chemosensory cues and the neural pathways mediating their physiological and behavioural effects. Despite the complexities of mammalian societies, there are instances where single molecules can act as classical pheromones attracting interest and approach behaviour. Chemosignals with relatively high volatility can be used to signal at a distance and are sensed by the main olfactory system. Most mammals also possess a vomeronasal system, which is specialized to detect relatively non-volatile chemosensory cues following direct contact. Single attractant molecules are sensed by highly specific receptors using a labelled line pathway. These act alongside more complex mixtures of signals that are required to signal individual identity. There are multiple sources of such individuality chemosignals, based on the highly polymorphic genes of the major histocompatibility complex (MHC) or lipocalins such as the mouse major urinary proteins. The individual profile of volatile components that make up an individual odour signature can be sensed by the main olfactory system, as the pattern of activity across an array of broadly tuned receptor types. In addition, the vomeronasal system can respond highly selectively to non-volatile peptide ligands associated with the MHC, acting at the V2r class of vomeronasal receptor. The ability to recognize individuals or their genetic relatedness plays an important role in mammalian social behaviour. Thus robust systems for olfactory

  3. The Evolution of Mammalian Olfactory Receptor Genes

    PubMed Central

    Issel-Tarver, L.; Rine, J.

    1997-01-01

    We performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans. The human subfamilies were localized to chromosomes 7, 11, and 19. The two subfamilies that were tightly linked in the dog genome were also tightly linked in the human genome. The four subfamilies were compared in human (primate), horse (perissodactyl), and a variety of artiodactyls and carnivores. Some changes in gene number were detected, but overall subfamily size appeared to have been established before the divergence of these mammals 60-100 million years ago. PMID:9017400

  4. Mechanism of Protein Biosynthesis in Mammalian Mitochondria

    PubMed Central

    Christian, Brooke E.; Spremulli, Linda L.

    2011-01-01

    Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. PMID:22172991

  5. Cenozoic climate change influences mammalian evolutionary dynamics

    PubMed Central

    Figueirido, Borja; Janis, Christine M.; Pérez-Claros, Juan A.; De Renzi, Miquel; Palmqvist, Paul

    2012-01-01

    Global climate change is having profound impacts on the natural world. However, climate influence on faunal dynamics at macroevolutionary scales remains poorly understood. In this paper we investigate the influence of climate over deep time on the diversity patterns of Cenozoic North American mammals. We use factor analysis to identify temporally correlated assemblages of taxa, or major evolutionary faunas that we can then study in relation to climatic change over the past 65 million years. These taxa can be grouped into six consecutive faunal associations that show some correspondence with the qualitative mammalian chronofaunas of previous workers. We also show that the diversity pattern of most of these chronofaunas can be correlated with the stacked deep-sea benthic foraminiferal oxygen isotope (δ18O) curve, which strongly suggests climatic forcing of faunal dynamics over a large macroevolutionary timescale. This study demonstrates the profound influence of climate on the diversity patterns of North American terrestrial mammals over the Cenozoic. PMID:22203974

  6. Regulation of Rap GTPases in mammalian neurons.

    PubMed

    Shah, Bhavin; Püschel, Andreas W

    2016-10-01

    Small GTPases are central regulators of many cellular processes. The highly conserved Rap GTPases perform essential functions in the mammalian nervous system during development and in mature neurons. During neocortical development, Rap1 is required to regulate cadherin- and integrin-mediated adhesion. In the adult nervous system Rap1 and Rap2 regulate the maturation and plasticity of dendritic spine and synapses. Although genetic studies have revealed important roles of Rap GTPases in neurons, their regulation by guanine nucleotide exchange factors (GEFs) that activate them and GTPase activating proteins (GAPs) that inactivate them by stimulating their intrinsic GTPase activity is just beginning to be explored in vivo. Here we review how GEFs and GAPs regulate Rap GTPases in the nervous system with a focus on their in vivo function.

  7. Studies on the mammalian toxicity of fenthion*

    PubMed Central

    Francis, Jean I.; Barnes, J. M.

    1963-01-01

    This paper constitutes a report on mammalian toxicological investigations of fen hion, carried out as part of the WHO malaria eradication programme, and on the conclusions drawn from them. Fenthion is found to be of intermediate toxicity to the four rodent species studied. In rats the signs of poisoning develop rather slowly but persist for several days, male rats being more susceptible than females, whereas for most phosphorothionates the converse is true. The results suggest that fenthion is not simply oxidized from the P=S compound to the P=O. It has been stated that the sulfoxide and sulfone are produced before the P=S→P=O oxidation takes place, but experiments suggest that further changes are involved. The findings are discussed in relation to the possible health hazard that might be encountered by those who have to apply fenthion as a residual spray. PMID:14056272

  8. Crystal structure of mammalian acid sphingomyelinase

    PubMed Central

    Gorelik, Alexei; Illes, Katalin; Heinz, Leonhard X.; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann–Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann–Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase. PMID:27435900

  9. Mechanism of protein biosynthesis in mammalian mitochondria.

    PubMed

    Christian, Brooke E; Spremulli, Linda L

    2012-01-01

    Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Mammalian synthetic biology for studying the cell

    PubMed Central

    Mathur, Melina; Xiang, Joy S.

    2017-01-01

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

  11. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  12. Peromyscus as a Mammalian Epigenetic Model

    PubMed Central

    Shorter, Kimberly R.; Crossland, Janet P.; Webb, Denessia; Szalai, Gabor; Felder, Michael R.; Vrana, Paul B.

    2012-01-01

    Deer mice (Peromyscus) offer an opportunity for studying the effects of natural genetic/epigenetic variation with several advantages over other mammalian models. These advantages include the ability to study natural genetic variation and behaviors not present in other models. Moreover, their life histories in diverse habitats are well studied. Peromyscus resources include genome sequencing in progress, a nascent genetic map, and >90,000 ESTs. Here we review epigenetic studies and relevant areas of research involving Peromyscus models. These include differences in epigenetic control between species and substance effects on behavior. We also present new data on the epigenetic effects of diet on coat-color using a Peromyscus model of agouti overexpression. We suggest that in terms of tying natural genetic variants with environmental effects in producing specific epigenetic effects, Peromyscus models have a great potential. PMID:22567379

  13. Mammalian Autophagy: How Does It Work?

    PubMed

    Bento, Carla F; Renna, Maurizio; Ghislat, Ghita; Puri, Claudia; Ashkenazi, Avraham; Vicinanza, Mariella; Menzies, Fiona M; Rubinsztein, David C

    2016-06-02

    Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.

  14. Fundamentals of Expression in Mammalian Cells.

    PubMed

    Dyson, Michael R

    2016-01-01

    Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions.

  15. Signaling Mechanisms in Mammalian Myoblast Fusion

    PubMed Central

    Hindi, Sajedah M.; Tajrishi, Marjan M.; Kumar, Ashok

    2013-01-01

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion. PMID:23612709

  16. Signaling mechanisms in mammalian myoblast fusion.

    PubMed

    Hindi, Sajedah M; Tajrishi, Marjan M; Kumar, Ashok

    2013-04-23

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion.

  17. New consensus nomenclature for mammalian keratins

    PubMed Central

    Schweizer, Jürgen; Bowden, Paul E.; Coulombe, Pierre A.; Langbein, Lutz; Lane, E. Birgitte; Magin, Thomas M.; Maltais, Lois; Omary, M. Bishr; Parry, David A.D.; Rogers, Michael A.; Wright, Mathew W.

    2006-01-01

    Keratins are intermediate filament–forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species. PMID:16831889

  18. Mammalian synthetic biology for studying the cell.

    PubMed

    Mathur, Melina; Xiang, Joy S; Smolke, Christina D

    2017-01-02

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

  19. [Thiamine triphosphatase activity in mammalian mitochondria].

    PubMed

    Rusina, I M; Makarchikov, A F

    2003-01-01

    Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC 3.6.1.28). The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.

  20. Mammalian Metallothionein-2A and Oxidative Stress

    PubMed Central

    Ling, Xue-Bin; Wei, Hong-Wei; Wang, Jun; Kong, Yue-Qiong; Wu, Yu-You; Guo, Jun-Li; Li, Tian-Fa; Li, Ji-Ke

    2016-01-01

    Mammalian metallothionein-2A (MT2A) has received considerable attention in recent years due to its crucial pathophysiological role in anti-oxidant, anti-apoptosis, detoxification and anti-inflammation. For many years, most studies evaluating the effects of MT2A have focused on reactive oxygen species (ROS), as second messengers that lead to oxidative stress injury of cells and tissues. Recent studies have highlighted that oxidative stress could activate mitogen-activated protein kinases (MAPKs), and MT2A, as a mediator of MAPKs, to regulate the pathogenesis of various diseases. However, the molecule mechanism of MT2A remains elusive. A deeper understanding of the functional, biochemical and molecular characteristics of MT2A would be identified, in order to bring new opportunities for oxidative stress therapy. PMID:27608012

  1. Cellular and Chemical Neuroscience of Mammalian Sleep

    PubMed Central

    Datta, Subimal

    2010-01-01

    Extraordinary strides have been made toward understanding the complexities and regulatory mechanisms of sleep over the past two decades, thanks to the help of rapidly evolving technologies. At its most basic level, mammalian sleep is a restorative process of the brain and body. Beyond its primary restorative purpose, sleep is essential for a number of vital functions. Our primary research interest is to understand the cellular and molecular mechanisms underlying the regulation of sleep and its cognitive functions. Here I will reflect on our own research contributions to fifty years of extraordinary advances in the neurobiology of slow-wave sleep (SWS) and rapid eye movement (REM) sleep regulation. I conclude this review by suggesting some potential future directions to further our understanding of the neurobiology of sleep. PMID:20359944

  2. Predictive chromatin signatures in the mammalian genome

    PubMed Central

    Hon, Gary C.; Hawkins, R. David; Ren, Bing

    2009-01-01

    The DNA sequence of an organism is a blueprint of life: it harbors not only the information about proteins and other molecules produced in each cell, but also instructions on when and where such molecules are made. Chromatin, the structure of histone and DNA that has co-evolved with eukaryotic genome, also contains information that indicates the function and activity of the underlying DNA sequences. Such information exists in the form of covalent modifications to the histone proteins that comprise the nucleosome. Thanks to the development of high throughput technologies such as DNA microarrays and next generation DNA sequencing, we have begun to associate the various combinations of chromatin modification patterns with functional sequences in the human genome. Here, we review the rapid progress from descriptive observations of histone modification profiles to highly predictive models enabling use of chromatin signatures to enumerate novel functional sequences in mammalian genomes that have escaped previous detection. PMID:19808796

  3. Mammalian protein glycosylation--structure versus function.

    PubMed

    Defaus, S; Gupta, P; Andreu, D; Gutiérrez-Gallego, R

    2014-06-21

    Carbohydrates fulfil many common as well as extremely important functions in nature. They show a variety of molecular displays--e.g., free mono-, oligo-, and polysaccharides, glycolipids, proteoglycans, glycoproteins, etc.--with particular roles and localizations in living organisms. Structure-specific peculiarities are so many and diverse that it becomes virtually impossible to cover them all from an analytical perspective. Hence this manuscript, focused on mammalian glycosylation, rather than a complete list of analytical descriptors or recognized functions for carbohydrate structures, comprehensively reviews three central issues in current glycoscience, namely (i) structural analysis of glycoprotein glycans, covering both classical and novel approaches for teasing out the structural puzzle as well as potential pitfalls of these processes; (ii) an overview of functions attributed to carbohydrates, covering from monosaccharide to complex, well-defined epitopes and full glycans, including post-glycosylational modifications, and (iii) recent technical advances allowing structural identification of glycoprotein glycans with simultaneous assignation of biological functions.

  4. Cenozoic climate change influences mammalian evolutionary dynamics.

    PubMed

    Figueirido, Borja; Janis, Christine M; Pérez-Claros, Juan A; De Renzi, Miquel; Palmqvist, Paul

    2012-01-17

    Global climate change is having profound impacts on the natural world. However, climate influence on faunal dynamics at macroevolutionary scales remains poorly understood. In this paper we investigate the influence of climate over deep time on the diversity patterns of Cenozoic North American mammals. We use factor analysis to identify temporally correlated assemblages of taxa, or major evolutionary faunas that we can then study in relation to climatic change over the past 65 million years. These taxa can be grouped into six consecutive faunal associations that show some correspondence with the qualitative mammalian chronofaunas of previous workers. We also show that the diversity pattern of most of these chronofaunas can be correlated with the stacked deep-sea benthic foraminiferal oxygen isotope (δ(18)O) curve, which strongly suggests climatic forcing of faunal dynamics over a large macroevolutionary timescale. This study demonstrates the profound influence of climate on the diversity patterns of North American terrestrial mammals over the Cenozoic.

  5. Genetic reassortment of mammalian reoviruses in mice.

    PubMed Central

    Wenske, E A; Chanock, S J; Krata, L; Fields, B N

    1985-01-01

    Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo. PMID:4057359

  6. Clues to the functions of mammalian sleep.

    PubMed

    Siegel, Jerome M

    2005-10-27

    The functions of mammalian sleep remain unclear. Most theories suggest a role for non-rapid eye movement (NREM) sleep in energy conservation and in nervous system recuperation. Theories of REM sleep have suggested a role for this state in periodic brain activation during sleep, in localized recuperative processes and in emotional regulation. Across mammals, the amount and nature of sleep are correlated with age, body size and ecological variables, such as whether the animals live in a terrestrial or an aquatic environment, their diet and the safety of their sleeping site. Sleep may be an efficient time for the completion of a number of functions, but variations in sleep expression indicate that these functions may differ across species.

  7. Mammalian telomeres and their partnership with lamins

    PubMed Central

    Burla, Romina; La Torre, Mattia; Saggio, Isabella

    2016-01-01

    ABSTRACT Chromosome ends are complex structures, which require a panel of factors for their elongation, replication, and protection. We describe here the mechanics of mammalian telomeres, dynamics and maintainance in relation to lamins. Multiple biochemical connections, including association of telomeres to the nuclear envelope and matrix, of telomeric proteins to lamins, and of lamin-associated proteins to chromosome ends, underline the interplay between lamins and telomeres. Paths toward senescence, such as defective telomere replication, altered heterochromatin organization, and impaired DNA repair, are common to lamins' and telomeres' dysfunction. The convergence of phenotypes can be interpreted through a model of dynamic, lamin-controlled functional platforms dedicated to the function of telomeres as fragile sites. The features of telomeropathies and laminopathies, and of animal models underline further overlapping aspects, including the alteration of stem cell compartments. We expect that future studies of basic biology and on aging will benefit from the analysis of this telomere-lamina interplay. PMID:27116558

  8. Differential Light Scattering from Spherical Mammalian Cells

    PubMed Central

    Brunsting, Albert; Mullaney, Paul F.

    1974-01-01

    The differential scattered light intensity patterns of spherical mammalian cells were measured with a new photometer which uses high-speed film as the light detector. The scattering objects, interphase and mitotic Chinese hamster ovary cells and HeLa cells, were modeled as (a) a coated sphere, accounting for nucleus and cytoplasm, and (b) a homogeneous sphere when no cellular nucleus was present. The refractive indices and size distribution of the cells were measured for an accurate comparison of the theoretical model with the light-scattering measurements. The light scattered beyond the forward direction is found to contain information about internal cellular morphology, provided the size distribution of the cells is not too broad. ImagesFIGURE 1 PMID:4134589

  9. Cooperative breeding and monogamy in mammalian societies

    PubMed Central

    Lukas, Dieter; Clutton-Brock, Tim

    2012-01-01

    Comparative studies of social insects and birds show that the evolution of cooperative and eusocial breeding systems has been confined to species where females mate completely or almost exclusively with a single male, indicating that high levels of average kinship between group members are necessary for the evolution of reproductive altruism. In this paper, we show that in mammals, the evolution of cooperative breeding has been restricted to socially monogamous species which currently represent 5 per cent of all mammalian species. Since extra-pair paternity is relatively uncommon in socially monogamous and cooperatively breeding mammals, our analyses support the suggestion that high levels of average kinship between group members have played an important role in the evolution of cooperative breeding in non-human mammals, as well as in birds and insects. PMID:22279167

  10. Chemical analysis of individual mammalian cells

    SciTech Connect

    Tan, W.; Yeung, E.S.

    1994-12-31

    The extremely small size of mammalian cells creates an unusual challenge for the analytical chemist, both in terms of separation and detection. Under a microscope, it is possible to confirm the injection of individual cells such as erythrocyte into capillaries with 10-{mu}m i.d. by hydrostatic pressure. The ionic contents can then be separated by capillary electrophoresis after the cell lyses. Enzymes at the zeptomole level can be monitored by on-column fluorescence enzyme assay. On-column particle-counting immunoassay can be applied to a broad range of analytes (antigens), also at the zeptomole level. The authors report here the simultaneous determination of the amounts of glucose-6-phosphate dehydrogenase (G6PDH) and their activities in individual erythrocytes by using a combination of the two detection schemes. Insights into the degradation of proteins as a function of cell age can be derived.

  11. Feeling the pressure in mammalian somatosensation

    PubMed Central

    Lumpkin, Ellen A; Bautista, Diana M

    2014-01-01

    Mechanoreceptor cells of the somatosensory system initiate the perception of touch and pain. Molecules required for mechanosensation have been identified from invertebrate neurons, and recent functional studies indicate that ion channels of the transient receptor potential and degenerin/epithelial Na+ channel families are likely to be transduction channels. The expression of related channels in mammalian somatosensory neurons has fueled the notion that these channels mediate mechanotransduction in vertebrates; however, genetic disruption and heterologous expression have not yet revealed a direct role for any of these candidates in somatosensory mechanotransduction. Thus, new systems are needed to define the function of these ion channels in somatosensation and to pinpoint molecules or signaling pathways that underlie mechanotransduction in vertebrates. PMID:19683913

  12. Cellular and chemical neuroscience of mammalian sleep.

    PubMed

    Datta, Subimal

    2010-05-01

    Extraordinary strides have been made toward understanding the complexities and regulatory mechanisms of sleep over the past two decades thanks to the help of rapidly evolving technologies. At its most basic level, mammalian sleep is a restorative process of the brain and body. Beyond its primary restorative purpose, sleep is essential for a number of vital functions. Our primary research interest is to understand the cellular and molecular mechanisms underlying the regulation of sleep and its cognitive functions. Here I will reflect on our own research contributions to 50 years of extraordinary advances in the neurobiology of slow-wave sleep (SWS) and rapid eye movement (REM) sleep regulation. I conclude this review by suggesting some potential future directions to further our understanding of the neurobiology of sleep.

  13. Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

    PubMed

    Champroux, A; Torres-Carreira, J; Gharagozloo, P; Drevet, J R; Kocer, A

    2016-01-01

    Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

  14. Global Epigenomic Reconfiguration During Mammalian Brain Development

    PubMed Central

    Nery, Joseph R.; Urich, Mark; Puddifoot, Clare A.; Johnson, Nicholas D.; Lucero, Jacinta; Huang, Yun; Dwork, Andrew J.; Schultz, Matthew D.; Yu, Miao; Tonti-Filippini, Julian; Heyn, Holger; Hu, Shijun; Wu, Joseph C.; Rao, Anjana; Esteller, Manel; He, Chuan; Haghighi, Fatemeh G.; Sejnowski, Terrence J.; Behrens, M. Margarita; Ecker, Joseph R.

    2013-01-01

    DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity. PMID:23828890

  15. Redox signaling during hypoxia in mammalian cells.

    PubMed

    Smith, Kimberly A; Waypa, Gregory B; Schumacker, Paul T

    2017-10-01

    Hypoxia triggers a wide range of protective responses in mammalian cells, which are mediated through transcriptional and post-translational mechanisms. Redox signaling in cells by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) occurs through the reversible oxidation of cysteine thiol groups, resulting in structural modifications that can change protein function profoundly. Mitochondria are an important source of ROS generation, and studies reveal that superoxide generation by the electron transport chain increases during hypoxia. Other sources of ROS, such as the NAD(P)H oxidases, may also generate oxidant signals in hypoxia. This review considers the growing body of work indicating that increased ROS signals during hypoxia are responsible for regulating the activation of protective mechanisms in diverse cell types. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Engineered Trehalose Permeable to Mammalian Cells.

    PubMed

    Abazari, Alireza; Meimetis, Labros G; Budin, Ghyslain; Bale, Shyam Sundhar; Weissleder, Ralph; Toner, Mehmet

    2015-01-01

    Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

  17. Scaling of the mammalian middle ear.

    PubMed

    Nummela, S

    1995-05-01

    This study considers the general question how animal size limits the size and information receiving capacity of sense organs. To clarify this in the case of the mammalian middle ear, I studied 63 mammalian species, ranging from a small bat to the Indian elephant. I determined the skull mass and the masses of the ossicles malleus, incus and stapes (M, I and S), and measured the tympanic membrane area, A1. The ossicular mass (in mg) is generally negatively allometric to skull mass (in g), the regression equation for the whole material (excluding true seals) being y = 1.373 x(0.513). However, for very small mammals the allometry approaches isometry. Within a group of large mammals no distinct allometry can be discerned. The true seals (Phocidae) are exceptional by having massive ossicles. The size relations within the middle ear are generally rather constant. However, the I/M relation is slightly positively allometric, y = 0.554 x(1.162). Two particularly isometric relations were found; the S/(M + I) relation for the ossicles characterized by the regression equation y = 0.054 x(0.993), and the relation between a two-dimensional measure of the ossicles and the tympanic membrane ares, (M + I)2/3 /A1. As in isometric ears the sound energy collected by the tympanic membrane is linearly related to its area, the latter isometry suggests that, regardless of animal size, a given ossicular cross-sectional area is exposed to a similar sound-induced stress. Possible morphological middle ear adaptations to particular acoustic environments are discussed.

  18. Cortical pathways to the mammalian amygdala.

    PubMed

    McDonald, A J

    1998-06-01

    The amygdaloid nuclear complex is critical for producing appropriate emotional and behavioral responses to biologically relevant sensory stimuli. It constitutes an essential link between sensory and limbic areas of the cerebral cortex and subcortical brain regions, such as the hypothalamus, brainstem, and striatum, that are responsible for eliciting emotional and motivational responses. This review summarizes the anatomy and physiology of the cortical pathways to the amygdala in the rat, cat and monkey. Although the basic anatomy of these systems in the cat and monkey was largely delineated in studies conducted during the 1970s and 1980s, detailed information regarding the cortico-amygdalar pathways in the rat was only obtained in the past several years. The purpose of this review is to describe the results of recent studies in the rat and to compare the organization of cortico-amygdalar projections in this species with that seen in the cat and monkey. In all three species visual, auditory, and somatosensory information is transmitted to the amygdala by a series of modality-specific cortico-cortical pathways ("cascades") that originate in the primary sensory cortices and flow toward higher order association areas. The cortical areas in the more distal portions of these cascades have stronger and more extensive projections to the amygdala than the more proximal areas. In all three species olfactory and gustatory/visceral information has access to the amygdala at an earlier stage of cortical processing than visual, auditory and somatosensory information. There are also important polysensory cortical inputs to the mammalian amygdala from the prefrontal and hippocampal regions. Whereas the overall organization of cortical pathways is basically similar in all mammalian species, there is anatomical evidence which suggests that there are important differences in the extent of convergence of cortical projections in the primate versus the nonprimate amygdala.

  19. Identification of mammalian orthologs using local synteny

    PubMed Central

    2009-01-01

    Background Accurate determination of orthology is central to comparative genomics. For vertebrates in particular, very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make inference of orthology between genes difficult. Many methods have been developed to identify orthologous genes, mostly based upon analysis of the inferred protein sequence of the genes. More recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment in vertebrates. Such methods have been most successfully implemented in fungal genomes and have long been used in prokaryotic genomes, where gene order is far less variable than in vertebrates. However, to our knowledge, no explicit comparison of synteny and sequence based definitions of orthology has been reported in vertebrates, or, more specifically, in mammals. Results We test a simple method for the measurement and utilization of gene order (local synteny) in the identification of mammalian orthologs by investigating the agreement between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny is a robust substitute to coding sequence for identifying orthologs. However, 7% of pairs were found to be discordant between local synteny and Inparanoid. These cases of discordance result from evolutionary events including retrotransposition and genome rearrangements. Conclusions By analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and might be used to identify cases of non-orthologous gene

  20. Defining the mammalian CArGome

    PubMed Central

    Sun, Qiang; Chen, Guang; Streb, Jeffrey W.; Long, Xiaochun; Yang, Yumei; Stoeckert, Christian J.; Miano, Joseph M.

    2006-01-01

    Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton. PMID:16365378

  1. An Analytical Study of Mammalian Bite Wounds Requiring Inpatient Management

    PubMed Central

    Lee, Young-Geun; Kim, Woo-Kyung

    2013-01-01

    Background Mammalian bite injuries create a public health problem because of their frequency, potential severity, and increasing number. Some researchers have performed fragmentary analyses of bite wounds caused by certain mammalian species. However, little practical information is available concerning serious mammalian bite wounds that require hospitalization and intensive wound management. Therefore, the purpose of this study was to perform a general review of serious mammalian bite wounds. Methods We performed a retrospective review of the medical charts of 68 patients who were referred to our plastic surgery department for the treatment of bite wounds between January 2003 and October 2012. The cases were analyzed according to the species, patient demographics, environmental factors, injury characteristics, and clinical course. Results Among the 68 cases of mammalian bite injury, 58 (85%) were caused by dogs, 8 by humans, and 2 by cats. Most of those bitten by a human and both of those bitten by cats were male. Only one-third of all the patients were children or adolescents. The most frequent site of injury was the face, with 40 cases, followed by the hand, with 16 cases. Of the 68 patients, 7 were treated with secondary intention healing. Sixty-one patients underwent delayed procedures, including delayed direct closure, skin graft, composite graft, and local flap. Conclusions Based on overall findings from our review of the 68 cases of mammalian bites, we suggest practical guidelines for the management of mammalian bite injuries, which could be useful in the treatment of serious mammalian bite wounds. PMID:24286042

  2. Adult Neurogenesis in the Mammalian Hippocampus: Why the Dentate Gyrus?

    ERIC Educational Resources Information Center

    Drew, Liam J.; Fusi, Stefano; Hen, René

    2013-01-01

    In the adult mammalian brain, newly generated neurons are continuously incorporated into two networks: interneurons born in the subventricular zone migrate to the olfactory bulb, whereas the dentate gyrus (DG) of the hippocampus integrates locally born principal neurons. That the rest of the mammalian brain loses significant neurogenic capacity…

  3. Adult Neurogenesis in the Mammalian Hippocampus: Why the Dentate Gyrus?

    ERIC Educational Resources Information Center

    Drew, Liam J.; Fusi, Stefano; Hen, René

    2013-01-01

    In the adult mammalian brain, newly generated neurons are continuously incorporated into two networks: interneurons born in the subventricular zone migrate to the olfactory bulb, whereas the dentate gyrus (DG) of the hippocampus integrates locally born principal neurons. That the rest of the mammalian brain loses significant neurogenic capacity…

  4. Rabconnectin-3 is a functional regulator of mammalian Notch signaling.

    PubMed

    Sethi, Nilay; Yan, Yan; Quek, Debra; Schupbach, Trudi; Kang, Yibin

    2010-11-05

    The Notch signaling pathway is important for cell fate decisions in embryonic development and adult life. Defining the functional importance of the Notch pathway in these contexts requires the elucidation of essential signal transduction components that have not been fully characterized. Here, we show that Rabconnectin-3B is required for the Notch pathway in mammalian cells. siRNA-mediated silencing of Rabconnectin-3B in mammalian cells attenuated Notch signaling and disrupted the activation and nuclear accumulation of the Notch target Hes1. Rabconnectin-3B knockdown also disrupted V-ATPase activity in mammalian cells, consistent with previous observations in Drosophila. Pharmacological inhibition of the V-ATPase complex significantly reduced Notch signaling in mammalian cells. Finally, Rabconnectin-3B knockdown phenocopied functional disruption of Notch signaling during osteoclast differentiation. Collectively, these findings define an important role for Rabconnectin-3 and V-ATPase activity in the Notch signaling pathway in mammalian cells.

  5. USE OF NON-MAMMALIAN ALTERNATIVE MODELS FOR NEUROTOXICOLOGICAL STUDY

    PubMed Central

    Peterson, Randall T.; Nass, Richard; Boyd, Windy A.; Freedman, Jonathan H.; Dong, Ke; Narahashi, Toshio

    2009-01-01

    The field of neurotoxicology needs to satisfy two opposing demands: the testing of a growing list of chemicals, and resource limitations and ethical concerns associated with testing using traditional mammalian species. National and international government agencies have defined a need to reduce, refine or replace mammalian species in toxicological testing with alternative testing methods and non-mammalian models. Toxicological assays using alternative animal models may relieve some of this pressure by allowing testing of more compounds while reducing expense and using fewer mammals. Recent advances in genetic technologies and the strong conservation between human and non-mammalian genomes allows for the dissection of the molecular pathways involved in neurotoxicological responses and neurological diseases using genetically tractable organisms. In this review, applications of four non-mammalian species, Zebrafish, cockroach, Drosophila, and Caenorhabditis elegans, in the investigation of neurotoxicology and neurological diseases are presented. PMID:18538410

  6. Retinal research using the perfused mammalian eye.

    PubMed

    Niemeyer, G

    2001-05-01

    The effort to isolate and maintain alive in vitro an intact mammalian eye is rewarded by the full control provided over the arterial input and exclusion of systemic regulatory or compensatory mechanisms. Electrical recording of typical light-evoked field potentials from retina and optic nerve can be complemented by single-cell recording. Thus, light-induced electrical activity reflects the function of the retinal pigment epithelium, of the layers of the retina and of the ganglion cells or their axons. Retinal function in vitro is documented by electrophysiological and morphological methods revealing subtle features of retinal information processing as well as optic nerve signals that approach-at threshold stimulus intensity-the human psychophysical threshold. Such sensitivity of third-order retinal neurons is described for the first time. This well controlled in vitro preparation has been used successfully for biophysical, metabolic and pharmacological studies. Examples are provided that demonstrate the marked sensibility of the rod system to changes in glucose supply. Moreover, histochemical identification of glycogen stores revealed labeling of the second- and third-order neurons subserving the rod system, in addition to labeling of Müller (glial) cells in the cat retina. The glycogen content of the cat retina is augmented by prolonged anesthesia, largely depleted by ischemia after enucleation and enhanced by insulin. Pharmacological experiments using agonists and antagonists of putative retinal neurotransmitters are summarized and outlined using the muscarinic cholinergic agonist QNB as an example. Actions and uptake of the neuromodulator adenosine are presented in detail, including inhibitory effects on physiologically characterized ganglion cells. Neuronal effects of adenosine are distinguished from those resulting from vasodilatation and from glycogenolysis induced by the neuromodulator. To open the blood-retina barrier, a hyperosmotic challenge can be

  7. The non-mammalian MIF superfamily.

    PubMed

    Sparkes, Amanda; De Baetselier, Patrick; Roelants, Kim; De Trez, Carl; Magez, Stefan; Van Ginderachter, Jo A; Raes, Geert; Bucala, Richard; Stijlemans, Benoît

    2017-03-01

    Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, ‎protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance.

  8. Mammalian prion amyloid formation in bacteria

    PubMed Central

    Macedo, Bruno; Cordeiro, Yraima; Ventura, Salvador

    2016-01-01

    ABSTRACT Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrPSc), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrPSc is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility. PMID:26910379

  9. Programmed cell senescence during mammalian embryonic development.

    PubMed

    Muñoz-Espín, Daniel; Cañamero, Marta; Maraver, Antonio; Gómez-López, Gonzalo; Contreras, Julio; Murillo-Cuesta, Silvia; Rodríguez-Baeza, Alfonso; Varela-Nieto, Isabel; Ruberte, Jesús; Collado, Manuel; Serrano, Manuel

    2013-11-21

    Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Angiogenesis is inhibitory for mammalian digit regeneration

    PubMed Central

    Yu, Ling; Yan, Mingquan; Simkin, Jennifer; Ketcham, Paulina D.; Leininger, Eric; Han, Manjong

    2014-01-01

    Abstract The regenerating mouse digit tip is a unique model for investigating blastema formation and epimorphic regeneration in mammals. The blastema is characteristically avascular and we previously reported that blastema expression of a known anti‐angiogenic factor gene, Pedf, correlated with a successful regenerative response (Yu, L., Han, M., Yan, M., Lee, E. C., Lee, J. & Muneoka, K. (2010). BMP signaling induces digit regeneration in neonatal mice. Development, 137, 551–559). Here we show that during regeneration Vegfa transcripts are not detected in the blastema but are expressed at the onset of differentiation. Treating the amputation wound with vascular endothelial growth factor enhances angiogenesis but inhibits regeneration. We next tested bone morphogenetic protein 9 (BMP9), another known mediator of angiogenesis, and found that BMP9 is also a potent inhibitor of digit tip regeneration. BMP9 induces Vegfa expression in the digit stump suggesting that regenerative failure is mediated by enhanced angiogenesis. Finally, we show that BMP9 inhibition of regeneration is completely rescued by treatment with pigment epithelium‐derived factor. These studies show that precocious angiogenesis is inhibitory for regeneration, and provide compelling evidence that the regulation of angiogenesis is a critical factor in designing therapies aimed at stimulating mammalian regeneration. PMID:27499862

  11. Myocardial ischemic protection in natural mammalian hibernation.

    PubMed

    Yan, Lin; Kudej, Raymond K; Vatner, Dorothy E; Vatner, Stephen F

    2015-03-01

    Hibernating myocardium is an important clinical syndrome protecting the heart with chronic myocardial ischemia, named for its assumed resemblance to hibernating mammals in winter. However, the effects of myocardial ischemic protection have never been studied in true mammalian hibernation, which is a unique strategy for surviving extreme winter environmental stress. The goal of this investigation was to test the hypothesis that ischemic stress may also be protected in woodchucks as they hibernate in winter. Myocardial infarction was induced by coronary occlusion followed by reperfusion in naturally hibernating woodchucks in winter with and without hibernation and in summer, when not hibernating. The ischemic area at risk was similar among groups. Myocardial infarction was significantly less in woodchucks in winter, whether hibernating or not, compared with summer, and was similar to that resulting after ischemic preconditioning. Whereas several genes were up or downregulated in both hibernating woodchuck and with ischemic preconditioning, one mechanism was unique to hibernation, i.e., activation of cAMP-response element binding protein (CREB). When CREB was upregulated in summer, it induced protection similar to that observed in the woodchuck heart in winter. The cardioprotection in hibernation was also mediated by endothelial nitric oxide synthase, rather than inducible nitric oxide synthase. Thus, the hibernating woodchuck heart is a novel model to study cardioprotection for two major reasons: (1) powerful cardioprotection occurs naturally in winter months in the absence of any preconditioning stimuli, and (2) it resembles ischemic preconditioning, but with novel mechanisms, making this model potentially useful for clinical translation.

  12. Nitric oxide negatively regulates mammalian adult neurogenesis

    NASA Astrophysics Data System (ADS)

    Packer, Michael A.; Stasiv, Yuri; Benraiss, Abdellatif; Chmielnicki, Eva; Grinberg, Alexander; Westphal, Heiner; Goldman, Steven A.; Enikolopov, Grigori

    2003-08-01

    Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system.

  13. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  14. A synthetic low-frequency mammalian oscillator

    PubMed Central

    Tigges, Marcel; Dénervaud, Nicolas; Greber, David; Stelling, Joerg; Fussenegger, Martin

    2010-01-01

    Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives. PMID:20197318

  15. Ventricular Fibrillation in Mammalian Hearts: Simulation Results

    NASA Astrophysics Data System (ADS)

    Fenton, Flavio H.

    2002-03-01

    The computational approach to understanding the initiation and evolution of cardiac arrhythmias forms a necessary link between experiment and theory. Numerical simulations combine useful mathematical models and complex geometry while offering clean and comprehensive data acquisition, reproducible results that can be compared to experiments, and the flexibility of exploring parameter space systematically. However, because cardiac dynamics occurs on many scales (on the order of 10^9 cells of size 10-100 microns with more than 40 ionic currents and time scales as fast as 0.01ms), roughly 10^17 operations are required to simulate just one second of real time. These intense computational requirements lead to significant implementation challenges even on existing supercomputers. Nevertheless, progress over the last decade in understanding the effects of some spatial scales and spatio-temporal dynamics on cardiac cell and tissue behavior justifies the use of certain simplifications which, along with improved models for cellular dynamics and detailed digital models of cardiac anatomy, are allowing simulation studies of full-size ventricles and atria. We describe this simulation problem from a combined numerical, physical and biological point of view, with an emphasis on the dynamics and stability of scroll waves of electrical activity in mammalian hearts and their relation to tachycardia, fibrillation and sudden death. Detailed simulations of electrical activity in ventricles including complex anatomy, anisotropic fiber structure, and electrophysiological effects of two drugs (DAM and CytoD) are presented and compared with experimental results.

  16. Movement Symmetries and the Mammalian Vestibular System

    NASA Astrophysics Data System (ADS)

    McCollum, Gin; Boyle, Richard

    2000-03-01

    Unity of movement requires vertebrates to have an ability to symmetrize along the midline. For example, human erect stance involves symmetry with respect to gravity. The mammalian vestibular system provides a mechanism for maintaining symmetries, which is also open to influence and adaptation by the rest of the organism. The vestibular system includes the inner ear endorgans and central nuclei, along with projections to oculomotor, cerebellar, thalamic, and spinal motor centers. The vestibular endorgans - the semicircular canals and the otoliths - use sensory hairs to register inertia. The vestibular endorgans are right-left symmetric and the semicircular canals form an approximately orthogonal coordinate system for angular motion. Primary afferent axons project from the endorgans to the vestibular nuclei (and a few other places). The vestibular nuclei integrate vestibular, visual, and somatosensory signals, along with a proposed copy of the voluntary motor command and signals from other central structures. The relationship between the canals and the otoliths gives rise to symmetries among neurons, in the organization among the several vestibular nuclei, and in the projections from the vestibular nuclei. These symmetries organize the space of body movements so that functional relationships are maintained in spite of the many free variables of body movement. They also provide a foundation for adaptive reinterpretation of the relationship between canal and otolith signals, for example in freefall.

  17. Multiple Functions and Regulation of Mammalian Peroxiredoxins.

    PubMed

    Rhee, Sue Goo; Kil, In Sup

    2017-06-20

    Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

  18. Tubulin dynamics in cultured mammalian cells

    PubMed Central

    1984-01-01

    Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells. PMID:6501419

  19. Activation of mammalian tyrosinase by ferrous ions.

    PubMed

    Palumbo, A; d'Ischia, M; Misuraca, G; Carratú, L; Prota, G

    1990-03-26

    Kinetic experiments are reported showing that mammalian tyrosinase from B16 mouse melanoma is significantly activated by catalytic amounts of ferrous ions. Monitoring of tyrosine oxidation by both dopachrome formation and oxygen consumption showed that ferrous ions at micromolar concentrations induce a marked enzymatic activity with 0.01 U/ml of highly purified tyrosinase, whereas no detectable reaction occurs in the absence of metal over a sufficiently prolonged period of time. The extent of the activating effect, which is specific for the reduced form of iron, is proportional to the concentration of the added metal with a typical saturation profile, no further effect being observed beyond a threshold value. Changing the buffer system from phosphate to hepes or tris results in a marked decrease of the Fe2(+)-induced activation. Scavengers of active oxygen species, such as superoxide dismutase, catalase, formate and mannitol have no detectable effect on the tyrosinase activity. These results are accounted for in terms of an activation mechanism involving reduction of the cupric ions at the active site of the resting enzyme.

  20. Melanopsin signalling in mammalian iris and retina.

    PubMed

    Xue, T; Do, M T H; Riccio, A; Jiang, Z; Hsieh, J; Wang, H C; Merbs, S L; Welsbie, D S; Yoshioka, T; Weissgerber, P; Stolz, S; Flockerzi, V; Freichel, M; Simon, M I; Clapham, D E; Yau, K-W

    2011-11-02

    Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

  1. Mammalian cyanide detoxification with sulphane sulphur.

    PubMed

    Westley, J

    1988-01-01

    It has been understood for nearly a century that most of the cyanide entering mammals by either ingestion or inhalation of sublethal quantities is detoxified by combination with cyanide-reactive sulphur. The detoxifying reaction is primarily enzymic, and the enzymes that catalyse it, as well as those that catalyse reactions which generate the cyanide-reactive sulphane sulphur, have been isolated and studied extensively. However, analytical methods suitable for quantitative characterization of the physiological pool of sulphur available for this detoxification have only recently come under development. A new method is based on the evaluation of cyanide depletion under controlled, mild reaction conditions in the presence of a sulphurtransferase and cofactor. The use of differential pulse polarography with nested internal standards makes this a practical procedure for samples containing as little as a few nanomoles of cyanide-reactive material. As measured by this method, the sulphane sulphur concentration in mammalian liver tissue is in the fractional millimolar range, with even less in the bloodstream. Nevertheless, the specific binding of sulphane sulphur by serum albumin, and the rapid cyanolysis of the albumin-bound sulphur, may yet indicate a significant role for the sulphurtransferase capability of serum albumin in cyanide detoxification.

  2. Cholesterol, the central lipid of mammalian cells

    PubMed Central

    Maxfield, Frederick R.; van Meer, Gerrit

    2010-01-01

    Summary of recent advances Despite its importance for mammalian cell biology and human health, there are many basic aspects of cholesterol homeostasis that are not well understood. Even for the well-characterized delivery of cholesterol to cells via lipoproteins, a novel regulatory mechanism has been discovered recently, involving a serum protein called PCSK9, which profoundly affects lipoproteins and their receptors. Cells can export cholesterol by processes that require the activity of ABC transporters, but the molecular mechanisms for cholesterol transport remain unclear. Cholesterol levels in different organelles vary by 5–10 fold, and the mechanisms for maintaining these differences are now partially understood. Several proteins have been proposed to play a role in the inter-organelle movement of cholesterol, but many aspects of the mechanisms for regulating intracellular transport and distribution of cholesterol remain to be worked out. The endoplasmic reticulum is the main organelle responsible for regulation of cholesterol synthesis, and careful measurements have shown that the proteins responsible for sterol sensing respond over a very narrow range of cholesterol concentrations to provide very precise, switch-like control over cholesterol synthesis. PMID:20627678

  3. Cell fate regulation in early mammalian development

    NASA Astrophysics Data System (ADS)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  4. Mitochondrial dynamics in mammalian health and disease.

    PubMed

    Liesa, Marc; Palacín, Manuel; Zorzano, Antonio

    2009-07-01

    The meaning of the word mitochondrion (from the Greek mitos, meaning thread, and chondros, grain) illustrates that the heterogeneity of mitochondrial morphology has been known since the first descriptions of this organelle. Such a heterogeneous morphology is explained by the dynamic nature of mitochondria. Mitochondrial dynamics is a concept that includes the movement of mitochondria along the cytoskeleton, the regulation of mitochondrial architecture (morphology and distribution), and connectivity mediated by tethering and fusion/fission events. The relevance of these events in mitochondrial and cell physiology has been partially unraveled after the identification of the genes responsible for mitochondrial fusion and fission. Furthermore, during the last decade, it has been identified that mutations in two mitochondrial fusion genes (MFN2 and OPA1) cause prevalent neurodegenerative diseases (Charcot-Marie Tooth type 2A and Kjer disease/autosomal dominant optic atrophy). In addition, other diseases such as type 2 diabetes or vascular proliferative disorders show impaired MFN2 expression. Altogether, these findings have established mitochondrial dynamics as a consolidated area in cellular physiology. Here we review the most significant findings in the field of mitochondrial dynamics in mammalian cells and their implication in human pathologies.