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Sample records for ajuba lim proteins

  1. The LIM Protein Ajuba Restricts the Second Heart Field Progenitor Pool by Regulating Isl1 Activity

    PubMed Central

    Witzel, Hagen R.; Jungblut, Benno; Choe, Chong Pyo; Crump, J. Gage; Braun, Thomas; Dobreva, Gergana

    2013-01-01

    SUMMARY Morphogenesis of the heart requires tight control of cardiac progenitor cell specification, expansion, and differentiation. Retinoic acid (RA) signaling restricts expansion of the second heart field (SHF), serving as an important morphogen in heart development. Here, we identify the LIM domain protein Ajuba as a crucial regulator of the SHF progenitor cell specification and expansion. Ajuba-deficient zebra-fish embryos show an increased pool of Isl1+ cardiac progenitors and, subsequently, dramatically increased numbers of cardiomyocytes at the arterial and venous poles. Furthermore, we show that Ajuba binds Isl1, represses its transcriptional activity, and is also required for autorepression of Isl1 expression in an RA-dependent manner. Lack of Ajuba abrogates the RA-dependent restriction of Isl1+ cardiac cells. We conclude that Ajuba plays a central role in regulating the SHF during heart development by linking RA signaling to the function of Isl1, a key transcription factor in cardiac progenitor cells. PMID:22771034

  2. AJUBA LIM Proteins Limit Hippo Activity in Proliferating Cells by Sequestering the Hippo Core Kinase Complex in the Cytosol

    PubMed Central

    Jagannathan, Radhika; Schimizzi, Gregory V.; Zhang, Kun; Loza, Andrew J.; Yabuta, Norikazu; Nojima, Hitoshi

    2016-01-01

    The Hippo pathway controls organ growth and is implicated in cancer development. Whether and how Hippo pathway activity is limited to sustain or initiate cell growth when needed is not understood. The members of the AJUBA family of LIM proteins are negative regulators of the Hippo pathway. In mammalian epithelial cells, we found that AJUBA LIM proteins limit Hippo regulation of YAP, in proliferating cells only, by sequestering a cytosolic Hippo kinase complex in which LATS kinase is inhibited. At the plasma membranes of growth-arrested cells, AJUBA LIM proteins do not inhibit or associate with the Hippo kinase complex. The ability of AJUBA LIM proteins to inhibit YAP regulation by Hippo and to associate with the kinase complex directly correlate with their capacity to limit Hippo signaling during Drosophila wing development. AJUBA LIM proteins did not influence YAP activity in response to cell-extrinsic or cell-intrinsic mechanical signals. Thus, AJUBA LIM proteins limit Hippo pathway activity in contexts where cell proliferation is needed. PMID:27457617

  3. AJUBA promotes the migration and invasion of esophageal squamous cell carcinoma cells through upregulation of MMP10 and MMP13 expression.

    PubMed

    Shi, Xuejiao; Chen, Zhaoli; Hu, Xueda; Luo, Mei; Sun, Zengmiao; Li, Jiagen; Shi, Susheng; Feng, Xiaoli; Zhou, Chengcheng; Li, Zitong; Yang, Wenhui; Li, Yuan; Wang, Pan; Zhou, Fang; Gao, Yibo; He, Jie

    2016-06-14

    The LIM-domain protein AJUBA has been reported to be involved in cell-cell adhesion, proliferation, migration and cell fate decision by acting as a scaffold or adaptor protein. We previously identified AJUBA as a putative cancer gene in esophageal squamous cell carcinoma (ESCC). However, the function and underlying mechanisms of AJUBA in ESCC remain largely unknown. In the present study, we detected AJUBA levels in ESCC tumor tissues and in corresponding adjacent non-tumor tissues by immunohistochemistry (IHC) and investigated the function and mechanism of AJUBA in ESCC cells. The IHC results showed that AJUBA levels were significantly higher in ESCC tissues compared with corresponding adjacent non-tumor tissues (P < 0.001). Both in vitro and in vivo experiments showed that AJUBA promoted cell growth and colony formation, inhibited cisplatin-induced apoptosis of ESCC cells, and promoted ESCC cell migration and invasion. RNA sequencing was used to reveal the oncogenic pathways of AJUBA that were involved, and MMP10 and MMP13 were identified as two of the downstream targets of AJUBA. Thus, AJUBA upregulates the levels of MMP10 and MMP13 by activating ERK1/2. Taken together, these findings revealed that AJUBA serves as oncogenic gene in ESCC and may serve as a new target for ESCC therapy.

  4. Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

    PubMed Central

    Jurata, L W; Kenny, D A; Gill, G N

    1996-01-01

    LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876198

  5. Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity.

    PubMed Central

    Johnson, J D; Zhang, W; Rudnick, A; Rutter, W J; German, M S

    1997-01-01

    LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues. PMID:9199284

  6. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

  7. Nucleo-cytoplasmic functions of the PDZ-LIM protein family: new insights in organ development

    PubMed Central

    Krcmery, Jennifer; Camarata, Troy; Kulisz, Andre; Simon, Hans-Georg

    2010-01-01

    Summary Recent work on the PDZ-LIM protein family has revealed important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ-LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ-LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ-LIM proteins have wide-ranging and multi-compartmental cell functions during development and homeostasis while, in contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T-box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ-LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture and control gene transcription. PMID:20091751

  8. The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

    PubMed

    Kim, Jinmahn; Yeon, Jihye; Choi, Seong-Kyoon; Huh, Yang Hoon; Fang, Zi; Park, Seo Jin; Kim, Myoung Ok; Ryoo, Zae Young; Kang, Kyeongjin; Kweon, Hee-Seok; Jeon, Won Bae; Li, Chris; Kim, Kyuhyung

    2015-08-01

    The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

  9. The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type

    PubMed Central

    Choi, Seong-Kyoon; Huh, Yang Hoon; Fang, Zi; Park, Seo Jin; Kim, Myoung Ok; Ryoo, Zae Young; Kang, Kyeongjin; Kweon, Hee-Seok; Jeon, Won Bae; Li, Chris; Kim, Kyuhyung

    2015-01-01

    The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes. PMID:26305787

  10. A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity

    PubMed Central

    Xu, Xiaojie; Fan, Zhongyi; Liang, Chaoyang; Li, Ling; Wang, Lili; Liang, Yingchun; Wu, Jun; Chang, Shaohong; Yan, Zhifeng; Lv, Zhaohui; Fu, Jing; Liu, Yang; Jin, Shuai; Wang, Tao; Hong, Tian; Dong, Yishan; Ding, Lihua; Cheng, Long; Liu, Rui; Fu, Shenbo; Jiao, Shunchang; Ye, Qinong

    2017-01-01

    Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2–CDC25 and CDC25–14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity. PMID:28094252

  11. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  12. The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

    PubMed Central

    Guy, Pamela M.; Kenny, Daryn A.; Gill, Gordon N.

    1999-01-01

    PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein α-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal β-TM). The interaction between Enigma and skeletal β-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal β-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal β-TM in transfected cells. The association of Enigma with skeletal β-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells. PMID:10359609

  13. ALP/Enigma PDZ-LIM domain proteins in the heart.

    PubMed

    Zheng, Ming; Cheng, Hongqiang; Banerjee, Indroneal; Chen, Ju

    2010-04-01

    Actinin-associated LIM protein (ALP) and Enigma are two subfamilies of Postsynaptic density 95, discs large and zonula occludens-1 (PDZ)-Lin-11, Isl1 and Mec-3 (LIM) domain containing proteins. ALP family members have one PDZ and one LIM domain, whereas Enigma proteins contain one PDZ and three LIM domains. Four ALP and three Enigma proteins have been identified in mammals, each having multiple splice variants and unique expression patterns. Functionally, these proteins bind through their PDZ domains to alpha-actinin and bind through their LIM domains or other internal protein interaction domains to other proteins, including signaling molecules. ALP and Enigma proteins have been implicated in cardiac and skeletal muscle structure, function and disease, neuronal function, bipolar disorder, tumor growth, platelet and epithelial cell motility and bone formation. This review will focus on recent advances in the biological roles of ALP/Enigma PDZ-LIM domain proteins in cardiac muscle and provide insights into mechanisms by which mutations in these proteins are related to human cardiac disease.

  14. Actinin-associated LIM protein-deficient mice maintain normal development and structure of skeletal muscle.

    PubMed

    Jo, K; Rutten, B; Bunn, R C; Bredt, D S

    2001-03-01

    The actinin-associated LIM protein, ALP, is the prototype of a large family of proteins containing an N-terminal PDZ domain and a C-terminal LIM domain. These PDZ-LIM proteins are components of the muscle cytoskeleton and occur along the Z lines owing to interaction of the PDZ domain with the spectrin-like repeats of alpha-actinin. Because PDZ and LIM domains are typically found in proteins that mediate cellular signaling, PDZ-LIM proteins are suspected to participate in muscle development. Interestingly the ALP gene occurs at 4q35 near the heterochromatic region mutated in facioscapulohumeral muscular dystrophy, indicating a possible role for ALP in this disease. Here, we describe the generation and analysis of mice lacking the ALP gene. Surprisingly, the ALP knockout mice show no gross histological abnormalities and maintain sarcolemmal integrity as determined by serum pyruvate kinase assays. The absence of a dystrophic phenotype in these mice suggests that down-regulation of ALP does not participate in facioscapulohumeral muscular dystrophy. These data suggest that ALP does not participate in muscle development or that an alternative PDZ-LIM protein can compensate for the lack of ALP.

  15. The LIM motif defines a specific zinc-binding protein domain.

    PubMed

    Michelsen, J W; Schmeichel, K L; Beckerle, M C; Winge, D R

    1993-05-15

    The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

  16. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton

    PubMed Central

    1992-01-01

    Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847- 5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2- terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine- rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene

  17. A novel muscle LIM-only protein is generated from the paxillin gene locus in Drosophila

    PubMed Central

    Yagi, Ryohei; Ishimaru, Satoshi; Yano, Hajime; Gaul, Ulrike; Hanafusa, Hidesaburo; Sabe, Hisataka

    2001-01-01

    Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musclature, largely utilizing the Paxillin LIM domains. PMID:11520860

  18. Molecular characterization of a PDZ-LIM protein in Atlantic salmon (Salmo salar): a fish ortholog of the alpha-actinin-associated LIM-protein (ALP).

    PubMed

    Andersen, Øivind; Østbye, Tone-Kari; Gabestad, Irene; Nielsen, Christer; Bardal, Tora; Galloway, Trina Falck

    2004-01-01

    A protein containing both PDZ and LIM protein-protein interaction motifs has for the first time been identified in a lower vertebrate species. A full-length cDNA encoding the ortholog of the alpha-actinin-associated LIM protein (ALP) was isolated from white skeletal muscle of Atlantic salmon (Salmo salar). Whereas ALP is expressed as two muscle specific isoforms in mammals and chicken as the result of alternative splicing, a single ALP transcript was found in both muscle and non-muscular tissues of Atlantic salmon. On the other hand, Western blot analysis revealed several immunoreactive ALP variants in salmon muscle tissues, including a 45 kDa protein in white and red skeletal muscle and a 37-40 kDa protein in heart and smooth muscle. Salmon ALP and alpha-actinin showed similar striated patterns in serial longitudinal sections of white and red skeletal muscle and heart muscle. Expression of ALP was initiated at the 45-somite stage of the salmon embryogenesis contemporary with the first appearance of alpha-actinin transcripts. The similarities in both the spatial and temporal expression patterns of salmon ALP and alpha-actinin strongly indicate that the two proteins are associated as in higher vertebrates, and that the assumed involvement of ALP in the organization and/or maintenance of the Z-lines in striated muscle has been conserved during vertebrate evolution. However, in contrast to the restricted expression of ALP in higher vertebrates, the ubiquitous expression of salmon ALP suggest that this factor is involved in the assembly of additional multi-protein complexes in fish.

  19. Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.

    PubMed

    Greening, David W; Ji, Hong; Kapp, Eugene A; Simpson, Richard J

    2013-11-01

    Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6

  20. Regulation of myocardin factor protein stability by the LIM-only protein FHL2

    PubMed Central

    Hinson, Jeremiah S.; Medlin, Matt D.; Taylor, Joan M.; Mack, Christopher P.

    2008-01-01

    Extensive evidence indicates that serum response factor (SRF) regulates muscle-specific gene expression and that myocardin family SRF cofactors are critical for smooth muscle cell differentiation. In a yeast two hybrid screen for novel SRF binding partners expressed in aortic SMC, we identified four and a half LIM domain protein 2 (FHL2) and confirmed this interaction by GST pull-down and coimmunoprecipitation assays. FHL2 also interacted with all three myocardin factors and enhanced myocardin and myocardin-related transcription factor (MRTF)-A-dependent transactivation of smooth muscle α-actin, SM22, and cardiac atrial natriuretic factor promoters in 10T1/2 cells. The expression of FHL2 increased myocardin and MRTF-A protein levels, and, importantly, this effect was due to an increase in protein stability not due to an increase in myocardin factor mRNA expression. Treatment of cells with proteasome inhibitors MG-132 and lactacystin strongly upregulated endogenous MRTF-A protein levels and resulted in a substantial increase in ubiquitin immunoreactivity in MRTF-A immunoprecipitants. Interestingly, the expression of FHL2 attenuated the effects of RhoA and MRTF-B on promoter activity, perhaps through decreased MRTF-B nuclear localization or decreased SRF-CArG binding. Taken together, these data indicate that myocardin factors are regulated by proteasome-mediated degradation and that FHL2 regulates SRF-dependent transcription by multiple mechanisms, including stabilization of myocardin and MRTF-A. PMID:18586895

  1. An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes.

    PubMed

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-08-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

  2. The LIM domain protein nTRIP6 recruits the mediator complex to AP-1-regulated promoters.

    PubMed

    Diefenbacher, Markus E; Reich, Daniela; Dahley, Oliver; Kemler, Denise; Litfin, Margarethe; Herrlich, Peter; Kassel, Olivier

    2014-01-01

    Several LIM domain proteins regulate transcription. They are thought to act through their LIM protein-protein interaction domains as adaptors for the recruitment of transcriptional co-regulators. An intriguing example is nTRIP6, the nuclear isoform of the focal adhesion protein TRIP6. nTRIP6 interacts with AP-1 and enhances its transcriptional activity. nTRIP6 is also essential for the transrepression of AP-1 by the glucocorticoid receptor (GR), by mediating GR tethering to promoter-bound AP-1. Here we report on the molecular mechanism by which nTRIP6 exerts these effects. Both the LIM domains and the pre-LIM region of nTRIP6 are necessary for its co-activator function for AP-1. Discrete domains within the pre-LIM region mediate the dimerization of nTRIP6 at the promoter, which enables the recruitment of the Mediator complex subunits THRAP3 and Med1. This recruitment is blocked by GR, through a competition between GR and THRAP3 for the interaction with the LIM domains of nTRIP6. Thus, nTRIP6 both positively and negatively regulates transcription by orchestrating the recruitment of the Mediator complex to AP-1-regulated promoters.

  3. Muscle Lim Protein isoform negatively regulates striated muscle actin dynamics and differentiation

    PubMed Central

    Vafiadaki, Elizabeth; Arvanitis, Demetrios A.; Papalouka, Vasiliki; Terzis, Gerasimos; Roumeliotis, Theodoros I.; Spengos, Konstantinos; Garbis, Spiros D.; Manta, Panagiota; Kranias, Evangelia G.; Sanoudou, Despina

    2015-01-01

    Muscle Lim Protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, while aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/CFL2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components including α-actinin, T-cap and MLP. Our findings unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, and in differentiated striated muscles as a contributor to sarcomeric integrity. PMID:24860983

  4. ZNF185, an actin-cytoskeleton-associated growth inhibitory LIM protein in prostate cancer.

    PubMed

    Zhang, J-S; Gong, A; Young, C Y F

    2007-01-04

    We have recently identified ZNF185 as a gene that is downregulated in prostate cancer (PCa), in part via epigenetic alteration, and maybe associated with disease progression. In this study, we cloned the ZNF185 cDNA from normal human prostate tissues and investigated its biological function. We show that ZNF185 is a novel actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein that localizes to F-actin structures, and is enriched at focal adhesions. We find that the NH(2)-terminal region, which we designate the actin-targeting domain, facilitates ZNF185 binding to actin in vitro and is both necessary and sufficient to mediate actin-cytoskeleton targeting of ZNF185, whereas the LIM domain, which is localized in the COOH-terminus is dispensable for this phenomenon. Interestingly, ectopic expression of full-length ZNF185, but not a mutant lacking the actin-targeting domain, could suppress proliferation and anchorage-independent growth of PCa cells. Together, our data suggest that ZNF185 may function as a tumor-suppressor protein by associating with the actin-cytoskeleton.

  5. Expression, purification and mass spectrometric analysis of LIM mineralization protein-1 in human lung epithelial cells.

    PubMed

    Sangadala, Sreedhara; Titus, Louisa; Boden, Scott D

    2008-11-01

    LIM mineralization protein-1 (LMP-1) is a novel osteoinductive protein that has been cloned and shown to induce bone formation both in vitro and in vivo. Detection and evaluation of the possible presence of carbohydrate structures in LMP-1 is an important regulatory consideration for the therapeutic use of recombinantly expressed protein. The sequence of LMP-1 contains a highly conserved N-terminal PDZ domain and three C-terminal LIM domains. The sequence analysis of LMP-1 predicts two potential N-glycosylation sites and several O-glycosylation sites. Here, we report the cloning and overexpression of LMP-1 in human lung carcinoma (A549) cells. Even though our group already reported the sequence of LMP-1 cDNA, we undertook this work to clarify whether or not the overexpressed protein undergoes any glycosylation in vivo. The expressed full-length recombinant protein was purified and subjected to chemical analysis and internal sequencing. The absence of any hexosamines (N-acetyl glucosamine or N-acetyl galactosamine) in chemical composition analysis of LMP-1 protein revealed that there is little or no post-translational glycosylation of the LMP-1 polypeptide in lung carcinoma cells (A549). We performed in-gel trypsin digestion on purified LMP-1, and the resulting peptide digests were analyzed further using matrix-assisted laser desorption and ionization mass spectrometry for peptide mass finger printing, which produced several exact matches with the corresponding LMP-1 peptides. Separation by high performance liquid chromatography and purification of the desired peptides followed by N-terminal sequencing resulted in many exact LMP-1 matches for several purified peptides, thus establishing the identity of the purified protein as LMP-1.

  6. Paired and LIM class homeodomain proteins coordinate differentiation of the C. elegans ALA neuron.

    PubMed

    Van Buskirk, Cheryl; Sternberg, Paul W

    2010-06-01

    The ancient origin of sleep is evidenced by deeply conserved signaling pathways regulating sleep-like behavior, such as signaling through the Epidermal growth factor receptor (EGFR). In Caenorhabditis elegans, a sleep-like state can be induced at any time during development or adulthood through conditional expression of LIN-3/EGF. The behavioral response to EGF is mediated by EGFR activity within a single cell, the ALA neuron, and mutations that impair ALA differentiation are expected to confer EGF-resistance. Here we describe three such EGF-resistant mutants. One of these corresponds to the LIM class homeodomain (HD) protein CEH-14/Lhx3, and the other two correspond to Paired-like HD proteins CEH-10/Chx10 and CEH-17/Phox2. Whereas CEH-14 is required for ALA-specific gene expression throughout development, the Prd-like proteins display complementary temporal contributions to gene expression, with the requirement for CEH-10 decreasing as that of CEH-17 increases. We present evidence that CEH-17 participates in a positive autoregulatory loop with CEH-14 in ALA, and that CEH-10, in addition to its role in ALA differentiation, functions in the generation of the ALA neuron. Similarly to CEH-17, CEH-10 is required for the posterior migration of the ALA axons, but CEH-14 appears to regulate an aspect of ALA axon outgrowth that is distinct from that of the Prd-like proteins. Our findings reveal partial modularity among the features of a neuronal differentiation program and their coordination by Prd and LIM class HD proteins.

  7. A Structural Basis for the Regulation of the LIM-Homeodomain Protein Islet 1 (Isl1) by Intra- and Intermolecular Interactions*

    PubMed Central

    Gadd, Morgan S.; Jacques, David A.; Nisevic, Ivan; Craig, Vanessa J.; Kwan, Ann H.; Guss, J. Mitchell; Matthews, Jacqueline M.

    2013-01-01

    Islet 1 (Isl1) is a transcription factor of the LIM-homeodomain (LIM-HD) protein family and is essential for many developmental processes. LIM-HD proteins all contain two protein-interacting LIM domains, a DNA-binding homeodomain (HD), and a C-terminal region. In Isl1, the C-terminal region also contains the LIM homeobox 3 (Lhx3)-binding domain (LBD), which interacts with the LIM domains of Lhx3. The LIM domains of Isl1 have been implicated in inhibition of DNA binding potentially through an intramolecular interaction with or close to the HD. Here we investigate the LBD as a candidate intramolecular interaction domain. Competitive yeast-two hybrid experiments indicate that the LIM domains and LBD from Isl1 can interact with apparently low affinity, consistent with no detection of an intermolecular interaction in the same system. Nuclear magnetic resonance studies show that the interaction is specific, whereas substitution of the LBD with peptides of the same amino acid composition but different sequence is not specific. We solved the crystal structure of a similar but higher affinity complex between the LIM domains of Isl1 and the LIM interaction domain from the LIM-HD cofactor protein LIM domain-binding protein 1 (Ldb1) and used these coordinates to generate a homology model of the intramolecular interaction that indicates poorer complementarity for the weak intramolecular interaction. The intramolecular interaction in Isl1 may provide protection against aggregation, minimize unproductive DNA binding, and facilitate cofactor exchange within the cell. PMID:23750000

  8. Competition between LIM-binding domains.

    PubMed

    Matthews, Jacqueline M; Bhati, Mugdha; Craig, Vanessa J; Deane, Janet E; Jeffries, Cy; Lee, Christopher; Nancarrow, Amy L; Ryan, Daniel P; Sunde, Margaret

    2008-12-01

    LMO (LIM-only) and LIM-HD (LIM-homeodomain) proteins form a family of proteins that is required for myriad developmental processes and which can contribute to diseases such as T-cell leukaemia and breast cancer. The four LMO and 12 LIM-HD proteins in mammals are expressed in a combinatorial manner in many cell types, forming a transcriptional 'LIM code'. The proteins all contain a pair of closely spaced LIM domains near their N-termini that mediate protein-protein interactions, including binding to the approximately 30-residue LID (LIM interaction domain) of the essential co-factor protein Ldb1 (LIM domain-binding protein 1). In an attempt to understand the molecular mechanisms behind the LIM code, we have determined the molecular basis of binding of LMO and LIM-HD proteins for Ldb1(LID) through a series of structural, mutagenic and biophysical studies. These studies provide an explanation for why Ldb1 binds the LIM domains of the LMO/LIM-HD family, but not LIM domains from other proteins. The LMO/LIM-HD family exhibit a range of affinities for Ldb1, which influences the formation of specific functional complexes within cells. We have also identified an additional LIM interaction domain in one of the LIM-HD proteins, Isl1. Despite low sequence similarity to Ldb1(LID), this domain binds another LIM-HD protein, Lhx3, in an identical manner to Ldb1(LID). Through our and other studies, it is emerging that the multiple layers of competitive binding involving LMO and LIM-HD proteins and their partner proteins contribute significantly to cell fate specification and development.

  9. Out on a LIM: chronic kidney disease, podocyte phenotype and the Wilm's tumor interacting protein (WTIP).

    PubMed

    Sedor, John R; Madhavan, Sethu M; Kim, Jane H; Konieczkowski, Martha

    2011-01-01

    Normal function of the glomerular filtration barrier requires wild-type differentiation of the highly specialized glomerular epithelial cell, the podocyte. Podocytes express three distinct domains, consisting of a cell body, primary processes, and secondary foot processes (FP). These FP express slit diaphragms, which are highly specialized cell-cell contacts critical for filtration-barrier function. Foot processes are dynamic structures that reorganize within minutes through actin cytoskeletal rearrangement. Glomerular diseases are characterized by a persistent simplification in podocyte domain structure with loss of FP, a phenotype described as FP effacement. The generation of such phenotypic plasticity requires that signaling pathways in subcellular compartments be integrated dynamically for a cell to respond appropriately to information flow from its microenvironment. We have identified a LIM-domain-containing protein, Wilm's tumor interacting protein (WTIP), that regulates podocyte actin dynamics to maintain stable cell contacts. After glomerular injury, the WTIP molecule shuttles to the podocyte nucleus in response to changes in slit-diaphragm assembly, and changes gene transcription to permit podocyte remodeling. Defining regulatory pathways of podocyte differentiation identifies novel, druggable targets for chronic kidney diseases characterized by glomerular scarring.

  10. Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease.

    PubMed

    Tran, M Khang; Kurakula, Kondababu; Koenis, Duco S; de Vries, Carlie J M

    2016-02-01

    FHL2 belongs to the LIM-domain only proteins and contains four and a half LIM domains, each of which are composed of two zinc finger structures. FHL2 exhibits specific interaction with proteins exhibiting diverse functions, including transmembrane receptors, transcription factors and transcription co-regulators, enzymes, and structural proteins. The function of these proteins is regulated by FHL2, which modulates intracellular signal transduction pathways involved in a plethora of cellular tasks. The present review summarizes the current knowledge on the protein interactome of FHL2 and provides an overview of the functional implication of these interactions in apoptosis, migration, and regulation of nuclear receptor function. FHL2 was originally identified in the heart and there is extensive literature available on the role of FHL2 in the cardiovascular system, which is also summarized in this review.

  11. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4-negative mouse hearts.

    PubMed

    Straubinger, Julia; Boldt, Karsten; Kuret, Anna; Deng, Lisa; Krattenmacher, Diana; Bork, Nadja; Desch, Matthias; Feil, Robert; Feil, Susanne; Nemer, Mona; Ueffing, Marius; Ruth, Peter; Just, Steffen; Lukowski, Robert

    2017-04-01

    LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. Significant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.

  12. Deficiency in the LIM-only protein Fhl2 impairs skin wound healing.

    PubMed

    Wixler, Viktor; Hirner, Stephanie; Müller, Judith M; Gullotti, Lucia; Will, Carola; Kirfel, Jutta; Günther, Thomas; Schneider, Holm; Bosserhoff, Anja; Schorle, Hubert; Park, Jung; Schüle, Roland; Buettner, Reinhard

    2007-04-09

    After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359-369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736-748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of alpha-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

  13. Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation.

    PubMed

    Clark, Kathleen A; Lesage-Horton, Heather; Zhao, Cuiping; Beckerle, Mary C; Swank, Douglas M

    2011-08-01

    Muscle LIM protein (MLP) can be found at the Z-disk of sarcomeres where it is hypothesized to be involved in sensing muscle stretch. Loss of murine MLP results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac hypertrophy, indicating a critical role for MLP in maintaining normal cardiac function. Loss of MLP in Drosophila (mlp84B) also leads to muscle dysfunction, providing a model system to examine MLP's mechanism of action. Mlp84B-null flies that survive to adulthood are not able to fly or beat their wings. Transgenic expression of the mlp84B gene in the Mlp84B-null background rescues flight ability and restores wing beating ability. Mechanical analysis of skinned flight muscle fibers showed a 30% decrease in oscillatory power production and a slight increase in the frequency at which maximum power is generated for fibers lacking Mlp84B compared with rescued fibers. Mlp84B-null muscle fibers displayed a 25% decrease in passive, active, and rigor stiffness compared with rescued fibers, but no significant decrease in isometric tension generation was observed. Muscle ultrastructure of Mlp84B-null muscle fibers is grossly normal; however, the null fibers have a slight decrease, 11%, in thick filament number per unit cross-sectional area. Our data indicate that MLP contributes to muscle stiffness and is necessary for maximum work and power generation.

  14. Decoding the LIM development code.

    PubMed

    Gill, Gordon N

    2003-01-01

    During development a vast number of distinct cell types arise from dividing progenitor cells. Concentration gradients of ligands that act via cell surface receptors signal transcriptional regulators that repress and activate particular genes. LIM homeodomain proteins are an important class of transcriptional regulators that direct cell fate. Although in C. elegans only a single LIM homeodomain protein is expressed in a particular cell type, in vertebrates combinations of LIM homeodomain proteins are expressed in cells that determine cell fates. We have investigated the molecular basis of the LIM domain "code" that determines cell fates such as wing formation in Drosophilia and motor neuron formation in chicks. The basic code is a homotetramer of 2 LIM homeodomain proteins bridged by the adaptor protein, nuclear LIM interactor (NLI). A more complex molecular language consisting of a hexamer complex involving NLI and 2 LIM homeodomain proteins, Lhx3 and Isl1 determines ventral motor neuron formation. The same molecular "words" adopt different meanings depending on the context of expression of other molecular "words."

  15. Actin/alpha-actinin-dependent transport of AMPA receptors in dendritic spines: role of the PDZ-LIM protein RIL.

    PubMed

    Schulz, Torsten W; Nakagawa, Terunaga; Licznerski, Pawel; Pawlak, Verena; Kolleker, Alexander; Rozov, Andrei; Kim, Jinhyun; Dittgen, Tanjew; Köhr, Georg; Sheng, Morgan; Seeburg, Peter H; Osten, Pavel

    2004-09-29

    The efficacy of excitatory transmission in the brain depends to a large extent on synaptic AMPA receptors, hence the importance of understanding the delivery and recycling of the receptors at the synaptic sites. Here we report a novel regulation of the AMPA receptor transport by a PDZ (postsynaptic density-95/Drosophila disc large tumor suppressor zona occludens 1) and LIM (Lin11/rat Isl-1/Mec3) domain-containing protein, RIL (reversion-induced LIM protein). We show that RIL binds to the AMPA glutamate receptor subunit GluR-A C-terminal peptide via its LIM domain and to alpha-actinin via its PDZ domain. RIL is enriched in the postsynaptic density fraction isolated from rat forebrain, strongly localizes to dendritic spines in cultured neurons, and coprecipitates, together with alpha-actinin, in a protein complex isolated by immunoprecipitation of AMPA receptors from forebrain synaptosomes. Functionally, in heterologous cells, RIL links AMPA receptors to the alpha-actinin/actin cytoskeleton, an effect that appears to apply selectively to the endosomal surface-internalized population of the receptors. In cultured neurons, an overexpression of recombinant RIL increases the accumulation of AMPA receptors in dendritic spines, both at the total level, as assessed by immunodetection of endogenous GluR-A-containing receptors, and at the synaptic surface, as assessed by recording of miniature EPSCs. Our results thus indicate that RIL directs the transport of GluR-A-containing AMPA receptors to and/or within dendritic spines, in an alpha-actinin/actin-dependent manner, and that such trafficking function promotes the synaptic accumulation of the receptors.

  16. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton.

    PubMed

    Crawford, A W; Pino, J D; Beckerle, M C

    1994-01-01

    LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated.

  17. The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

    PubMed Central

    Wolfram, Verena; Southall, Tony D.; Brand, Andrea H.; Baines, Richard A.

    2012-01-01

    Summary Neuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes. PMID:22920257

  18. The LIM protein complex establishes a retinal circuitry of visual adaptation by regulating Pax6 α-enhancer activity

    PubMed Central

    Kim, Yeha; Lim, Soyeon; Ha, Taejeong; Song, You-Hyang; Sohn, Young-In; Park, Dae-Jin; Paik, Sun-Sook; Kim-Kaneyama, Joo-ri; Song, Mi-Ryoung; Leung, Amanda; Levine, Edward M; Kim, In-Beom; Goo, Yong Sook; Lee, Seung-Hee; Kang, Kyung Hwa; Kim, Jin Woo

    2017-01-01

    The visual responses of vertebrates are sensitive to the overall composition of retinal interneurons including amacrine cells, which tune the activity of the retinal circuitry. The expression of Paired-homeobox 6 (PAX6) is regulated by multiple cis-DNA elements including the intronic α-enhancer, which is active in GABAergic amacrine cell subsets. Here, we report that the transforming growth factor ß1-induced transcript 1 protein (Tgfb1i1) interacts with the LIM domain transcription factors Lhx3 and Isl1 to inhibit the α-enhancer in the post-natal mouse retina. Tgfb1i1-/- mice show elevated α-enhancer activity leading to overproduction of Pax6ΔPD isoform that supports the GABAergic amacrine cell fate maintenance. Consequently, the Tgfb1i1-/- mouse retinas show a sustained light response, which becomes more transient in mice with the auto-stimulation-defective Pax6ΔPBS/ΔPBS mutation. Together, we show the antagonistic regulation of the α-enhancer activity by Pax6 and the LIM protein complex is necessary for the establishment of an inner retinal circuitry, which controls visual adaptation. DOI: http://dx.doi.org/10.7554/eLife.21303.001 PMID:28139974

  19. Protein expression of phospho-lim kinase-1 in patients and an experimental rat model with intractable temporal lobe epilepsy

    PubMed Central

    Huang, Hao; Wang, Heng; Yuan, Jinxian; Wu, Xuling; Huang, Yunyi; Zhou, Xin; Chen, Yangmei

    2015-01-01

    Lim kinase-1 (LIMK1) plays a critical role in dendritic spine morphogenesis and brain function. The protein expression pattern of phospho-LIMK1 (p-LIMK1), the active form of LIMK1, in intractable temporal lobe epilepsy (TLE), however, is unknown. Here we measured p-LIMK1 protein expression in thirty temporal neocortex tissue samples from intractable TLE patients, fifteen histologically normal temporal neocortex tissue samples from trauma patients without epilepsy, in the hippocampi of lithium chloride/pilocarpine-induced TLE rats, and in controls. We found that p-LIMK1 was expressed mainly in the cytoplasm of neurons. The protein expression of p-LIMK1 was significantly higher in the TLE patients and rats than in the control groups. Our results suggest that p-LIMK1 might be involved in the pathogenesis of intractable TLE. PMID:25785037

  20. Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1

    PubMed Central

    Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D.

    2014-01-01

    Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquiti-nation and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

  1. Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.

    PubMed

    Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D

    2014-01-01

    Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

  2. LIM homeobox protein 5 (Lhx5) is essential for mamillary body development

    PubMed Central

    Miquelajáuregui, Amaya; Sandoval-Schaefer, Teresa; Martínez-Armenta, Miriam; Pérez-Martínez, Leonor; Cárabez, Alfonso; Zhao, Yangu; Heide, Michael; Alvarez-Bolado, Gonzalo; Varela-Echavarría, Alfredo

    2015-01-01

    The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM. PMID:26578897

  3. LIM homeobox protein 5 (Lhx5) is essential for mamillary body development.

    PubMed

    Miquelajáuregui, Amaya; Sandoval-Schaefer, Teresa; Martínez-Armenta, Miriam; Pérez-Martínez, Leonor; Cárabez, Alfonso; Zhao, Yangu; Heide, Michael; Alvarez-Bolado, Gonzalo; Varela-Echavarría, Alfredo

    2015-01-01

    The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.

  4. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton

    PubMed Central

    1994-01-01

    LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated. PMID:8294495

  5. Carbonic anhydrase III and four-and-a-half LIM protein 1 are preferentially oxidized with muscle unloading.

    PubMed

    Chen, Chiao-nan; Ferrington, Deborah A; Thompson, LaDora V

    2008-11-01

    The identities of proteins that show disuse-related changes in the content of oxidative modification are unknown. Furthermore, it is unknown whether the global accumulation of oxidized proteins is greater in aged animals with muscle disuse. The purposes of this study are 1) to identify the exact proteins that show disuse-related changes in oxidation levels and 2) to test the hypothesis that the global accumulation of oxidized proteins with muscle disuse would be greater in aged animals. Adult and old rats were randomized into four groups: weight bearing and 3, 7, or 14 days of hindlimb unloading. Soleus muscles were harvested to investigate the protein oxidation with unloading. Slot blot, SDS-PAGE, and Western blot analyses were used to detect the accumulation of 4-hydroxy-2-nonenol (HNE)- and nitrotyrosine (NT)-modified proteins. Matrix-assisted laser desorption ionization-time of flight and tandem mass spectroscopy were used to identify modified proteins. We found that global HNE- and NT-modified proteins accumulated significantly with aging but not with muscle unloading. Two HNE and NT target proteins, four-and-a-half LIM protein 1 (FHL1) and carbonic anhydrase III (CAIII), showed changes in the oxidation levels with muscle unloading. The changes in the oxidation levels happened to adult rats but not old rats. However, old rats had higher baseline levels of HNE-modified FHL1. In summary, the data suggest that the muscle unloading-related changes of protein oxidation are more significant in specific proteins and that the changes are age related.

  6. Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein.

    PubMed

    Sangadala, Sreedhara; Boden, Scott D; Metpally, Raghu Prasad Rao; Reddy, Boojala Vijay B

    2007-08-15

    LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.

  7. The LIM-Only Protein PINCH Directly Interacts with Integrin-Linked Kinase and Is Recruited to Integrin-Rich Sites in Spreading Cells

    PubMed Central

    Tu, Yizeng; Li, Fugang; Goicoechea, Silvia; Wu, Chuanyue

    1999-01-01

    PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the α5β1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways. PMID:10022929

  8. Protein Inhibitor of Activated STAT Y (PIASy) Regulates Insulin Secretion by Interacting with LIM Homeodomain Transcription Factor Isl1

    PubMed Central

    Yan, Chengzhi; Yu, Chulin; Zhang, Di; Cui, Yan; Zhou, Jinlian; Cui, Sheng

    2016-01-01

    It is known that the LIM homeodomain transcription factor Isl1 is highly expressed in all pancreatic endocrine cells and functions in regulating pancreatic development and insulin secretion. The Isl1 mutation has been found to be associated with type 2 diabetes, but the mechanism responsible for Isl1 regulation of insulin synthesis and secretion still needs to be elucidated. In the present study, the protein inhibitor of activated STAT Y (PIASy) was identified as a novel Isl1-interacting protein with a yeast two-hybrid system, and its interaction with Isl1 was further confirmed by a co-immunoprecipitation experiment. PIASy and Isl1 colocalize in human and mouse pancreas and NIT beta cells. Furthermore, PIASy and Isl1 upregulate insulin gene expression and insulin secretion in a dose-dependent manner by activating the insulin promoter. PIASy and Isl1 mRNA expression levels were also increased in type 2 diabetic db/db mice. In addition, our results demonstrate that PIASy and Isl1 cooperate to activate the insulin promoter through the Isl1 homeodomain and PIASy ring domain. These data suggest that that PIASy regulates insulin synthesis and secretion by interacting with Isl1 and provide new insight into insulin regulation, although the detailed molecular mechanism needs to be clarified in future studies. PMID:28000708

  9. Deficiency in four and one half LIM domain protein 2 (FHL2) aggravates liver fibrosis in mice

    PubMed Central

    2013-01-01

    Background Four and one half LIM domain protein 2 (FHL2) has been reported to be a key regulator in many cellular processes being associated with fibrogenesis such as cell migration and contraction. Moreover, hepatic FHL2 is involved in regulation pathways mediating proliferation and cell death machineries. We here investigated the role of FHL2 in the setting of experimental and clinical liver fibrosis. Methods FHL2−/− and wild type (wt) mice were challenged with CCl4. Fibrotic response was assessed by quantitative real time PCR (qRT-PCR) of fibrotic marker genes, measurement of hydroxyproline content and histological methods. Murine FHL2−/− and hepatic stellate cells (HSC) were isolated and investigated via immunofluorescence. Human fibrotic and normal liver samples were analysed immunohistochemically using antibodies directed against FHL2. Results FHL2−/− mice displayed aggravated liver fibrosis compared to wt mice. However, immunofluorescence revealed no significant morphological changes in cultured FHL2−/− and wt myofibroblasts (MFB). In human liver samples, FHL2 was strongly expressed both in the nucleus and cytoplasm in MFB of fibrotic livers. In contrast, FHL2 expression was absent in normal liver tissue. Conclusions Deficiency of FHL2 results in aggravation of murine liver fibrosis. In human liver samples, FHL2 is expressed in activated HSCs and portal fibroblasts in human fibrotic livers, pointing to a central role of FHL2 for human hepatic fibrogenesis as well. PMID:23311569

  10. Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

    PubMed

    Friedberg, Felix

    2009-05-01

    In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

  11. Functional Analysis of LIM Domain Proteins and Co-Factors in Breast Cancer

    DTIC Science & Technology

    2002-10-01

    by tetracyclin . The LMO-4 protein is tagged with Myc and the Clim-2 protein is tagged with HA, thus allowing specific immunoprecipitation of these...western blot detecting expression of DN-Climn and LM0-4 in pools of MCF-7 cells in response to tetracyclin . The middle panel shows tet-inducible...binding proteins and transcriptional co-regulators. To search for such factors, we have screened a human breast cDNA library with LMO-4 as bait in the yeast

  12. LIM-only protein FHL2 activates NF-κB signaling in the control of liver regeneration and hepatocarcinogenesis.

    PubMed

    Dahan, Jennifer; Nouët, Yann; Jouvion, Grégory; Levillayer, Florence; Adib-Conquy, Minou; Cassard-Doulcier, Anne-Marie; Tebbi, Ali; Blanc, Fany; Remy, Lauriane; Chen, Ju; Cairo, Stefano; Werts, Catherine; Si-Tahar, Mustapha; Tordjmann, Thierry; Buendia, Marie-Annick; Wei, Yu

    2013-08-01

    Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor-NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2(-/-) macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2(-/-) mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

  13. ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain.

    PubMed

    Nakagawa, N; Hoshijima, M; Oyasu, M; Saito, N; Tanizawa, K; Kuroda, S

    2000-06-07

    The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

  14. Functional Analysis of LIM Domain Proteins and Co-Factors in Breast Cancer

    DTIC Science & Technology

    2004-10-01

    expressing vectors that can be induced by tetracycline (Tet-on system). However, expression in these cell lines was not stable and therefore, we have...breast cDNA library with LMO-4 as bait in the yeast two hybrid system and found several potential interacting partners, including DNA-binding proteins

  15. Expression patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system.

    PubMed

    Fujii, T; Pichel, J G; Taira, M; Toyama, R; Dawid, I B; Westphal, H

    1994-01-01

    We report the cloning, sequence analysis, and developmental expression pattern of lim1, a member of the LIM class homeobox gene family in the mouse. lim1 cDNA encodes a predicted 406 amino acid protein that is 93% identical with the product of the Xenopus LIM class homeobox gene Xlim1. We have characterized lim1 expression from day 8.5 post coitum onward. Northern blot analysis of RNA transcripts indicates that lim1 is expressed both during embryogenesis and in the adult brain. Analysis by whole-mount and section in situ hybridization shows lim1 expression in the central nervous system from the telencephalon through the spinal cord and in the developing excretory system including pronephric region, mesonephros, nephric duct, and metanephros. In the metanephros, lim1 is strongly expressed in renal vesicles and S-shaped bodies, and transcripts are also detected in the ureteric branches.

  16. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    SciTech Connect

    Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou . E-mail: mcutler@usuhs.mil

    2005-05-15

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.

  17. LIMS user acceptance testing.

    PubMed

    Klein, Corbett S

    2003-01-01

    Laboratory Information Management Systems (LIMS) play a key role in the pharmaceutical industry. Thorough and accurate validation of such systems is critical and is a regulatory requirement. LIMS user acceptance testing is one aspect of this testing and enables the user to make a decision to accept or reject implementation of the system. This paper discusses key elements in facilitating the development and execution of a LIMS User Acceptance Test Plan (UATP).

  18. Decreased interactions of mutant muscle LIM protein (MLP) with N-RAP and alpha-actinin and their implication for hypertrophic cardiomyopathy.

    PubMed

    Gehmlich, Katja; Geier, Christian; Osterziel, Karl Josef; Van der Ven, Peter F M; Fürst, Dieter O

    2004-08-01

    Previous work has shown that mutations in muscle LIM protein (MLP) can cause hypertrophic cardiomyopathy (HCM). In order to gain an insight into the molecular basis of the disease phenotype, we analysed the binding characteristics of wild-type MLP and of the (C58G) mutant MLP that causes hypertrophic cardiomyopathy. We show that MLP can form a ternary complex with two of its previously documented myofibrillar ligand proteins, N-RAP and alpha-actinin, which indicates the presence of distinct, non-overlapping binding sites. Our data also show that, in comparison to wild-type MLP, the capacity of the mutated MLP protein to bind both N-RAP and alpha-actinin is significantly decreased. In addition, this single point mutation prevents zinc coordination and proper folding of the second zinc-finger in the first LIM domain, which consequently renders the protein less stable and more susceptible to proteolysis. The molecular basis for HCM-causing mutations in the MLP gene might therefore be an alteration in the equilibrium of interactions of the ternary complex MLP-N-RAP-alpha-actinin. This assumption is supported by the previous observation that in the pathological situation accompanied by MLP down regulation, cardiomyocytes try to compensate for the decreased stability of MLP protein by increasing the expression of its ligand N-RAP, which might finally result in the development of myocyte disarray that is characteristic of this disease.

  19. LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads.

    PubMed

    Sangadala, Sreedhara; Boden, Scott D; Viggeswarapu, Manjula; Liu, Yunshan; Titus, Louisa

    2006-06-23

    Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.

  20. The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans

    PubMed Central

    Luo, Shuo; Schaefer, Anneliese M.; Dour, Scott; Nonet, Michael L.

    2014-01-01

    We describe the identification of zyxin as a regulator of synapse maintenance in mechanosensory neurons in C. elegans. zyx-1 mutants lacked PLM mechanosensory synapses as adult animals. However, most PLM synapses initially formed during development but were subsequently lost as the animals developed. Vertebrate zyxin regulates cytoskeletal responses to mechanical stress in culture. Our work provides in vivo evidence in support of such a role for zyxin. In particular, zyx-1 mutant synaptogenesis phenotypes were suppressed by disrupting locomotion of the mutant animals, suggesting that zyx-1 protects mechanosensory synapses from locomotion-induced forces. In cultured cells, zyxin is recruited to focal adhesions and stress fibers via C-terminal LIM domains and modulates cytoskeletal organization via the N-terminal domain. The synapse-stabilizing activity was mediated by a short isoform of ZYX-1 containing only the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. Our results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development. PMID:25252943

  1. The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans.

    PubMed

    Luo, Shuo; Schaefer, Anneliese M; Dour, Scott; Nonet, Michael L

    2014-10-01

    We describe the identification of zyxin as a regulator of synapse maintenance in mechanosensory neurons in C. elegans. zyx-1 mutants lacked PLM mechanosensory synapses as adult animals. However, most PLM synapses initially formed during development but were subsequently lost as the animals developed. Vertebrate zyxin regulates cytoskeletal responses to mechanical stress in culture. Our work provides in vivo evidence in support of such a role for zyxin. In particular, zyx-1 mutant synaptogenesis phenotypes were suppressed by disrupting locomotion of the mutant animals, suggesting that zyx-1 protects mechanosensory synapses from locomotion-induced forces. In cultured cells, zyxin is recruited to focal adhesions and stress fibers via C-terminal LIM domains and modulates cytoskeletal organization via the N-terminal domain. The synapse-stabilizing activity was mediated by a short isoform of ZYX-1 containing only the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. Our results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development.

  2. The LIM homeobox protein mLIM3/Lhx3 induces expression of the prolactin gene by a Pit-1/GHF-1-independent pathway in corticotroph AtT20 cells.

    PubMed

    Girardin, S E; Benjannet, S; Barale, J C; Chrétien, M; Seidah, N G

    1998-07-24

    mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides we determined the DNA sequence ANNAGGAAA(T/C)GA(CIG)AA as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15-mer stretch from nucleotides (nts) -218 to -204 which is highly conserved between human, rat, and bovine. In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tumor cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity towards the 15-mer element of the PRL promoter. From these results, we propose that the PRL promoter element (nts -218 to -204) could be functional in vivo. Finally, we demonstrate that in AtT20 cells prolactin mRNA expression is not induced by the Pit-1/GHF-1 pathway and that growth hormone mRNA is not detected concomitantly with prolactin. We conclude that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit-1/GHF-1 site within the proximal PRL promoter.

  3. PARPs database: A LIMS systems for protein-protein interaction data mining or laboratory information management system

    PubMed Central

    Droit, Arnaud; Hunter, Joanna M; Rouleau, Michèle; Ethier, Chantal; Picard-Cloutier, Aude; Bourgais, David; Poirier, Guy G

    2007-01-01

    Background In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteins and the rapid advancement of this technique, in combination with other proteomics methods, results in an increasing amount of proteome data. This data must be archived and analysed using specialized bioinformatics tools. Description We herein describe "PARPs database," a data analysis and management pipeline for liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. PARPs database is a web-based tool whose features include experiment annotation, protein database searching, protein sequence management, as well as data-mining of the peptides and proteins identified. Conclusion Using this pipeline, we have successfully identified several interactions of biological significance between PARP-1 and other proteins, namely RFC-1, 2, 3, 4 and 5. PMID:18093328

  4. The seven-pass transmembrane cadherin Flamingo controls dendritic self-avoidance via its binding to a LIM domain protein, Espinas, in Drosophila sensory neurons.

    PubMed

    Matsubara, Daisuke; Horiuchi, Shin-Ya; Shimono, Kohei; Usui, Tadao; Uemura, Tadashi

    2011-09-15

    Members of the Flamingo cadherin family are required in a number of different in vivo contexts of neural development. Even so, molecular identities downstream from the family have been poorly understood. Here we show that a LIM domain protein, Espinas (Esn), binds to an intracellular juxtamembrane domain of Flamingo (Fmi), and that this Fmi-Esn interplay elicits repulsion between dendritic branches of Drosophila sensory neurons. In wild-type larvae, branches of the same class IV dendritic arborization neuron achieve efficient coverage of its two-dimensional receptive field with minimum overlap with each other. However, this self-avoidance was disrupted in a fmi hypomorphic mutant, in an esn knockout homozygote, and in the fmi/esn trans-heterozygote. A functional fusion protein, Fmi:3eGFP, was localized at most of the branch tips, and in a heterologous system, assembly of Esn at cell contact sites required its LIM domain and Fmi. We further show that genes controlling epithelial planar cell polarity (PCP), such as Van Gogh (Vang) and RhoA, are also necessary for the self-avoidance, and that fmi genetically interacts with these loci. On the basis of these and other results, we propose that the Fmi-Esn complex, together with the PCP regulators and the Tricornered (Trc) signaling pathway, executes the repulsive interaction between isoneuronal dendritic branches.

  5. 1H, 15N and 13C assignments of an intramolecular Lmo2-LIM2/Ldb1-LID complex.

    PubMed

    Wilkinson-White, Lorna E; Dastmalchi, Siavoush; Kwan, Ann H; Ryan, Daniel P; Mackay, Joel P; Matthews, Jacqueline M

    2010-10-01

    Lmo2 is a LIM-only protein involved in hematopoiesis and the development of T-cell acute lymphoblastic leukaemia. Here we report backbone and side chain NMR assignments for an engineered intramolecular complex of the C-terminal LIM domain from Lmo2 tethered to the LIM interaction domain (LID) from LIM domain binding protein 1 (Ldb1).

  6. LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum

    PubMed Central

    Zhao, Yangu; Kwan, Kin-Ming; Mailloux, Christina M.; Lee, Woon-Kyu; Grinberg, Alexander; Wurst, Wolfgang; Behringer, Richard R.; Westphal, Heiner

    2007-01-01

    Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum. PMID:17664423

  7. Functional analysis of the nuclear LIM domain interactor NLI.

    PubMed Central

    Jurata, L W; Gill, G N

    1997-01-01

    LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions. PMID:9315627

  8. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta.

    PubMed Central

    Birkenfeld, Jörg; Betz, Heinrich; Roth, Dagmar

    2003-01-01

    Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3 zeta isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3 zeta. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3 zeta inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3 zeta-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1. PMID:12323073

  9. The LIM homeodomain protein Lhx6 regulates maturation of interneurons and network excitability in the mammalian cortex.

    PubMed

    Neves, Guilherme; Shah, Mala M; Liodis, Petros; Achimastou, Angeliki; Denaxa, Myrto; Roalfe, Grant; Sesay, Abdul; Walker, Matthew C; Pachnis, Vassilis

    2013-08-01

    Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

  10. Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel Kv1.5

    PubMed Central

    Poparic, Ivana; Schreibmayer, Wolfgang; Schoser, Benedikt; Desoye, Gernot; Gorischek, Astrid; Miedl, Heidi; Hochmeister, Sonja; Binder, Josepha; Quasthoff, Stefan; Wagner, Klaus; Windpassinger, Christian; Malle, Ernst

    2011-01-01

    Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G0/G1 phase. Furthermore, low expression of Kv1.5, a voltage-gated potassium channel known to alter myoblast proliferation during the G1 phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between Kv1.5 and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and Kv1.5 within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of Kv1.5 with FHL1C in Xenopus laevis oocytes markedly reduced K+ currents when compared to oocytes expressing Kv1.5 only. We here present the first evidence on a biological relevance of FHL1C. PMID:22053194

  11. Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain.

    PubMed

    Sangadala, Sreedhara; Okada, Motohiro; Liu, Yunshan; Viggeswarapu, Manjula; Titus, Louisa; Boden, Scott D

    2009-06-01

    Short peptide sequences known as protein transduction domains have become increasingly prevalent as tools to internalize molecules that would otherwise remain extracellular. Here, we determine whether a purified recombinant mammalian intracellular osteogenic factor delivered by a HIV-derived TAT-peptide tag is indeed capable of intracellular localization in a form accessible to interaction with other proteins. We engineered and bacterially expressed a TAT-fusion-cDNA construct of a known osteogenic factor, LIM mineralization protein-1 (LMP-1) involved in the bone morphogenetic protein (BMP) pathway that has the potential to serve as an enhancer of BMP-2 efficacy. The expressed recombinant protein contains an N-terminal (His)(6)-tag, a hemagglutinin(HA)-tag, and an 11-amino acid HIV-derived TAT-membrane transduction domain and was purified to homogeneity by Sephacryl S-100 molecular exclusion and Ni(2+)-affinity chromatography. The purified TAT-LMP-1 protein was chemically labeled with fluorescein, and its time and concentration dependent entry into rabbit blood cells was monitored by flow cytometry. We demonstrate the accumulation of TAT-tagged LMP-1 both in cytoplasmic and nuclear compartments. By performing affinity pull-down assays, we confirm our earlier findings that the recombinant TAT-LMP-1, when used as molecular bait to identify the intracellular binding proteins, interacts with Smurf1, a known binding partner of LMP-1. We also show potentiation of BMP-2 activity using the purified TAT-LMP-1 in mouse muscle C2C12 cells by assaying a heterologous luciferase-reporter construct containing multiple copies of a BMP-responsive sequence motif. Finally, we also confirm the biological activity of the purified TAT-LMP-1 by showing enhancement of BMP-2 induced increase of alkaline phosphatase mRNA and protein by RT-PCR and enzyme activity, respectively.

  12. Proteomics LIMS: A caBIG™ Project, Year 1

    PubMed Central

    Tchuvatkina, Olga; Shimoni, Liat; Ochs, Michael F.; Moloshok, Thomas

    2006-01-01

    protLIMS is a web-based proteomics laboratory information management system. In February, we released version 1 of protLIMS, completed to the year one goal: The web-interface provides for recording of the biological material, protein mixture preparation, 2D-PAGE, gel image files and spot and plug mapping. Associated files may be uploaded and retrieved through the web-interface to the file system. In year two, protLIMS will be extended to accommodate mass spectrometric analyses and protein identification. PMID:17238735

  13. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    SciTech Connect

    Venkitachalam, Srividya; Chueh, Fu-Yu; Yu, Chao-Lan

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  14. Osteoinductive LIM Mineralization Protein-1 Suppresses Activation of NF-κB and Selectively Regulates MAPK Pathways in Pre-osteoclasts

    PubMed Central

    Liu, Hui; Bargouti, Maggie; Zughaier, Susu; Zheng, Zhaomin; Liu, Yunshan; Sangadala, Sreedhara; Boden, Scott D.; Titus, Louisa

    2009-01-01

    LIM Mineralization Protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible Nitric Oxide Synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-κB), and prevented the phosphorylation of inhibitor of kappa B (IκB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-κB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-κB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways. PMID:19931434

  15. Osteoinductive LIM mineralization protein-1 suppresses activation of NF-kappaB and selectively regulates MAPK pathways in pre-osteoclasts.

    PubMed

    Liu, Hui; Bargouti, Maggie; Zughaier, Susu; Zheng, Zhaomin; Liu, Yunshan; Sangadala, Sreedhara; Boden, Scott D; Titus, Louisa

    2010-05-01

    LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible nitric oxide synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-kappaB), and prevented the phosphorylation of inhibitor of kappa B (IkappaB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-kappaB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-kappaB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways.

  16. Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

    PubMed Central

    Tu, Yizeng; Li, Fugang; Wu, Chuanyue

    1998-01-01

    Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways. PMID:9843575

  17. Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

    SciTech Connect

    Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.; Lee, Mihwa; Craig, Vanessa J.; Bach, Ingolf; Guss, J. Mitchell; Mackay, Joel P.; Matthews, Jacqueline M.

    2008-09-03

    LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}). Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

  18. LIM mineralization protein-1 suppresses TNF-α induced intervertebral disc degeneration by maintaining nucleus pulposus extracellular matrix production and inhibiting matrix metalloproteinases expression.

    PubMed

    Liu, Hui; Pan, Hehai; Yang, Hao; Wang, Jianru; Zhang, Kuibo; Li, Xiang; Wang, Hua; Ding, Wenbin; Li, Bingxue; Zheng, Zhaomin

    2015-03-01

    Imbalanced metabolism of Nucleus pulposus (NP) extracellular matrix (ECM) is closely correlated to Intervertebral Disc Degenerative Disease. LIM mineralization protein-1 (LMP-1) has been proven to induce sulfated glycosaminoglycan (sGAG) production in NP and have an anti-inflammatory effect in pre-osteoclast. However, whether it has any effect on the NP ECM production and degradation under inflammatory stimulation has not been studied. In the current study, a TNF-α induced cell model was established in vitro. Lentivirus encoding LMP-1 (LV-LMP-1) and short heparin LMP-1 (LV-shLMP-1) were constructed to overexpress and knockdown LMP-1 expression in NP cells. LMP-1 mRNA level was regulated in a dose-dependent manner after transfection. LV-LMP-1 increased whereas LV-shLMP-1 decreased collagen II, aggrecan, versican expression, and sGAG production. LV-LMP-1 abolished while LV-shLMP-1 aggravated TNF-α mediated down-regulation of the above matrix genes via ERK1/2 activation. Moreover, LV-LMP-1 abrogated TNF-α induced MMP-3 and MMP-13 expression via inhibiting p65 translocation and MMP-3 and MMP-13 promoter activity. These results indicated that LMP-1 had an ECM production maintenance effect under inflammatory stimulation. This effect was via up-regulation of matrix genes expression at least partially through ERK1/2 activation, and down-regulation of MMPs expression through NF-κB inhibition.

  19. LIMS and Clinical Data Management.

    PubMed

    Chen, Yalan; Lin, Yuxin; Yuan, Xuye; Shen, Bairong

    2016-01-01

    In order to achieve more accurate disease prevention, diagnosis, and treatment, clinical and genetic data need extensive and systematically associated study. As one way to achieve precision medicine, a laboratory information management system (LIMS) can effectively associate clinical data in a macrocosmic aspect and genomic data in a microcosmic aspect. This chapter summarizes the application of the LIMS in a clinical data management and implementation mode. It also discusses the principles of a LIMS in clinical data management, as well as the opportunities and challenges in the context of medical informatics.

  20. [Expression of LIM and SH3 protein 1 in renal clear cell carcinoma and its effects on invasion and migration of renal clear cell carcinoma 786-O cells].

    PubMed

    Jin, B; Gao, L; Li, W; Chen, J C; Wen, R M; Wang, J Q

    2017-03-23

    Objective: To investigate the expression of LIM and SH3 protein 1 (LASP1) in renal cell carcinoma and its significance in the invasion and migration of renal clear cell carcinoma 786-O cell line. Methods: The expression level of LASP1 in 41 cases of renal cell carcinoma tissues and normal renal tissues was analyzed by immunohistochemistry. The relationship between the expression level of LASP1 and clinical characteristics was further analyzed. Expression of LASP1 in 10 cases of tumor tissues with or without lymph node metastasis was analyzed by Western blot. Furthermore, small interfering RNA (siRNA) targeting LASP1 was constructed and transfected into 786-O cells to downregulate LASP1 expression. The interference effect of LASP1 siRNA on LASP1 protein and the expression of related proteins in epithelial mesenchymal transition (EMT) pathway were detected by Western blot. The effects of LASP1 knockdown on cell proliferation, migration and invasion and gene expression were then assessed using CCK8 assay, transwell cell migration system and western blot analysis, respectively. Results: The positive rate of LASP1 expression in renal clear cell carcinoma tissues was 90.2% (37/41), which was significantly higher than that in the adjacent tissues (29.3%, P=0.002). The expression of LASP1 in renal cell carcinoma was positively correlated with lymph node metastasis and TNM stage of renal cell carcinoma (P<0.05). The results of Western blot showed that LASP1 (0.696±0.053) was highly expressed in renal cell carcinoma (1.459±0.628), especially in cases with lymph node metastasis (2.692±0.186, P<0.05). The LASP1 siRNA remarkably down-regulated the expression of LASP1 protein in 786-O cells. The abilities of proliferation, invasion and migration of 786-O cells were decreased significantly in the LASP1 siRNA groups.The relative expression of E-cadherin protein in the siRNA group (0.848±0.020) was significantly higher than those in the siRNA-NC group (0.671±0.018) and control

  1. Expression analysis of LIM gene family in poplar, toward an updated phylogenetic classification

    PubMed Central

    2012-01-01

    Background Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied. Findings In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues. Conclusions The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins. PMID:22339987

  2. LIMS: from theory to practice.

    PubMed

    Cardot, J M; Hulot, T; Le Bricon, C; Stockis, A

    1998-01-01

    This paper gives a definition and some basic knowledge about Laboratory Information Management Systems (LIMS) as well as their impact on the organisation, the laboratory and the co-workers. The major advantages and disadvantages of LIMS are pointed out. Two practical experiences are described. The first is related to an in house development of a PC based system which has to integrate a Vax VMS system (Multichrom) and PC based analytical and analysis softwares. The second experience is dealing with the selection and implementation of a commercial package in a pharmacokinetic laboratory. In both cases the human and time aspects were important.

  3. Solution structure of a tethered Lmo2(LIM2) /Ldb1(LID) complex.

    PubMed

    Dastmalchi, Siavoush; Wilkinson-White, Lorna; Kwan, Ann H; Gamsjaeger, Roland; Mackay, Joel P; Matthews, Jacqueline M

    2012-11-01

    LIM-only protein 2, Lmo2, is a regulatory protein that is essential for hematopoietic development and inappropriate overexpression of Lmo2 in T-cells contributes to T-cell leukemia. It exerts its functions by mediating protein-protein interactions and nucleating multicomponent transcriptional complexes. Lmo2 interacts with LIM domain binding protein 1 (Ldb1) through the tandem LIM domains of Lmo2 and the LIM interaction domain (LID) of Ldb1. Here, we present the solution structure of the LIM2 domain of Lmo2 bound to Ldb1(LID) . The ordered regions of Ldb1 in this complex correspond well with binding hotspots previously defined by mutagenic studies. Comparisons of this Lmo2(LIM2) -Ldb1(LID) structure with previously determined structures of the Lmo2/Ldb1(LID) complexes lead to the conclusion that modular binding of tandem LIM domains in Lmo2 to tandem linear motifs in Ldb1 is accompanied by several disorder-to-order transitions and/or conformational changes in both proteins.

  4. Solution structure of a tethered Lmo2LIM2/Ldb1LID complex

    PubMed Central

    Dastmalchi, Siavoush; Wilkinson-White, Lorna; Kwan, Ann H; Gamsjaeger, Roland; Mackay, Joel P; Matthews, Jacqueline M

    2012-01-01

    LIM-only protein 2, Lmo2, is a regulatory protein that is essential for hematopoietic development and inappropriate overexpression of Lmo2 in T-cells contributes to T-cell leukemia. It exerts its functions by mediating protein–protein interactions and nucleating multicomponent transcriptional complexes. Lmo2 interacts with LIM domain binding protein 1 (Ldb1) through the tandem LIM domains of Lmo2 and the LIM interaction domain (LID) of Ldb1. Here, we present the solution structure of the LIM2 domain of Lmo2 bound to Ldb1LID. The ordered regions of Ldb1 in this complex correspond well with binding hotspots previously defined by mutagenic studies. Comparisons of this Lmo2LIM2–Ldb1LID structure with previously determined structures of the Lmo2/Ldb1LID complexes lead to the conclusion that modular binding of tandem LIM domains in Lmo2 to tandem linear motifs in Ldb1 is accompanied by several disorder-to-order transitions and/or conformational changes in both proteins. PMID:22936624

  5. Solution structure of the focal adhesion adaptor PINCH LIM1 domain and characterization of its interaction with the integrin-linked kinase ankyrin repeat domain.

    PubMed

    Velyvis, A; Yang, Y; Wu, C; Qin, J

    2001-02-16

    PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interactions that are involved in cell adhesion, growth, and differentiation. The LIM1 domain of PINCH interacts with integrin-linked kinase (ILK), thereby mediating focal adhesions via a specific integrin/ILK signaling pathway. We have solved the NMR structure of the PINCH LIM1 domain and characterized its binding to ILK. LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine-rich protein and cysteine-rich intestinal protein families with CCHC and CCCC zinc finger types. Gel-filtration and NMR experiments demonstrated a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment identified regions in PINCH LIM1 that are important for interaction with ILK. Comparison of surface features between PINCH LIM1 and other functionally different LIM domains indicated that the LIM motif might have a highly variable mode in recognizing various target proteins.

  6. Cloning, characterization and subcellular localization of Nuclear LIM interactor interacting factor gene from Leishmania donovani.

    PubMed

    Ravinder, R; Goyal, N

    2017-05-05

    LIM domains are zinc-binding motifs that mediate protein-protein interactions and are found in a wide variety of cytoplasmic and nuclear proteins. The nuclear LIM domain family members have a number of different functions including transcription factors, gene regulation, cell fate determination, organization of the cytoskeleton and tumour formation exerting their function through various LIM domain interacting protein partners/cofactors. Nuclear LIM domain interacting proteins/factors have not been reported in any protozoan parasites including Leishmania. Here, we report for the first time cloning, characterization and subcellular localization of nuclear LIM interactor-interacting factor (NLI) like protein from Leishmania donovani, the causative agent of Indian Kala-azar. Primary sequence analysis of LdNLI revealed presence of characteristic features of nuclear LIM interactor-interacting factor. However, leishmanial NLI represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. The sub-cellular distribution of LdNLI revealed the discreet localization in nucleus and kinetoplast only, suggesting that the gene may have a role in parasite gene expression.

  7. Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility.

    PubMed

    Brown, Michael C; Turner, Christopher E

    2002-07-01

    Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.

  8. Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1)

    PubMed Central

    Endres, Marcel; Kneitz, Susanne; Orth, Martin F.; Perera, Ruwan K.; Zernecke, Alma; Butt, Elke

    2016-01-01

    The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines. By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines. In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown. Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression. The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies. PMID:27588391

  9. Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

    SciTech Connect

    Hamada, Fumika N.; Koshiyama, Akiyo; Namekawa, Satoshi H.; Ishii, Satomi; Iwabata, Kazuki; Sugawara, Hiroko; Nara, Takayuki Y.; Sakaguchi, Kengo . E-mail: kengo@rs.noda.tus.ac.jp; Sawado, Tomoyuki

    2007-01-26

    PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg{sup 2+} ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

  10. WIST: toolkit for rapid, customized LIMS development

    PubMed Central

    Huang, Y. Wayne; Arkin, Adam P.; Chandonia, John-Marc

    2011-01-01

    Summary: Workflow Information Storage Toolkit (WIST) is a set of application programming interfaces and web applications that allow for the rapid development of customized laboratory information management systems (LIMS). WIST provides common LIMS input components, and allows them to be arranged and configured using a flexible language that specifies each component's visual and semantic characteristics. WIST includes a complete set of web applications for adding, editing and viewing data, as well as a powerful setup tool that can build new LIMS modules by analyzing existing database schema. Availability and implementation: WIST is implemented in Perl and may be obtained from http://vimss.sf.net under the BSD license. Contact: jmchandonia@lbl.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21258060

  11. LIMS Version 6 Level 3 Dataset

    NASA Technical Reports Server (NTRS)

    Remsberg, Ellis E.; Lingenfelser, Gretchen

    2010-01-01

    This report describes the Limb Infrared Monitor of the Stratosphere (LIMS) Version 6 (V6) Level 3 data products and the assumptions used for their generation. A sequential estimation algorithm was used to obtain daily, zonal Fourier coefficients of the several parameters of the LIMS dataset for 216 days of 1978-79. The coefficients are available at up to 28 pressure levels and at every two degrees of latitude from 64 S to 84 N and at the synoptic time of 12 UT. Example plots were prepared and archived from the data at 10 hPa of January 1, 1979, to illustrate the overall coherence of the features obtained with the LIMS-retrieved parameters.

  12. SIGLa: an adaptable LIMS for multiple laboratories

    PubMed Central

    2010-01-01

    Background The need to manage large amounts of data is a clear demand for laboratories nowadays. The use of Laboratory Information Management Systems (LIMS) to achieve this is growing each day. A LIMS is a complex computational system used to manage laboratory data with emphasis in quality assurance. Several LIMS are available currently. However, most of them have proprietary code and are commercialized with a high cost. Moreover, due to its complexity, LIMS are usually designed to comply with the needs of one kind of laboratory, making it very difficult to reuse a LIMS. In this work we describe the Sistema Integrado de Gerência de Laboratórios (SIGLa), an open source LIMS with a new approach designed to allow it to adapt its activities and processes to various types of laboratories. Results SIGLa incorporates a workflow management system, making it possible to create and manage customized workflows. For each new laboratory a workflow is defined with its activities, rules and procedures. During the execution, for each workflow created, the values of attributes defined in a XPDL file (which describe the workflow) are stored in SIGLa’s database, allowing then to be managed and retrieved upon request. These characteristics increase system’s flexibility and extend its usability to include the needs of multiple types of laboratories. To construct the main functionalities of SIGLa a workflow of a proteomic laboratory was first defined. To validate the SIGLa capability of adapting to multiples laboratories, on this paper we study theprocess and the needs of a microarray laboratory and define its workflow. This workflow has been defined in a period of about two weeks, showing the efficiency and flexibility of the tool. Conclusions Using SIGLa it has been possible to construct a microarray LIMS in a few days illustrating the flexibility and power of the method proposed. With SIGLa’s development we hope to contribute positively to the area of management of complex

  13. Comparing sea ice, hydrography and circulation between NEMO3.6 LIM3 and LIM2

    NASA Astrophysics Data System (ADS)

    Uotila, Petteri; Iovino, Doroteaciro; Vancoppenolle, Martin; Lensu, Mikko; Rousset, Clement

    2017-03-01

    A set of hindcast simulations with the new version 3.6 of the Nucleus for European Modelling of the Ocean (NEMO) ocean-ice model in the ORCA1 configuration and forced by the DRAKKAR Forcing Set version 5.2 (DFS5.2) atmospheric data was performed from 1958 to 2012. Simulations differed in their sea-ice component: the old standard version Louvain-la-Neuve Sea Ice Model (LIM2) and its successor LIM3. Main differences between these sea-ice models are the parameterisations of sub-grid-scale sea-ice thickness distribution, ice deformation, thermodynamic processes, and sea-ice salinity. Our main objective was to analyse the response of the ocean-ice system sensitivity to the change in sea-ice physics. Additional sensitivity simulations were carried out for the attribution of observed differences between the two main simulations.In the Arctic, NEMO-LIM3 compares better with observations by realistically reproducing the sea-ice extent decline during the last few decades due to its multi-category sea-ice thickness. In the Antarctic, NEMO-LIM3 more realistically simulates the seasonal evolution of sea-ice extent than NEMO-LIM2. In terms of oceanic properties, improvements are not as evident, although NEMO-LIM3 reproduces a more realistic hydrography in the Labrador Sea and in the Arctic Ocean, including a reduced cold temperature bias of the Arctic Intermediate Water at 250 m. In the extra-polar regions, the oceanographic conditions of the two NEMO-LIM versions remain relatively similar, although they slowly drift apart over decades. This drift is probably due to a stronger deep water formation around Antarctica in LIM3.

  14. Principles and application of LIMS in mouse clinics.

    PubMed

    Maier, Holger; Schütt, Christine; Steinkamp, Ralph; Hurt, Anja; Schneltzer, Elida; Gormanns, Philipp; Lengger, Christoph; Griffiths, Mark; Melvin, David; Agrawal, Neha; Alcantara, Rafael; Evans, Arthur; Gannon, David; Holroyd, Simon; Kipp, Christian; Raj, Navis Pretheeba; Richardson, David; LeBlanc, Sophie; Vasseur, Laurent; Masuya, Hiroshi; Kobayashi, Kimio; Suzuki, Tomohiro; Tanaka, Nobuhiko; Wakana, Shigeharu; Walling, Alison; Clary, David; Gallegos, Juan; Fuchs, Helmut; de Angelis, Martin Hrabě; Gailus-Durner, Valerie

    2015-10-01

    Large-scale systemic mouse phenotyping, as performed by mouse clinics for more than a decade, requires thousands of mice from a multitude of different mutant lines to be bred, individually tracked and subjected to phenotyping procedures according to a standardised schedule. All these efforts are typically organised in overlapping projects, running in parallel. In terms of logistics, data capture, data analysis, result visualisation and reporting, new challenges have emerged from such projects. These challenges could hardly be met with traditional methods such as pen & paper colony management, spreadsheet-based data management and manual data analysis. Hence, different Laboratory Information Management Systems (LIMS) have been developed in mouse clinics to facilitate or even enable mouse and data management in the described order of magnitude. This review shows that general principles of LIMS can be empirically deduced from LIMS used by different mouse clinics, although these have evolved differently. Supported by LIMS descriptions and lessons learned from seven mouse clinics, this review also shows that the unique LIMS environment in a particular facility strongly influences strategic LIMS decisions and LIMS development. As a major conclusion, this review states that there is no universal LIMS for the mouse research domain that fits all requirements. Still, empirically deduced general LIMS principles can serve as a master decision support template, which is provided as a hands-on tool for mouse research facilities looking for a LIMS.

  15. Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts

    PubMed Central

    Irum, Bushra; Khan, Shahid Y.; Ali, Muhammad; Kaul, Haiba; Kabir, Firoz; Rauf, Bushra; Fatima, Fareeha; Nadeem, Raheela; Khan, Arif O.; Al Obaisi, Saif; Naeem, Muhammad Asif; Nasir, Idrees A.; Khan, Shaheen N.; Husnain, Tayyab; Riazuddin, Sheikh; Akram, Javed; Eghrari, Allen O.; Riazuddin, S. Amer

    2016-01-01

    Purpose To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family. Methods All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model. Results Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15

  16. Principles of Contour Information: Reply to Lim and Leek (2012)

    ERIC Educational Resources Information Center

    Singh, Manish; Feldman, Jacob

    2012-01-01

    Lim and Leek (2012) presented a formalization of information along object contours, which they argued was an alternative to the approach taken in our article (Feldman & Singh, 2005). Here, we summarize the 2 approaches, showing that--notwithstanding Lim and Leek's (2012) critical rhetoric--their approach is substantially identical to ours,…

  17. Mapping of four mouse genes encoding eye lens-specific structural, gap junction, and integral membrane proteins: Cryba1 (crystallin{beta}A3/A1), Crybb2 (crystallin{beta}B2), Gja8 (MP70), and Lim2 (MP19)

    SciTech Connect

    Kerscher, S.; Boyd, Y.; Lyon, M.F.

    1995-09-20

    Four genes encoding eye lens-specific proteins, potential candidate genes for congenital cataract (CC) mutations, were mapped in the mouse genome using a panel of somatic cell hybrids and DNAs from the EUCIB (European Collaborative Interspecific Backcross). Two of them are lens fiber cell structural proteins: the Cryba1 locus encoding crystallin{beta}A3/A1 maps to chromosome 11, 2.5 {+-} 2.5 cM distal to D11Mit31, and the Crybb2 locus encoding crystallin{beta}B2 maps to chromosome 5, 9.1 {+-} 4.3 cM distal to D5Mit88. The other two genes encode lens-specific gap junction and integral membrane proteins, respectively: the Gja8 locus encoding gap junction membrane channel protein {alpha}8, also called connexin50 or MP70, maps to chromosome 3, 11.9 {+-} 5.0 cM distal to D3Mit22, and the Lim2 locus encoding lens intrinsic membrane protein 2, also call MP19, maps to chromosome 7, 2.5 {+-} 2.5 cM proximal to Ngfg. All four map positions, when compared with the corresponding positions in human, lie within known regions of conserved synteny between mouse and human chromosomes. 43 refs., 2 figs., 1 tab.

  18. Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development.

    PubMed

    Huang, Mingqian; Sage, Cyrille; Li, Huawei; Xiang, Mengquig; Heller, Stefan; Chen, Zheng-Yi

    2008-11-01

    LIM-homeodomain transcription factors (LIM-HDs) are essential in tissue patterning and differentiation. But their expression patterns in the inner ear are largely unknown. Here we report on a study of twelve LIM-HDs, by their tempo-spatial patterns that imply distinct yet overlapping roles, in the developing mouse inner ear. Expression of Lmx1a and Isl1 begins in the otocyst stage, with Lmx1a exclusively in the non-sensory and Isl1 in the prosensory epithelia. The second wave of expression at E12.5 includes Lhx3, 5, 9, Isl2, and Lmx1b in the differentiating sensory epithelia with cellular specificities. With the exception of Lmx1a and Lhx3, all LIM-HDs are expressed in ganglion neurons. Expression of multiple LIM-HDs within a cell type suggests their redundant function.

  19. Laboratory Information Management System (LIMS): A case study

    NASA Technical Reports Server (NTRS)

    Crandall, Karen S.; Auping, Judith V.; Megargle, Robert G.

    1987-01-01

    In the late 70's, a refurbishment of the analytical laboratories serving the Materials Division at NASA Lewis Research Center was undertaken. As part of the modernization efforts, a Laboratory Information Management System (LIMS) was to be included. Preliminary studies indicated a custom-designed system as the best choice in order to satisfy all of the requirements. A scaled down version of the original design has been in operation since 1984. The LIMS, a combination of computer hardware, provides the chemical characterization laboratory with an information data base, a report generator, a user interface, and networking capabilities. This paper is an account of the processes involved in designing and implementing that LIMS.

  20. The accuracy of Nimbus 7 LIMS temperatures in the mesosphere

    NASA Astrophysics Data System (ADS)

    Remsberg, E. E.

    1986-04-01

    Zonal-mean temperatures from the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) experiment are compared with a temperature 'climatology' derived from 4 years of Rayleigh backscatter lidar measurements at 44 N and for the months of March, April, and May, when wave activity and thus atmospheric variability is very weak. The mean difference between the two data sets is less than 3.5 K between 37 and 64 km and is consistent with the theoretical estimates of accuracy reported for the LIMS data. The effects of longitudinal and year-to-year variaiations in this comparison were considered, but they do not change the conclusions significantly. These validation results extend the altitude range of accurate LIMS temperatures in the mesosphere some 10 km higher than previously reported. It also means the LIMS ozone and NO2 retrievals in the lower mesosphere should be free of any major temperature bias error effects.

  1. It takes two to tango: the structure and function of LIM, RING, PHD and MYND domains.

    PubMed

    Matthews, J M; Bhati, M; Lehtomaki, E; Mansfield, R E; Cubeddu, L; Mackay, J P

    2009-01-01

    LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.

  2. Energetic Particle Precipitation Effects Observed in LIMS Data

    NASA Astrophysics Data System (ADS)

    Holt, L. A.; Randall, C. E.; Harvey, V. L.; Stiller, G. P.; Funke, B.; López-Puertas, M.; Remsberg, E. E.

    2008-12-01

    The Limb Infrared Monitor of the Stratosphere (LIMS) observed stratospheric enhancements in NO2 inside the Arctic polar vortex during the winter of 1978-1979. These enhancements were attributed to the descent of NOx originally produced by precipitating energetic particles in the upper atmosphere. Although few observations of such stratospheric NOx enhancements were made during the decade succeeding the LIMS measurements, investigations in the last decade have shown abundant evidence for these enhancements. Interannual variability in the enhancements appears to be controlled both by the amount of particle precipitation and the prevailing meteorological conditions, which dictate the efficiency with which NOx is transported from the upper atmosphere into the stratosphere. In this presentation, recent satellite measurements of the temporal evolution of NOx in the polar vortex are compared to the LIMS measurements. Our goal is to investigate whether the enhancements were observed by LIMS because of enhanced geomagnetic activity and/or anomalous dynamical conditions, or whether the nighttime observing capability of LIMS simply enabled it to detect the NOx enhancements under nominal conditions.

  3. LimsPortal and BonsaiLIMS: development of a lab information management system for translational medicine

    PubMed Central

    2011-01-01

    Background Laboratory Information Management Systems (LIMS) are an increasingly important part of modern laboratory infrastructure. As typically very sophisticated software products, LIMS often require considerable resources to select, deploy and maintain. Larger organisations may have access to specialist IT support to assist with requirements elicitation and software customisation, however smaller groups will often have limited IT support to perform the kind of iterative development that can resolve the difficulties that biologists often have when specifying requirements. Translational medicine aims to accelerate the process of treatment discovery by bringing together multiple disciplines to discover new approaches to treating disease, or novel applications of existing treatments. The diverse set of disciplines and complexity of processing procedures involved, especially with the use of high throughput technologies, bring difficulties in customizing a generic LIMS to provide a single system for managing sample related data within a translational medicine research setting, especially where limited IT support is available. Results We have designed and developed a LIMS, BonsaiLIMS, around a very simple data model that can be easily implemented using a variety of technologies, and can be easily extended as specific requirements dictate. A reference implementation using Oracle 11 g database and the Python framework, Django is presented. Conclusions By focusing on a minimal feature set and a modular design we have been able to deploy the BonsaiLIMS system very quickly. The benefits to our institute have been the avoidance of the prolonged implementation timescales, budget overruns, scope creep, off-specifications and user fatigue issues that typify many enterprise software implementations. The transition away from using local, uncontrolled records in spreadsheet and paper formats to a centrally held, secured and backed-up database brings the immediate benefits of

  4. Alternative Splicing of the LIM-Homeodomain Transcription Factor Isl1 in the Mouse Retina

    PubMed Central

    Whitney, Irene E.; Kautzman, Amanda G.; Reese, Benjamin E.

    2015-01-01

    Islet-1 (Isl1) is a LIM-homeodomain (LIM-HD) transcription factor that functions in a combinatorial manner with other LIM-HD proteins to direct the differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that Isl1 is alternatively spliced generating a second isoform, Isl1β, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative Isl1 transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of Isl1 is shown to occur in multiple tissues, but the relative abundance of Isl1α and Isl1 β expression varies greatly across them. In most tissues, Isl1α is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice variant is dominant. Within the retina, differential expression of the two Isl1 transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system. PMID:25752730

  5. SMITH: a LIMS for handling next-generation sequencing workflows

    PubMed Central

    2014-01-01

    Background Life-science laboratories make increasing use of Next Generation Sequencing (NGS) for studying bio-macromolecules and their interactions. Array-based methods for measuring gene expression or protein-DNA interactions are being replaced by RNA-Seq and ChIP-Seq. Sequencing is generally performed by specialized facilities that have to keep track of sequencing requests, trace samples, ensure quality and make data available according to predefined privileges. An integrated tool helps to troubleshoot problems, to maintain a high quality standard, to reduce time and costs. Commercial and non-commercial tools called LIMS (Laboratory Information Management Systems) are available for this purpose. However, they often come at prohibitive cost and/or lack the flexibility and scalability needed to adjust seamlessly to the frequently changing protocols employed. In order to manage the flow of sequencing data produced at the Genomic Unit of the Italian Institute of Technology (IIT), we developed SMITH (Sequencing Machine Information Tracking and Handling). Methods SMITH is a web application with a MySQL server at the backend. Wet-lab scientists of the Centre for Genomic Science and database experts from the Politecnico of Milan in the context of a Genomic Data Model Project developed SMITH. The data base schema stores all the information of an NGS experiment, including the descriptions of all protocols and algorithms used in the process. Notably, an attribute-value table allows associating an unconstrained textual description to each sample and all the data produced afterwards. This method permits the creation of metadata that can be used to search the database for specific files as well as for statistical analyses. Results SMITH runs automatically and limits direct human interaction mainly to administrative tasks. SMITH data-delivery procedures were standardized making it easier for biologists and analysts to navigate the data. Automation also helps saving time. The

  6. 77 FR 6782 - In the Matter of: Kok Tong Lim, a/k/a Thomas Lim Blk 258A Compassvale Road #07-551 Singapore...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-09

    ...)) (``IEEPA''). Specifically, Lim conspired to illegally export wound carbon fiber, an item subject to the... this provision may be for a period of up to 10 years from the date of the conviction. 15 CFR 766.25(d... IEEPA, and have provided notices and opportunities for Lim to make a written submission to BIS,...

  7. Hydroxyl radical regeneration in isoprene oxidation: upgraded mechanism LIM1

    NASA Astrophysics Data System (ADS)

    Peeters, Jozef; Son Nguyen, Vinh; Nguyen, Thanh Lam; Stravrakou, Trissevgeni; Muller, Jean-Francois

    2013-04-01

    The OH regeneration known to occur in isoprene oxidation at low/moderate NO is attributed in the Leuven Isoprene Mechanism to novel, theoretically characterized chemical pathways (LIM0: Peeters et al. 2009; Peeters and Muller 2010). Its key new features are (i) quasi-equilibration of the thermally labile beta-OH- and delta-OH-isoprenylperoxy isomers; (ii) 1,6-H shift isomerisation of the Z-delta-OH-peroxy isomers to yield hydroperoxy-methyl-butenals (HPALDs); (iii) fast photolysis of the HPALDs resulting overall in several OH radicals per HPALD. The OH-regeneration through photolabile HPALDs has recently found experimental support, but the peroxy isomerisation rate, HPALD yield and extent of OH recycling are still uncertain (Crounse et al. 2011; Wolfe et al. 2012). In this work, the upgraded LIM1 mechanism is presented. Based on much higher levels of theory that fully account for dispersion effects, the crucial equilibrium ratio of the isomerising Z-delta-OH-peroxys over the majority beta-OH-isoprenylperoxys is reduced by a factor ≈5 and the isomerisation rate of the Z-delta-OH-peroxys by a factor ≈1.5 compared to LIM0. The chemistry following the 1,6-H shift of the Z-delta-OH-peroxys is also much expanded and extended. Firstly, LIM1 introduces other pathways beside HPALD formation following the Z-delta-OH-peroxy isomerisation, but resulting likewise in OH recycling. This, together with the revised Z-delta-OH- equilibrium and isomerisation data above, affords a fair model-reproduction of the HPALD and other product yields observed by Crounse et al. (2011). Secondly, LIM1 proposes new fast reactions of HO2 with the alpha-oxoketene products from the peroxy isomerisation routes; these reactions are shown to efficiently convert HO2 into OH and are prime candidates for the unknown X + HO2 → OH + ... hydroxyl-recycling routes invoked in recent studies (Hofzumahaus et al.2009; Whalley et al. 2011). Modeling results using the IMAGES global CTM will be presented on

  8. Detection of cadherin-17 in human colon cancer LIM1215 cell secretome and tumour xenograft-derived interstitial fluid and plasma.

    PubMed

    Bernhard, Oliver K; Greening, David W; Barnes, Thomas W; Ji, Hong; Simpson, Richard J

    2013-11-01

    Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if detected early. However, 70% of CRC is detected at an advanced stage. This is largely due to the inadequacy of current faecal occult blood screening testing and costs involved in conducting population-based colonoscopy, the 'gold standard' for CRC detection. Another biomarker for CRC, carcinoembryonic antigen, while useful for monitoring CRC recurrence, is ineffective, lacking the specificity required early detection of CRC. For these reasons there is a need for more effective blood-based markers for early CRC detection. In this study we targeted glycoproteins secreted from the human colon carcinoma cell line LIM1215 as a source of potential CRC biomarkers. Secreted candidate glycoproteins were confirmed by MS and validated by Western blot analysis of tissue/tumour interstitial fluid (Tif) from LIM1215 xenograft tumours grown in immunocompromised mice. Overall, 39 glycoproteins were identified in LIM1215 culture media (CCM) and 5 glycoproteins in LIM1215 tumour xenograft Tif; of these, cadherin-17 (CDH17), galectin-3 binding protein (LGALS3BP), and tyrosine-protein kinase-like 7 (PTK7) were identified in both CM and glycosylation motifs. Swiss-Prot was used to annotate Tif. Many of the glycoproteins identified in this study (e.g., AREG, DSG2, EFNA1, EFNA3, EFNA4, EPHB4, ST14, and TIMP1) have been reported to be implicated in CRC biology. Interestingly, the cadherin-17 ectodomain, but not full length cadherin-17, was identified in CM, Tif and plasma derived from mice bearing the LIM1215 xenograft tumour. To our knowledge, this is the first report of the cadherin-17 ectodomain in plasma. In this study, we report for the first time that the presence of full-length cadherin-17 in exosomes released into the CM. This article is part of a Special Issue entitled: An Updated Secretome.

  9. Topics in Chemical Instrumentation: Information Management Systems in the Undergraduate Instrumental Analysis Laboratory: Part I. Introduction to LIMS.

    ERIC Educational Resources Information Center

    Merrer, Robert J.

    1985-01-01

    Provides an overview of Laboratory Information Management Systems (LIMS) and their implementation in undergraduate analytical laboratories. Basic components of a well-constructed LIMS system, hardware considerations, and software considerations are addressed. (JN)

  10. openBIS ELN-LIMS: an open-source database for academic laboratories

    PubMed Central

    Barillari, Caterina; Ottoz, Diana S. M.; Fuentes-Serna, Juan Mariano; Ramakrishnan, Chandrasekhar; Rinn, Bernd; Rudolf, Fabian

    2016-01-01

    Summary: The open-source platform openBIS (open Biology Information System) offers an Electronic Laboratory Notebook and a Laboratory Information Management System (ELN-LIMS) solution suitable for the academic life science laboratories. openBIS ELN-LIMS allows researchers to efficiently document their work, to describe materials and methods and to collect raw and analyzed data. The system comes with a user-friendly web interface where data can be added, edited, browsed and searched. Availability and implementation: The openBIS software, a user guide and a demo instance are available at https://openbis-eln-lims.ethz.ch. The demo instance contains some data from our laboratory as an example to demonstrate the possibilities of the ELN-LIMS (Ottoz et al., 2014). For rapid local testing, a VirtualBox image of the ELN-LIMS is also available. Contact: brinn@ethz.ch or fabian.rudolf@bsse.ethz.ch PMID:26508761

  11. Transcriptome and long noncoding RNA sequencing of three extracellular vesicle subtypes released from the human colon cancer LIM1863 cell line

    PubMed Central

    Chen, Maoshan; Xu, Rong; Ji, Hong; Greening, David W.; Rai, Alin; Izumikawa, Keiichi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Simpson, Richard J.

    2016-01-01

    Previously we reported that LIM1863 colorectal cancer (CRC) cells secrete three distinct extracellular vesicle subtypes – two subpopulations of exosomes (apical EpCAM-Exos and basolateral A33-Exos) and shed microvesicles (sMVs) – with distinct protein and miRNA signatures. Here, we extend our omics approach to understand the fundamental role of LIM1863-derived EVs by performing a comprehensive analysis of their mRNAs and long non-coding RNAs (lncRNAs) using RNA-Seq. We show that 2,389 mRNAs, 317 pseudogene transcripts, 1,028 lncRNAs and 206 short non-coding RNAs selectively distributed to (i.e., are enriched in) LIM1863 EVs, relative to the parent cell. An Ensembl/UniProtKB analysis revealed 1,937 mRNAs encode canonical proteins, 348 isoforms (including splice-variant proteins), and 119 ‘missing proteins’ (i.e., annotated in Ensembl but not UniProtKB). Further dissection of our protein/RNA data revealed that 6/151 observed RNA binding proteins have the potential to interact with ~75% of EV-enriched RNAs. Intriguingly, the co-existence of U1 and U2 ribonucleoproteins and their cognate snRNAs in LIM1863 EVs suggests a possible association of CRC EVs with recipient cell splicing events. Our data reveal several potential lncRNA CRC biomarkers and novel splicing/fusion genes that, collectively, will advance our understanding of EV biology in CRC and accelerate the development of EV-based diagnostics and therapeutics. PMID:27917920

  12. Early evolution of the LIM homeobox gene family

    SciTech Connect

    Srivastava, Mansi; Larroux, Claire; Lu, Daniel R; Mohanty, Kareshma; Chapman, Jarrod; Degnan, Bernard M; Rokhsar, Daniel S

    2010-01-01

    LIM homeobox (Lhx) transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known. We identified Lhx genes in two cnidarians and a placozoan and report the expression of Lhx genes during embryonic development in Nematostella and the demosponge Amphimedon. Members of the six major LIM homeobox subfamilies are represented in the genomes of the starlet sea anemone, Nematostella vectensis, and the placozoan Trichoplax adhaerens. The hydrozoan cnidarian, Hydra magnipapillata, has retained four of the six Lhx subfamilies, but apparently lost two others. Only three subfamilies are represented in the haplosclerid demosponge Amphimedon queenslandica. A tandem cluster of three Lhx genes of different subfamilies and a gene containing two LIM domains in the genome of T. adhaerens (an animal without any neurons) indicates that Lhx subfamilies were generated by tandem duplication. This tandem cluster in Trichoplax is likely a remnant of the original chromosomal context in which Lhx subfamilies first appeared. Three of the six Trichoplax Lhx genes are expressed in animals in laboratory culture, as are all Lhx genes in Hydra. Expression patterns of Nematostella Lhx genes correlate with neural territories in larval and juvenile polyp stages. In the aneural demosponge, A. queenslandica, the three Lhx genes are expressed widely during development, including in cells that are associated with the larval photosensory ring. The Lhx family expanded and diversified early in animal evolution, with all six subfamilies already diverged prior to the cnidarian-placozoan-bilaterian last common ancestor. In Nematostella, Lhx gene expression is correlated with neural

  13. Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog, Hic 5

    PubMed Central

    Ryan, Philip E.; Kales, Stephen C.; Yadavalli, Rajgopal; Nau, Marion M.; Zhang, Han; Lipkowitz, Stanley

    2012-01-01

    Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl-c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain. PMID:23145173

  14. The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

    PubMed

    Shirazi Fard, Shahrzad; Thyselius, Malin; All-Ericsson, Charlotta; Hallböök, Finn

    2014-01-01

    For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.

  15. The Nimbus 7 LIMS (Limb Infrared Monitor of the Stratosphere) water vapor measurements

    NASA Astrophysics Data System (ADS)

    Remsberg, Ellis E.; Russell, James M., III

    Earth orbital instruments, designed to measure the vertical and spatial distribution of atmospheric water vapor is discussed. Specifically, the operation of the Limb Infrared Monitor of the Stratosphere (LIMS) experiment is examined. The LIMS is a six channel limb scanning radiometer that was launched aboard Nimbus 7 in 1978. Profiles of stratospheric and mesospheric temperature, water vapor, and various other constituents were obtained by inverting the LIMS radiance measurements. This same technique was used in 1981 to analyze the data returned from another limb scanning radiometer aboard the Solar Mesosphere Explorer.

  16. LIM-homeodomain transcription factor Isl-1 mediates kisspeptin's effect on insulin secretion in mice.

    PubMed

    Chen, Juan; Fu, Rui; Cui, Yan; Pan, Jirong; Li, Yushan; Zhang, Xiaoxin; Evans, Sylvia M; Cui, Sheng; Liu, Jiali

    2014-08-01

    Kisspeptin and the G protein-coupled receptor 54 (GPR54) are highly abundant in the pancreas. In addition, circulating kisspeptin directly influences insulin secretion through GPR54. However, the mechanisms by which kisspeptin affects insulin release are unclear. The LIM-homeodomain transcription factor, Isl-1, is expressed in all pancreatic islet cells and is involved in regulating both islet development and insulin secretion. We therefore investigated potential interactions between kisspeptin and Isl-1. Our results demonstrate that Isl-1 and GPR54 are coexpressed in mouse pancreatic islet β-cells and NIT cells. Both in vitro and in vivo results demonstrate that kisspeptin-54 (KISS-54) inhibits Isl-1 expression and insulin secretion and both the in vivo and in vitro effects of KISS-54 on insulin gene expression and secretion are abolished when an Isl-1-inducible knockout model is used. Moreover, our results demonstrate that the direct action of KISS-54 on insulin secretion is mediated by Isl-1. Our results further show that KISS-54 influences Isl-1 expression and insulin secretion through the protein kinase C-ERK1/2 pathway. Conversely, insulin has a feedback loop via the Janus kinase-phosphatidylinositol 3-kinase pathway regulating kisspeptin expression and secretion. These findings are important in understanding mechanisms of insulin secretion and metabolism in diabetes.

  17. Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids.

    PubMed

    Tauro, Bow J; Greening, David W; Mathias, Rommel A; Mathivanan, Suresh; Ji, Hong; Simpson, Richard J

    2013-03-01

    Exosomes are naturally occurring biological nanomembranous vesicles (∼40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to clearly distinguish A33- and EpCAM-Exos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44

  18. Intercomparison of nitrogen-containing species in Nimbus 7 LIMS and SAMS Data

    SciTech Connect

    Jackman, C.H.; Guthrie, P.D.; Kaye, J.A.

    1987-01-20

    Nitrogen dioxide (NO/sub 2/) and nitric acid (HNO/sub 3/) distributions were derived with a two-dimensional time-dependent model using N/sub 2/O, CH/sub 4/, and temperature measurements from the stratospheric and mesospheric sounder (SAMS) along with H/sub 2/O measurements from the limb infrared monitor of the stratosphere (LIMS) and O/sub 3/ measurements from the solar backscatter ultraviolet (SBUV) instrument. All three instruments (SAMS, LIMS, and SBUV) were aboard the Nimbus 7 satellite. The computed NO/sub 2/ and HNO/sub 3/ were compared with LIMS NO/sub 2/ and HNO/sub 3/ measurements. Calculated NO/sub 2/ is lower than the LIMS NO/sub 2/ in much of the lower stratosphere by about a factor of 4 or more. Differences in the upper stratosphere between derived NO/sub 2/ and LIMS NO/sub 2/ are within the photochemical uncertainties of the computation. Derived NO/sub 2/ is much larger than LIMS NO/sub 2/ at high latitudes in the upper stratosphere in December, indicating that dynamics and/or photochemistry in the atmosphere are different from those used in the model. Derived HNO/sub 3/ is in fairly good agreement with LIMS HNO/sub 3/ in the upper stratosphere. Derived and LIMS HNO/sub 3/ gradually become more different through the middle stratosphere and reach a fairly substantial disagreement in the lower stratosphere. The high-latitude maxima observed in LIMS HNO/sub 3/ are present at lower altitudes and at higher concentrations than those derived in the model computations. More significantly, the derived HNO/sub 3/ at polar latitudes is highest in the summer while LIMS HNO/sub 3/ is highest in the winter. Disagreements in the lower stratosphere between the computed and LIMS NO/sub 2/ and HNO/sub 3/ can only be reduced if two changes are made in the model computations: (1) additional lower stratospheric sources of odd nitrogen (other than N/sub 2/O+O(/sup 1/D)) are included and (2) a modified chemistry to allow the formation of HNO/sub 3/ at the expense of N/sub 2

  19. Purification, crystallization and preliminary X-ray analysis of a fusion of the LIM domains of LMO2 and the LID domain of Ldb1.

    PubMed

    El Omari, Kamel; Porcher, Catherine; Mancini, Erika J

    2010-11-01

    LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, β = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Å and the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress.

  20. The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans.

    PubMed

    Cassata, G; Kagoshima, H; Andachi, Y; Kohara, Y; Dürrenberger, M B; Hall, D H; Bürglin, T R

    2000-03-01

    In Caenorhabditis elegans three pairs of neurons, AFD, AIY, and AIZ, play a key role in thermosensation. The LIM homeobox gene ceh-14 is expressed in the AFD thermosensory neurons. ceh-14 mutant animals display athermotactic behaviors, although the neurons are still present and differentiated. Two other LIM homeobox genes, ttx-3 and lin-11, function in the two interneurons AIY and AIZ, respectively. Thus, the three key thermosensory neurons are specified by three different LIM homeobox genes. ceh-14 ttx-3 lin-11 triple mutant animals have a basic cryophilic thermotaxis behavior indicative of a second thermotaxis pathway. Misexpression of ceh-14 in chemosensory neurons can restore thermotactic behavior without impairing the chemosensory function. Thus, ceh-14 confers thermosensory function to neurons.

  1. The geometry structures and electronic properties of LimBn (m + n = 12) clusters

    NASA Astrophysics Data System (ADS)

    Ruan, Wen; Xie, An-Dong; Wu, Dong-Lan; Luo, Wen-Lang; Yu, Xiao-Guang

    2014-03-01

    The geometric structures, electronic properties, total and binding energies, harmonic frequencies, the highest occupied molecular orbital to the lowest unoccupied molecular orbital energy gaps, and the vertical ionization potential energies of small LimBn (m+n = 12) clusters were investigated by the density functional theory B3LYP with a 6-311+G (2d, 2p) basis set. All the calculations were performed using the Gaussian09 program. For the study of the LimBn clusters, the global minimum of the B12 cluster was chosen as the starting point and the boron atoms were gradually replaced by Li atoms. The results showed that as the number of Li atoms increased, the stability of the LimBn cluster decreased and the physical and chemical properties became more active. In addition, on average there was a large charge transfer from the Li atoms to the B atoms.

  2. An intercomparison of ozone profile measurements from LIMS, SAGE, and SBUV

    NASA Technical Reports Server (NTRS)

    Fleig, A. J.; Bhartia, P. K.; Wong, C. K.; Klenk, K. F.

    1983-01-01

    Ozone profile measurements by the Limb IR Monitor of Stratosphere (LIMS) and the Solar Backscatter UV (SBUV) aboard the Nimbus satellite, and the Stratospheric Aerosol and Gas Experiment (SAGE) aboard the AEM-2 satellite are intercompared in an effort to assess the quality of satellite ozone retrieval techniques. Good correlation between LIMS and SBUV observations is noted in the ozone layer bounded by pressures of 31.2-15.6 mb; absolute differences are generally less than 10 percent, with similar zonal variations. Above 35 km SAGE values are systematically larger than SBUV or LIMS. The differences generally increase with height and are largest in the tropics. Finally, excellent agreement is noted to exist among the three data sets between 10 and 35 km of altitude with respect to the latitude dependence of the ozone profiles.

  3. Technical Considerations in Remote LIMS Access via the World Wide Web

    PubMed Central

    Schlabach, David M.

    2005-01-01

    The increased dependency on the World Wide Web by both laboratories and their customers has led LIMS developers to take advantage of thin-client web applications that provide both remote data entry and manipulation, along with remote reporting functionality. Use of an LIMS through a web browser allows a person to interact with a distant application, providing both remote administration and real-time analytical result delivery from virtually anywhere in the world. While there are many benefits of web-based LIMS applications, some concern must be given to these new methods of system architecture before justifying them as a suitable replacement for their traditional client-server systems. Developers and consumers alike must consider the security aspects of introducing a wide area network capable system into a production environment, as well as the concerns of data integrity and usability. PMID:18924736

  4. On the quality of the Nimbus 7 LIMS Version 6 water vapor profiles and distributions

    NASA Astrophysics Data System (ADS)

    Remsberg, E. E.; Natarajan, M.; Lingenfelser, G. S.; Thompson, R. E.; Marshall, B. T.; Gordley, L. L.

    2009-12-01

    This report describes the quality of the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) water vapor (H2O) profiles of 1978/79 that were processed with a Version 6 (V6) algorithm and archived in 2002. The V6 profiles incorporate a better knowledge of the instrument attitude for the LIMS measurements along its orbits, leading to improvements for its temperature profiles and for the registration of its water vapor radiances with pressure. As a result, the LIMS V6 zonal-mean distributions of H2O exhibit better hemispheric symmetry than was the case from the original Version 5 (V5) dataset that was archived in 1982. Estimates of the precision and accuracy of the V6 H2O profiles are developed and provided. Individual profiles have a precision of order 5% and an estimated accuracy of about 19% at 3 hPa, 14% at 10 hPa, and 26% at 50 hPa. Profile segments within about 2 km of the tropopause are often affected by emissions from clouds that appear in the finite field-of-view of the detector for the LIMS H2O channel. Zonally-averaged distributions of the LIMS V6 H2O are compared with those from the more recent Microwave Limb Sounder (MLS) satellite experiment for November, February, and May of 2004/05. The patterns and values of their respective distributions are similar in many respects. Effects of a strengthened Brewer-Dobson circulation are indicated in the MLS distributions of the recent decade versus those of LIMS from 1978/79. A tropical tape recorder signal is present in the 7-month time series of LIMS V6 H2O with lowest values in February 1979, and the estimated, annually-averaged "entry-level" H2O is 3.5 to 3.8 ppmv. It is judged that this historic LIMS water vapor dataset is of good quality for studies of the near global-scale chemistry and transport for pressure levels from 3 hPa to about 70 to 100 hPa.

  5. On the quality of the Nimbus 7 LIMS Version 6 water vapor profiles and distributions

    NASA Astrophysics Data System (ADS)

    Remsberg, E. E.; Natarajan, M.; Lingenfelser, G. S.; Thompson, R. E.; Marshall, B. T.; Gordley, L. L.

    2009-09-01

    This report describes the quality of the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) water vapor (H2O) profiles of 1978/1979 that were processed with a Version 6 (V6) algorithm and archived in 2002. The V6 profiles incorporate a better knowledge of the instrument attitude for the LIMS measurements along its orbits, leading to improvements for its temperature profiles and for the registration of its water vapor radiances with pressure. As a result, the LIMS V6 zonal-mean distributions of H2O exhibit better hemispheric symmetry than was the case from the original Version 5 (V5) dataset that was archived in 1982. Estimates of the precision and accuracy of the V6 H2O profiles are developed and provided. Individual profiles have a precision of order 5% and an estimated accuracy of about 19% at 3 hPa, 14% at 10 hPa, and 26% at 50 hPa. Profile segments within about 2 km of the tropopause are often affected by emissions from clouds that appear in the finite field-of-view of the detector for the LIMS H2O channel. Zonally-averaged distributions of the LIMS V6 H2O are compared with those from the more recent Microwave Limb Sounder (MLS) satellite experiment for November, February, and May of 2004/2005. The patterns and values of their respective distributions are similar in many respects. Effects of a strengthened Brewer-Dobson circulation are indicated in the MLS distributions of the recent decade versus those of LIMS from 1978/1979. A tropical tape recorder signal is present in the 7-month time series of LIMS V6 H2O with lowest values in February 1979, and the estimated, annually-averaged "entry-level" H2O is 3.5 to 3.8 ppmv. It is judged that this historic LIMS water vapor dataset is of good quality for studies of the near global-scale chemistry and transport for pressure levels from 3 hPa to about 70 to 100 hPa.

  6. On the Quality of the Nimbus 7 LIMS Version 6 Water Vapor Profiles and Distributions

    NASA Technical Reports Server (NTRS)

    Remsberg, E. E.; Natarajan, M.; Lingenfelser, G. S.; Thompson, R. E.; Marshall, B. T.; Gordley, L. L.

    2009-01-01

    This report describes the quality of the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) water vapor (H2O) profiles of 1978/79 that were processed with a Version 6 (V6) algorithm and archived in 2002. The V6 profiles incorporate a better knowledge of the instrument attitude for the LIMS measurements along its orbits, leading to improvements for its temperature profiles and for the registration of its water vapor radiances with pressure. As a result, the LIMS V6 zonal-mean distributions of H2O exhibit better hemispheric symmetry than was the case from the original Version 5 (V5) dataset that was archived in 1982. Estimates of the precision and accuracy of the V6 H2O profiles are developed and provided. Individual profiles have a precision of order 5% and an estimated accuracy of about 19% at 3 hPa, 14% at 10 hPa, and 26% at 50 hPa. Profile segments within about 2 km of the tropopause are often affected by emissions from clouds that appear in the finite field-of-view of the detector for the LIMS H2O channel. Zonally-averaged distributions of the LIMS V6 H2O are compared with those from the more recent Microwave Limb Sounder (MLS) satellite experiment for November, February, and May of 2004/2005. The patterns and values of their respective distributions are similar in many respects. Effects of a strengthened Brewer-Dobson circulation are indicated in the MLS distributions of the recent decade versus those of LIMS from 1978/79. A tropical tape recorder signal is present in the 7-month time series of LIMS V6 H2O with lowest values in February 1979, and the estimated, annually-averaged "entry-level" H2O is 3.5 to 3.8 ppmv. It is judged that this historic LIMS water vapor dataset is of good quality for studies of the near global-scale chemistry and transport for pressure levels from 3 hPa to about 70 to 100 hPa.

  7. Caldera-related gold mineralization of the El Limón mining district, western Nicaragua

    NASA Astrophysics Data System (ADS)

    Malone, Gary B.; Stoiber, Richard E.

    1987-08-01

    The town of El Limón, the center of a small gold mining district, lies 36 km northeast of León, Nicaragua. This paper reports on the sequence of volcanic rocks in the district, the structures in these volcanics and the relationship of the gold veins to them.

  8. Energetic particle precipitation effects on the Northern Hemisphere stratosphere observed by LIMS

    NASA Astrophysics Data System (ADS)

    Holt, L. A.; Randall, C. E.; Harvey, L.; Funke, B.; Stiller, G. P.

    2009-12-01

    Energetic particle precipitation (EPP) in the upper atmosphere contributes to polar stratospheric enhancements of NOx. The first experimental evidence of this emerged when the Limb Infrared Monitor of the Stratosphere (LIMS) observed stratospheric NOx enhancements during the Arctic winter of 1978/79. Such enhancements have since been observed on numerous occasions, but until recently were much less obvious in the Northern Hemisphere than in the Southern Hemisphere. It is now understood that the magnitude of these stratospheric NOx enhancements depends on both the level of EPP and dynamical conditions. Three out of the last six Arctic winters have seen much larger than average polar stratospheric NOx enhancements due to EPP that have been attributed to extraordinary meteorological events. These are unique events on record and affirm that even with low levels of EPP, the stratosphere can still be influenced to a large degree by EPP. In this study, data from the LIMS instrument is revisited in order to understand the NOx enhancement it observed with respect to meteorological conditions and EPP activity. The temporal evolution of NOx in the polar vortex as measured by LIMS is compared to more recent satellite data, including the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) instrument. Preliminary results indicate that the enhancements are caused neither by elevated EPP nor unusual dynamical conditions, but rather are seen because of the ability of LIMS to observe in the polar night.

  9. On the Validation of the Nimbus 7 LIMS Version 6 Stratospheric Ozone

    NASA Astrophysics Data System (ADS)

    Remsberg, E.; Natarajan, M.; Thomason, L.; Lingenfelser, G.

    2005-12-01

    The Nimbus 7 LIMS experiment obtained daily distributions of ozone from late October 1978 through late May 1979 and with good vertical resolution and spatial sampling along its orbital tangent tracks. The historic LIMS Version 5 (V5) dataset was archived in 1982. Studies of those data revealed detailed information about the roles of chemistry and transport on the distribution of ozone in the stratosphere for the latitudes of 64S to 84N. Although its profiles were of good quality throughout most of the stratosphere, both the simulation and validation studies indicated that the accuracy of the V5 ozone became less good below about the 30-hPa level at low latitudes and below the 50-hPa level at middle and high latitudes, especially during polar winter. A LIMS Version 6 (V6) algorithm was developed some years later, in part, to improve on the quality of the ozone profiles in the lower stratosphere. That V6 profile dataset was created in 2001 and archived in 2002. In this paper we present comparisons of the LIMS V6 ozone with the SBUV Version 8 (V8) data, with the preliminary SAGE I Version 6 data, and with balloon ECC ozonesonde soundings from several stations. The findings indicate significant improvements in accuracy for LIMS V6 ozone in the lower stratosphere and better vertical and horizontal sampling for the changes in ozone at polar latitudes and throughout the Arctic winter period. This revised ozone dataset should provide for a better assessment of the effects of chemistry and transport on polar and middle latitude ozone for the Northern Hemisphere winter of 1978-79, when the loss of ozone due to reactive chlorine from heterogeneous processes was not as significant as it is today.

  10. Hydroxyl Radical Regeneration in Isoprene Oxidation: the Upgraded Mechanism LIM1

    NASA Astrophysics Data System (ADS)

    Peeters, J.; Nguyen, S.; Nguyen, T.; Stavrakou, T.; Muller, J. J.

    2012-12-01

    Measured hydroxyl radical concentrations in isoprene-rich areas are much higher than predicted by existing chemical models, to the extent that the global oxidizing capacity of our atmosphere should be significantly revised upwards. The OH regeneration that clearly occurs in isoprene oxidation at low/moderate NO is attributed in the Leuven Isoprene Mechanism to novel, theoretically characterized chemical pathways (LIM0: Peeters et al. 2009; Peeters and Muller 2010). The key new features of LIM0 are (i) thermal equilibration of the labile beta-OH- and delta-OH-isoprenylperoxy isomers; (ii) 1,6-H shift isomerisation of the Z-delta-OH-peroxy isomers to yield hydroperoxy-methyl-butenals (HPALDs); (iii) fast photolysis of the HPALDs resulting overall in several OH radicals per HPALD. The OH-regeneration through photolabile HPALDs has recently found experimental support, but the peroxy isomerisation rate, HPALD yield and the extent of OH recycling are still uncertain (Crounse et al. 2011; Wolfe et al. 2012). In this work, the upgraded LIM1 mechanism is presented. Based on better levels of theory, the crucial equilibrium ratio of the isomerising Z-delta-OH-peroxys over the majority beta-OH-isoprenylperoxys had to be reduced by a factor of about 5 compared to LIM0, while the isomerisation rate of the Z-delta-OH-peroxys adopted from Taraborrelli et al. (2012) is about 3 times lower than in LIM0. The chemistry following the 1,6-H shift of the Z-delta-OH-peroxys is much expanded and extended. Firstly, LIM1 introduces other pathways beside HPALD formation following the Z-delta-OH-peroxy isomerisation, resulting likewise in OH recycling. This, together with the revised Z-delta-OH- equilibrium and isomerisation data above, affords a close model-reproduction of the HPALD and other product yields observed by Crounse et al. (2011). Secondly, LIM1 proposes new fast reactions of HO2 with the alpha-oxoketene products from the peroxy isomerisation routes; these reactions are shown to

  11. Elevated LIM kinase 1 in nonmetastatic prostate cancer reflects its role in facilitating androgen receptor nuclear translocation.

    PubMed

    Mardilovich, Katerina; Gabrielsen, Mads; McGarry, Lynn; Orange, Clare; Patel, Rachana; Shanks, Emma; Edwards, Joanne; Olson, Michael F

    2015-01-01

    Prostate cancer affects a large proportion of the male population, and is primarily driven by androgen receptor (AR) activity. First-line treatment typically consists of reducing AR signaling by hormone depletion, but resistance inevitably develops over time. One way to overcome this issue is to block AR function via alternative means, preferably by inhibiting protein targets that are more active in tumors than in normal tissue. By staining prostate cancer tumor sections, elevated LIM kinase 1 (LIMK1) expression and increased phosphorylation of its substrate Cofilin were found to be associated with poor outcome and reduced survival in patients with nonmetastatic prostate cancer. A LIMK-selective small molecule inhibitor (LIMKi) was used to determine whether targeted LIMK inhibition was a potential prostate cancer therapy. LIMKi reduced prostate cancer cell motility, as well as inhibiting proliferation and increasing apoptosis in androgen-dependent prostate cancer cells more effectively than in androgen-independent prostate cancer cells. LIMK inhibition blocked ligand-induced AR nuclear translocation, reduced AR protein stability and transcriptional activity, consistent with its effects on proliferation and survival acting via inhibition of AR activity. Furthermore, inhibition of LIMK activity increased αTubulin acetylation and decreased AR interactions with αTubulin, indicating that the role of LIMK in regulating microtubule dynamics contributes to AR function. These results indicate that LIMK inhibitors could be beneficial for the treatment of prostate cancer both by reducing nuclear AR translocation, leading to reduced proliferation and survival, and by inhibiting prostate cancer cell dissemination.

  12. Roles of LIM kinases in central nervous system function and dysfunction.

    PubMed

    Cuberos, H; Vallée, B; Vourc'h, P; Tastet, J; Andres, C R; Bénédetti, H

    2015-12-21

    LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin. In this review, we outline studies that have shown an involvement of LIMKs in neuronal function and we detail some of the pathways and molecular mechanisms involving LIMKs in neurodevelopment and synaptic plasticity. We also review the involvement of LIMKs in neuronal diseases and emphasize the differences in the regulation of LIMKs expression and mode of action. We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.

  13. Variability of stratospheric nitrogen compounds observed by LIMS in the winter of 1978-1979

    SciTech Connect

    Russell, J.M. III; Remsberg, E.E.; Gordley, L.L.

    1982-01-01

    The LIMS experiment was launched on the Nimbus 7 satellite for the purpose of sounding the vertical structure of temperature and key upper atmosphere trace gases on a global scale. The technique of thermal infrared limb sounding was used to obtain measurements of O3, H2O, NO2, and HNO3. LIMS collected data almost continuously from late October to late May over the latitude range from 64 deg S to 84 deg N. Two of the gases, NO2 and HNO3, are important elements in the NO(x) chain of chemical reactions leading to ozone destruction. Results for these gases are described in terms of zonal mean profiles and latitudinal distributions. The period selected for study is January-May 1979, when a major stratospheric warming occurred.

  14. Ozone budget in the upper stratosphere: Model studies using the reprocessed LIMS and the HALOE datasets

    NASA Astrophysics Data System (ADS)

    Natarajan, Murali; Remsberg, Ellis E.; Gordley, Larry L.

    2002-04-01

    Recently reprocessed LIMS dataset has been used with a contemporary photochemical model to study the balance between photochemical production and destruction of ozone in the upper stratosphere. Model results corresponding to January 1979 indicate that the ozone deficit is less than 15% in the pressure range of 5 to 0.5 mb between 50°S and 50°N latitude. The imbalance at 40 km is much smaller than reported by the earliest studies with the archived LIMS data. The same model, when initialized with HALOE (version 19) data for January, 1996, shows similar results with peak ozone deficits being less than 10%. For both cases, the model shows a near balance in the ozone budget above 1 mb, contrary to recent studies based on balloon-borne measurements. The magnitude of the ozone imbalance seen in this study is within the uncertainties of the data and model.

  15. Optimized conditions for selective gold flotation by ToF-SIMS and ToF-LIMS

    NASA Astrophysics Data System (ADS)

    Chryssoulis, S. L.; Dimov, S. S.

    2004-06-01

    This work describes a comprehensive characterization of the factors controlling the floatability of free gold from flotation test using reagents (collectors) at plant concentration levels. A relationship between the collectors loadings on gold particles and their surface composition has been established. The findings of this study show that silver activates gold flotation and there is a strong correlation between the surface concentration of silver and the loading of certain collectors. The organic surface analysis was done by ToF-SIMS while the inorganic surface analysis was carried out by time-of-flight laser ionization mass spectrometry (ToF-LIMS). The developed testing protocol based on ToF-LIMS and ToF-SIMS complementary surface analysis allows for optimization of the flotation scheme and hence improved gold recovery.

  16. Estimation of synoptic fields of middle atmosphere parameters from Nimbus-7 LIMS profile data

    NASA Technical Reports Server (NTRS)

    Remsberg, Ellis E.; Haggard, Kenneth V.; Russell, James M., III

    1990-01-01

    The most widely used version of the Nimbus-7 middle atmosphere dataset is the set of high quality, daily, and zonal Fourier coefficients that resolve information out to six wavenumbers at 12 UTC. A Kalman filter algorithm was applied to the original profile data in order to generate those fields for the data archive or LAMAT product. The characteristics and implementation of the algorithm are described in some detail, along with examples of the output for each of the LIMS parameters.

  17. ms_lims, a simple yet powerful open source laboratory information management system for MS-driven proteomics.

    PubMed

    Helsens, Kenny; Colaert, Niklaas; Barsnes, Harald; Muth, Thilo; Flikka, Kristian; Staes, An; Timmerman, Evy; Wortelkamp, Steffi; Sickmann, Albert; Vandekerckhove, Joël; Gevaert, Kris; Martens, Lennart

    2010-03-01

    MS-based proteomics produces large amounts of mass spectra that require processing, identification and possibly quantification before interpretation can be undertaken. High-throughput studies require automation of these various steps, and management of the data in association with the results obtained. We here present ms_lims (http://genesis.UGent.be/ms_lims), a freely available, open-source system based on a central database to automate data management and processing in MS-driven proteomics analyses.

  18. H2O concentration in the middle atmosphere: Processing of LIMS radiance measurements with a research algorithm

    NASA Astrophysics Data System (ADS)

    Fischer, H.

    Transmittance functions as well as inversion algorithms have been developed for deriving H2O profiles from radiometer measurements. These computer programs have been applied to evaluate own stratospheric balloon occultation measurements and LIMS (Limb Infrared Monitor of the Stratosphere) radiance measurements in the H2O channel. The results are compared with the H2O profiles in the LIMS data archive. The differences between corresponding H2O profiles are discussed in dependence of altitude and latitude.

  19. The LIM homeobox gene ceh-14 is required for phasmid function and neurite outgrowth.

    PubMed

    Kagoshima, Hiroshi; Cassata, Giuseppe; Tong, Yong Guang; Pujol, Nathalie; Niklaus, Gisela; Bürglin, Thomas R

    2013-08-15

    Transcription factors play key roles in cell fate specification and cell differentiation. Previously, we showed that the LIM homeodomain factor CEH-14 is expressed in the AFD neurons where it is required for thermotaxis behavior in Caenorhabditis elegans. Here, we show that ceh-14 is expressed in the phasmid sensory neurons, PHA and PHB, a number of neurons in the tail, i.e., PHC, DVC, PVC, PVN, PVQ, PVT, PVW and PVR, as well as the touch neurons. Analysis of the promoter region shows that important regulatory elements for the expression in most neurons reside from -4kb to -1.65kb upstream of the start codon. Further, within the first introns are elements for expression in the hypodermis. Phylogenetic footprinting revealed numerous conserved motifs in these regions. In addition to the existing deletion mutation ceh-14(ch3), we isolated a new allele, ceh-14(ch2), in which only one LIM domain is disrupted. The latter mutant allele is partially defective for thermosensation. Analysis of both mutant alleles showed that they are defective in phasmid dye-filling. However, the cell body, dendritic outgrowth and ciliated endings of PHA and PHB appear normal, indicating that ceh-14 is not required for growth. The loss of a LIM domain in the ceh-14(ch2) allele causes a partial loss-of-function phenotype. Examination of the neurites of ALA and tail neurons using a ceh-14::GFP reporter shows abnormal axonal outgrowth and pathfinding.

  20. Gravity Wave Variance in LIMS Temperatures. Part II: Comparison with the Zonal-Mean Momentum Balance.

    NASA Astrophysics Data System (ADS)

    Fetzer, Eric J.; Gille, John C.

    1996-02-01

    Zonal-mean gravity wave variance in the Limb Infrared Monitor of the Stratosphere (LIMS) temperature data is seen to correlate strongly with the residual term in the LIMS zonal-mean momentum budget throughout much of the observed mesosphere. This momentum residual is attributed to gravity wave momentum transport at scales that cannot be directly sampled by the LIMS instrument Correlation is highest in the vicinity of the fall and winter mesospheric jets, where both gravity wave variance and momentum residual reach their largest values. Correlation is also high in the Southern Hemisphere subtropical mesophere, where gravity wave variance and the momentum residual have broad temporal maxima during the easterly acceleration of the stratopause semi-annual oscillation (SAO). This subtropical correlation has important implications for the SAO eastward acceleration, which several studies suggest is forced by gravity wave momentum flux divergence. Correlation between gravity wave variance and inferred gravity wave momentum flux divergence is unexpected because variance is dominated by large scales and long periods (inertio-gravity waves), while both theoretical arguments and ground-based observations indicate that momentum transport is dominated by periods under 1 h. The results of this study suggest a broadband gravity wave field experiencing forcing and loss processes, which are largely independent of frequency.

  1. A Reassessment of the Middle Atmosphere Heat Budget Using Nimbus 7 LIMS Data

    NASA Astrophysics Data System (ADS)

    Mertens, C. J.; Remsberg, E. E.; Mlynczak, M. G.; Natarajan, M.; Gordley, L. L.

    2002-05-01

    A reprocessing of the Nimbus 7 LIMS Level 2 dataset (its Version 6 or V6 profiles) is now complete. The revised algorithm employs a number of changes that affect its temperature T(p) and species profiles. Comparisons with the archived (Version 5 or V5) dataset show that the V6 temperatures are warmer in the mesosphere and in better agreement with correlative rocket profiles. V6 T(p) is cooler by several degrees at high latitudes of the winter upper stratosphere and by about 1 K at all latitudes in the middle stratosphere. V6 ozone is larger than V5 by about 10 percent at 2 to 3 mb, but smaller by about 5 to 10 percent in the middle mesosphere. These changes in temperature and ozone reduce the radiative imbalances that were reported for the stratosphere by Mlynczak et al. (JGR, p. 6039, 1999). Imbalances decrease but are still present in the middle mesosphere because of the non-LTE effects in CO2 and O3 that have not been accounted for in the retrieval of daytime LIMS ozone, in particular. The radiative heating and cooling and the net radiative balance for the V6 dataset will be presented along with revised estimates of the induced diabatic circulation. These new findings are an excellent indication of the quality of LIMS V6 temperature and ozone profiles.

  2. LIM Kinase 1 Modulates Cortical Actin and CXCR4 Cycling and Is Activated by HIV-1 to Initiate Viral Infection*

    PubMed Central

    Vorster, Paul J.; Guo, Jia; Yoder, Alyson; Wang, Weifeng; Zheng, Yanfang; Xu, Xuehua; Yu, Dongyang; Spear, Mark; Wu, Yuntao

    2011-01-01

    Almost all viral pathogens utilize a cytoskeleton for their entry and intracellular transport. In HIV-1 infection, binding of the virus to blood resting CD4 T cells initiates a temporal course of cortical actin polymerization and depolymerization, a process mimicking the chemotactic response initiated from chemokine receptors. The actin depolymerization has been suggested to promote viral intracellular migration through cofilin-mediated actin treadmilling. However, the role of the virus-mediated actin polymerization in HIV infection is unknown, and the signaling molecules involved remain unidentified. Here we describe a pathogenic mechanism for triggering early actin polymerization through HIV-1 envelope-mediated transient activation of the LIM domain kinase (LIMK), a protein that phosphorylates cofilin. We demonstrate that HIV-mediated LIMK activation is through gp120-triggered transient activation of the Rack-PAK-LIMK pathway, and that knockdown of LIMK through siRNA decreases filamentous actin, increases CXCR4 trafficking, and diminishes viral DNA synthesis. These results suggest that HIV-mediated early actin polymerization may directly regulate the CXCR4 receptor during viral entry and is involved in viral DNA synthesis. Furthermore, we also demonstrate that in resting CD4 T cells, actin polymerization can be triggered through transient treatment with a pharmacological agent, okadaic acid, that activates LIMK and promotes HIV latent infection of resting CD4 T cells. Taken together, our results suggest that HIV hijacks LIMK to control the cortical actin dynamics for the initiation of viral infection of CD4 T cells. PMID:21321123

  3. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop.

    PubMed

    Ohashi, K; Nagata, K; Maekawa, M; Ishizaki, T; Narumiya, S; Mizuno, K

    2000-02-04

    LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.

  4. Role of the New South Wales Department of Primary Industries' Laboratory Information Management System (LIMS) in the 2007 equine influenza emergency animal disease response.

    PubMed

    Croft, M G; Fraser, G C; Gaul, W N

    2011-07-01

    A Laboratory Information Management System (LIMS) was used to manage the laboratory data and support planning and field activities as part of the response to the equine influenza outbreak in Australia in 2007. The database structure of the LIMS and the system configurations that were made to best handle the laboratory implications of the disease response are discussed. The operational aspects of the LIMS and the related procedures used at the laboratory to process the increased sample throughput are reviewed, as is the interaction of the LIMS with other corporate systems used in the management of the response. Outcomes from this tailored configuration and operation of the LIMS resulted in effective provision and control of the laboratory and laboratory information aspects of the response. The extent and immediate availability of the information provided from the LIMS was critical to some of the activities of key operatives involved in controlling the response.

  5. PP2A binds to the LIM domains of lipoma-preferred partner through its PR130/B″ subunit to regulate cell adhesion and migration

    PubMed Central

    Janssens, Veerle; Zwaenepoel, Karen; Rossé, Carine; Petit, Marleen M. R.; Goris, Jozef; Parker, Peter J.

    2017-01-01

    Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn2+-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP–PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130–LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics. PMID:26945059

  6. On the Quality of the Nimbus 7 LIMS Version 6 Ozone for Studies of the Middle Atmosphere

    NASA Technical Reports Server (NTRS)

    Remsberg, Ellis; Lingenfelser, Gretchen; Natarajan, Murali; Gordley, Larry; Thompson, Earl

    2006-01-01

    The Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) radiance profile dataset of 1978/79 was reconditioned and reprocessed to Version 6 (V6) profiles of temperature and species that are improved significantly over those from Version 5 (V5). The LIMS V6 dataset was archived for public use in 2002. Improvements for its ozone include: (1) a more accurate accounting for instrument and spacecraft motion effects in the radiances, (2) the use of better spectroscopic line parameters for its ozone forward model, (3) retrievals of all its scans, (4) more accurate and compatible temperature versus pressure profiles (or T(p)), which are needed for the registration of the ozone radiances and for the removal of temperature effects from them, and (5) a better accounting for interfering species in the lower stratosphere. The retrieved V6 ozone profiles extend from near cloud top altitudes to about 80 km and from 64S to 84N latitude with better sampling along the orbit than for the V5 dataset. Calculated estimates of the single-profile precision and accuracy are provided for the V6 ozone from this study. Precision estimates based on the data themselves are of order 3% or better from 1 to 30 hPa. Estimates of total systematic error for a single profile are hard to generalize because the separate sources of error may not all be of the same sign and they depend somewhat on the atmospheric state. It is estimated that the V6 zonal mean ozone distributions are accurate to within 9% to 7% from 50 hPa to 3 hPa, respectively. Effects of a temperature bias can be significant and may be present at 1 to 2 hPa though. There may be ozone biases of order 10% at those levels due to possible biases of up to +2 K, but there is no indication of a similar problem elsewhere in the stratosphere. Simulation studies show that the LIMS retrievals are also underestimating slightly the small amplitudes of the atmospheric temperature tides, which affect its retrieved day/night ozone differences

  7. adLIMS: a customized open source software that allows bridging clinical and basic molecular research studies

    PubMed Central

    2015-01-01

    Background Many biological laboratories that deal with genomic samples are facing the problem of sample tracking, both for pure laboratory management and for efficiency. Our laboratory exploits PCR techniques and Next Generation Sequencing (NGS) methods to perform high-throughput integration site monitoring in different clinical trials and scientific projects. Because of the huge amount of samples that we process every year, which result in hundreds of millions of sequencing reads, we need to standardize data management and tracking systems, building up a scalable and flexible structure with web-based interfaces, which are usually called Laboratory Information Management System (LIMS). Methods We started collecting end-users' requirements, composed of desired functionalities of the system and Graphical User Interfaces (GUI), and then we evaluated available tools that could address our requirements, spanning from pure LIMS to Content Management Systems (CMS) up to enterprise information systems. Our analysis identified ADempiere ERP, an open source Enterprise Resource Planning written in Java J2EE, as the best software that also natively implements some highly desirable technological advances, such as the high usability and modularity that grants high use-case flexibility and software scalability for custom solutions. Results We extended and customized ADempiere ERP to fulfil LIMS requirements and we developed adLIMS. It has been validated by our end-users verifying functionalities and GUIs through test cases for PCRs samples and pre-sequencing data and it is currently in use in our laboratories. adLIMS implements authorization and authentication policies, allowing multiple users management and roles definition that enables specific permissions, operations and data views to each user. For example, adLIMS allows creating sample sheets from stored data using available exporting operations. This simplicity and process standardization may avoid manual errors and

  8. Interaction of subway LIM vehicle with ballasted track in polygonal wheel wear development

    NASA Astrophysics Data System (ADS)

    Li, Ling; Xiao, Xin-Biao; Jin, Xue-Song

    2011-04-01

    This paper develops a coupled dynamics model for a linear induction motor (LIM) vehicle and a subway track to investigate the influence of polygonal wheels of the vehicle on the dynamic behavior of the system. In the model, the vehicle is modeled as a multi-body system with 35 degrees of freedom. A Timoshenko beam is used to model the rails which are discretely supported by sleepers. The sleepers are modeled as rigid bodies with their vertical, lateral, and rolling motions being considered. In order to simulate the vehicle running along the track, a moving sleeper support model is introduced to simulate the excitation by the discrete sleeper supporters, in which the sleepers are assumed to move backward at a constant speed that is the same as the train speed. The Hertzian contact theory and the Shen-Hedrick-Elkins' model are utilized to deal with the normal dynamic forces and the tangential forces between wheels and rails, respectively. In order to better characterize the linear metro system (LMS), Euler beam theory based on modal superposition method is used to model LIM and RP. The vertical electric magnetic force and the lateral restoring force between the LIM and RP are also taken into consideration. The former has gap-varying nonlinear characteristics, whilst the latter is considered as a constant restoring force of 1 kN. The numerical analysis considers the effect of the excitation due to polygonal wheels on the dynamic behavior of the system at different wear stages, in which the used data regarding the polygonal wear on the wheel tread are directly measured at the subway site.

  9. Description of data on the Nimbus 7 LIMS map archive tape: Water vapor and nitrogen dioxide

    NASA Technical Reports Server (NTRS)

    Haggard, Kenneth V.; Marshall, B. T.; Kurzeja, Robert J.; Remsberg, Ellis E.; Russell, James M., III

    1988-01-01

    Described is the process by which the analysis of the Limb Infrared Monitor of the Stratosphere (LIMS) experiment data were used to produce estimates of synoptic maps of water vapor and nitrogen dioxide. In addition to a detailed description of the analysis procedure, also discussed are several interesting features in the data which are used to demonstrate how the analysis procedure produced the final maps and how one can estimate the uncertainties in the maps. In addition, features in the analysis are noted that would influence how one might use, or interpret, the results. These include subjects such as smoothing and the interpretation of wave components.

  10. Effects of Polar Stratospheric Clouds in the Nimbus 7 LIMS Version 6 Data Set

    NASA Technical Reports Server (NTRS)

    Remsberg, Ellis; Harvey, V. Lynn

    2016-01-01

    The historic Limb Infrared Monitor of the Stratosphere (LIMS) measurements of 1978-1979 from the Nimbus 7 satellite were re-processed with Version 6 (V6) algorithms and archived in 2002. The V6 data set employs updated radiance registration methods, improved spectroscopic line parameters, and a common vertical resolution for all retrieved parameters. Retrieved profiles are spaced about every 1.6 of latitude along orbits and include the additional parameter of geopotential height. Profiles of O3 are sensitive to perturbations from emissions of polar stratospheric clouds (PSCs). This work presents results of implementing a first-order screening for effects of PSCs using simple algorithms based on vertical gradients of the O3 mixing ratio. Their occurrences are compared with the co-located, retrieved temperatures and related to the temperature thresholds needed for saturation of H2O and/or HNO3 vapor onto PSC particles. Observed daily locations where the major PSC screening criteria are satisfied are validated against PSCs observed with the Stratospheric Aerosol Monitor (SAM) II experiment also on Nimbus 7. Remnants of emissions from PSCs are characterized for O3 and HNO3 following the screening. PSCs may also impart a warm bias in the co-located LIMS temperatures, but by no more than 1-2K at the altitudes of where effects of PSCs are a maximum in the ozone; thus, no PSC screening was applied to the V6 temperatures. Minimum temperatures vary between 187 and 194K and often occur 1 to 2 km above where PSC effects are first identified in the ozone (most often between about 21 and 28 hPa). Those temperature-pressure values are consistent with conditions for the existence of nitric acid trihydrate (NAT) mixtures and to a lesser extent of super-cooled ternary solution (STS) droplets. A local, temporary uptake of HNO3 vapor of order 1-3 ppbv is indicated during mid-January for the 550K surface. Seven-month time series of the distributions of LIMS O3 and HNO3 are shown based

  11. Effects of polar stratospheric clouds in the Nimbus 7 LIMS Version 6 data set

    NASA Astrophysics Data System (ADS)

    Remsberg, Ellis; Harvey, V. Lynn

    2016-07-01

    The historic Limb Infrared Monitor of the Stratosphere (LIMS) measurements of 1978-1979 from the Nimbus 7 satellite were re-processed with Version 6 (V6) algorithms and archived in 2002. The V6 data set employs updated radiance registration methods, improved spectroscopic line parameters, and a common vertical resolution for all retrieved parameters. Retrieved profiles are spaced about every 1.6° of latitude along orbits and include the additional parameter of geopotential height. Profiles of O3 are sensitive to perturbations from emissions of polar stratospheric clouds (PSCs). This work presents results of implementing a first-order screening for effects of PSCs using simple algorithms based on vertical gradients of the O3 mixing ratio. Their occurrences are compared with the co-located, retrieved temperatures and related to the temperature thresholds needed for saturation of H2O and/or HNO3 vapor onto PSC particles. Observed daily locations where the major PSC screening criteria are satisfied are validated against PSCs observed with the Stratospheric Aerosol Monitor (SAM) II experiment also on Nimbus 7. Remnants of emissions from PSCs are characterized for O3 and HNO3 following the screening. PSCs may also impart a warm bias in the co-located LIMS temperatures, but by no more than 1-2 K at the altitudes of where effects of PSCs are a maximum in the ozone; thus, no PSC screening was applied to the V6 temperatures. Minimum temperatures vary between 187 and 194 K and often occur 1 to 2 km above where PSC effects are first identified in the ozone (most often between about 21 and 28 hPa). Those temperature-pressure values are consistent with conditions for the existence of nitric acid trihydrate (NAT) mixtures and to a lesser extent of super-cooled ternary solution (STS) droplets. A local, temporary uptake of HNO3 vapor of order 1-3 ppbv is indicated during mid-January for the 550 K surface. Seven-month time series of the distributions of LIMS O3 and HNO3 are shown

  12. The interannual variability of trace gases in the stratosphere: A comparative study of the LIMS and UARS measurement periods

    NASA Astrophysics Data System (ADS)

    Gray, L. J.; Ruth, S.

    1992-04-01

    The inter-annual variability of trace gases in the equatorial lower stratosphere is examined in order to explore the usefulness of comparisons between measurements from LIMS and the UARS satellite instruments. The quasi biennial oscillation (QBO) is simulated in a two dimensional model by relaxing the modelled equatorial winds towards observed values. It is shown that during the LIMS data period the QBO was in the opposite phase to that expected during the first winter of the UARS measurements. The model predicts that LIMS measurements of ozone, NO2, and HNO3 may have been up to 30% less than the long-term average in the equatorial lower stratosphere while the first few months of UARS measurements may observe abundances up to 30% more than the long-term average. The importance of taking this variability into account and the desirability of several complete cycles of the QBO in the derivation of climatological values is noted.

  13. ISL1-based LIM complexes control Slit2 transcription in developing cranial motor neurons

    PubMed Central

    Kim, Kyung-Tai; Kim, Namhee; Kim, Hwan-Ki; Lee, Hojae; Gruner, Hannah N.; Gergics, Peter; Park, Chungoo; Mastick, Grant S.; Park, Hae-Chul; Song, Mi-Ryoung

    2016-01-01

    LIM-homeodomain (HD) transcription factors form a multimeric complex and assign neuronal subtype identities, as demonstrated by the hexameric ISL1-LHX3 complex which gives rise to somatic motor (SM) neurons. However, the roles of combinatorial LIM code in motor neuron diversification and their subsequent differentiation is much less well understood. In the present study, we demonstrate that the ISL1 controls postmitotic cranial branchiomotor (BM) neurons including the positioning of the cell bodies and peripheral axon pathfinding. Unlike SM neurons, which transform into interneurons, BM neurons are normal in number and in marker expression in Isl1 mutant mice. Nevertheless, the movement of trigeminal and facial BM somata is stalled, and their peripheral axons are fewer or misrouted, with ectopic branches. Among genes whose expression level changes in previous ChIP-seq and microarray analyses in Isl1-deficient cell lines, we found that Slit2 transcript was almost absent from BM neurons of Isl1 mutants. Both ISL1-LHX3 and ISL1-LHX4 bound to the Slit2 enhancer and drove endogenous Slit2 expression in SM and BM neurons. Our findings suggest that combinations of ISL1 and LHX factors establish cell-type specificity and functional diversity in terms of motor neuron identities and/or axon development. PMID:27819291

  14. Description of data on the Nimbus 7 LIMS map archive tape: ozone and nitric acid

    SciTech Connect

    Remsberg, E.E.; Kurzeja, R.J.; Haggard, K.V.; Russell, J.M. III; Gordley, L.L.

    1986-12-01

    The Nimbus 7 Limb infrared monitor of the stratosphere (LIMS) data set has been processed into a Fourier coefficient representation with a Kalman filter algorithm applied to profile data at individual latitudes and pressure levels. The algorithm produces synoptic data at noon Greenwich Mean Time (GMT) from the asynoptic orbital profiles. This form of the data set is easy to use and is appropriate for time series analysis and further data manipulation and display. Ozone and nitric acid results are grouped together in this report because the LIMS vertical field of views (FOV's) and analysis characteristics for these species are similar. A comparison of the orbital input data with mixing ratios derived from Kalman filter coefficients indicates errors in mixing ratio of generally less than 5 percent, with 15 percent being a maximum error. The high quality of the mapped data was indicated by coherence of both the phases and the amplitudes of waves with latitude and pressure. Examples of the mapped fields are presented, and details are given concerning the importance of diurnal variations, the removal of polar stratospheric cloud signatures, and the interpretation of bias effects in the data near the tops of profiles.

  15. Description of data on the Nimbus 7 LIMS map archive tape: Ozone and nitric acid

    NASA Technical Reports Server (NTRS)

    Remsberg, E. E.; Kurzeja, R. J.; Haggard, K. V.; Russell, J. M., III; Gordley, L. L.

    1986-01-01

    The Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) data set has been processed into a Fourier coefficient representation with a Kalman filter algorithm applied to profile data at individual latitudes and pressure levels. The algorithm produces synoptic data at noon Greenwich Mean Time (GMT) from the asynoptic orbital profiles. This form of the data set is easy to use and is appropriate for time series analysis and further data manipulation and display. Ozone and nitric acid results are grouped together in this report because the LIMS vertical field of views (FOV's) and analysis characteristics for these species are similar. A comparison of the orbital input data with mixing ratios derived from Kalman filter coefficients indicates errors in mixing ratio of generally less than 5 percent, with 15 percent being a maximum error. The high quality of the mapped data was indicated by coherence of both the phases and the amplitudes of waves with latitude and pressure. Examples of the mapped fields are presented, and details are given concerning the importance of diurnal variations, the removal of polar stratospheric cloud signatures, and the interpretation of bias effects in the data near the tops of profiles.

  16. LIMS Instrument Package (LIP) balloon experiment: Nimbus 7 satellite correlative temperature, ozone, water vapor, and nitric acid measurements

    NASA Technical Reports Server (NTRS)

    Lee, R. B., III; Gandrud, B. W.; Robbins, D. E.; Rossi, L. C.; Swann, N. R. W.

    1982-01-01

    The Limb Infrared Monitor of the Stratosphere (LIMS) LIP balloon experiment was used to obtain correlative temperature, ozone, water vapor, and nitric acid data at altitudes between 10 and 36 kilometers. The performance of the LIMS sensor flown on the Nimbus 7 Satellite was assessed. The LIP consists of the modified electrochemical concentration cell ozonesonde, the ultraviolet absorption photometric of ozone, the water vapor infrared radiometer sonde, the chemical absorption filter instrument for nitric acid vapor, and the infrared radiometer for nitric acid vapor. The limb instrument package (LIP), its correlative sensors, and the resulting data obtained from an engineering and four correlative flights are described.

  17. FluxTransgenics: a flexible LIMS-based tool for management of plant transformation experimental data

    PubMed Central

    2014-01-01

    Background The production and commercial release of Genetically Modified Organisms (GMOs) are currently the focus of important discussions. In order to guarantee the quality and reliability of their trials, companies and institutions working on this subject must adopt new approaches on management, organization and recording of laboratory conditions where field studies are performed. Computational systems for management and storage of laboratory data known as Laboratory Information Management Systems (LIMS) are essential tools to achieve this. Results In this work, we have used the SIGLa system – a workflow based LIMS as a framework to develop the FluxTransgenics system for a GMOs laboratory of Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Maize and Sorghum (Sete Lagoas, MG - Brazil). A workflow representing all stages of the transgenic maize plants generation has been developed and uploaded in FluxTransgenics. This workflow models the activities involved in maize and sorghum transformation using the Agrobacterium tumefaciens method. By uploading this workflow in the SIGLa system we have created Fluxtransgenics, a complete LIMS for managing plant transformation data. Conclusions FluxTransgenics presents a solution for the management of the data produced by a laboratory of genetically modified plants that is efficient and supports different kinds of information. Its adoption will contribute to guarantee the quality of activities and products in the process of transgenic production and enforce the use of Good Laboratory Practices (GLP). The adoption of the transformation protocol associated to the use of FluxTransgenics has made it possible to increase productivity by at least 300%, increasing the efficiency of the experiments from between 0.5 and 1 percent to about 3%. This has been achieved by an increase in the number of experiments performed and a more accurate choice of parameters, all of which have been made possible because it became easier to

  18. [The mangrove and others vegetation associations in de Gandoca lagoon, Limón, Costa Rica].

    PubMed

    Coll, M; Fonseca, A C; Cortés, J

    2001-12-01

    Six plant associations were identified at Gandoca Lagoon by photointerpretation and field verification: a) mangroves, b) palm trees swamp, and palm trees with Acrostichum aureum and A. danaefolium, c) mixed palm trees, d) very humid tropical rain forest, and e) tropical beach vegetation. The mangroves cover 12.5 ha surrounding the lagoon and extend 2 km up the Gandoca River. Rhizophora mangle (red mangrove) was the dominant species, with Avicennia germinans (black mangrove), Laguncularia racemosa (white mangrove) and Conocarpus erectus (buttonwood) also present. Moving inland the mangroves grade into a tropical rain forest. Gandoca, the largest and best preserved mangrove of Caribbean Costa Rica, tripled its area from 1976 to 2000. Possible causes include sedimentation and the Limón earthquake, which may have subside the lagoon area.

  19. Estimates of the stratospheric distribution of odd nitrogen from the LIMS data

    NASA Astrophysics Data System (ADS)

    Callis, L. B.; Russell, J. M., III; Natarajan, M.

    1985-05-01

    Reasonable lower-limit estimates of the latitude-altitude distribution of stratospheric NO(x) for December 1978 and March 1979 are presented. The lower-limit estimates are based on nighttime measurements of NO2 and HNO3 taken by the LIMS instrument aboard the NIMBUS 7 satellite. It is shown that the estimates do not depend upon model calculations or a priori knowledge of the nature of the stratospheric photochemical system. The results indicate that the measured sum of nighttime NO2 nd HNO3 is as high as 22.5 + or - ppbv and that atmospheric NO(x) levels may be as high as 26 + or - 4.5 ppbv at 37 km, which is larger than most calculated NO(x) levels.

  20. Description of data on the Nimbus 7 LIMS map archive tape: Temperature and geopotential height

    NASA Technical Reports Server (NTRS)

    Haggard, K. V.; Remsberg, E. E.; Grose, W. L.; Russell, J. M., III; Marshall, B. T.; Lingenfelser, G.

    1986-01-01

    The process by which the analysis of the Limb Infared Monitor of the Stratosphere (LIMS) experiment data were used to produce estimates of synoptic maps of temperature and geopotential height is described. In addition to a detailed description of the analysis procedure, several interesting features in the data are discussed and these features are used to demonstrate how the analysis procedure produced the final maps and how one can estimate the uncertainties in the maps. In addition, features in the analysis are noted that would influence how one might use, or interpret, the results. These include subjects such as smoothing and the interpretation of wave components. While some suggestions are made for an improved analysis of the data, it is shown that, in general, the maps are an excellent estimation of the synoptic fields.

  1. On the quality of the Nimbus 7 LIMS version 6 ozone for studies of the middle atmosphere

    NASA Astrophysics Data System (ADS)

    Remsberg, Ellis; Lingenfelser, Gretchen; Natarajan, Murali; Gordley, Larry; Marshall, B. Thomas; Thompson, Earl

    2007-07-01

    The Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) radiance profile dataset of 1978/79 was reconditioned and reprocessed to Version 6 (V6) profiles of temperature and species that are improved significantly over those from Version 5 (V5). The LIMS V6 dataset was archived for public use in 2002. Improvements for its ozone include: (1) a more accurate accounting for instrument and spacecraft motion effects in the radiances, (2) the use of better spectroscopic line parameters for its ozone forward model, (3) retrievals of all its scans, (4) more accurate and compatible temperature versus pressure profiles (or T(p)) that are needed for the registration of the ozone radiances and for the removal of temperature effects from them, and (5) a better accounting for interfering species in the lower stratosphere. The retrieved V6 ozone profiles extend from near cloud top altitudes to about 80 km and from 64S to 84N latitude with better sampling along the orbit than for the V5 dataset. Calculated estimates of the single-profile precision and accuracy are provided; precision estimates based on the data themselves are of order 3% or better from 1 to 30 hPa. Estimates of total systematic error are hard to generalize because the separate sources of error may not all be of the same sign, and they depend somewhat on the atmospheric state. It is estimated that the accuracy of the V6 zonal mean ozone distribution is within ±9% from 50 10 hPa, improving to ±7% in the uppermost stratosphere. Simulation studies show that the LIMS T(p) retrievals are underestimating slightly the small amplitudes of the atmospheric temperature tides, which affect the retrieved day/night ozone differences. There are also small biases in the middle to lower stratosphere for the ascending versus descending node LIMS ozone, due principally to not accounting for the asymmetric weighting of its radiance within the tangent layer. The total accuracy for the LIMS ozone was assessed by comparing its

  2. LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury

    PubMed Central

    Wang, Weiwei; Townes-Anderson, Ellen

    2015-01-01

    Purpose Rod photoreceptors retract their axon terminals and develop neuritic sprouts in response to retinal detachment and reattachment, respectively. This study examines the role of LIM kinase (LIMK), a component of RhoA and Rac pathways, in the presynaptic structural remodeling of rod photoreceptors. Methods Phosphorylated LIMK (p-LIMK), the active form of LIMK, was examined in salamander retina with Western blot and confocal microscopy. Axon length within the first 7 hours and process growth after 3 days of culture were assessed in isolated rod photoreceptors treated with inhibitors of upstream regulators ROCK and p21-activated kinase (Pak) (Y27632 and IPA-3) and a direct LIMK inhibitor (BMS-5). Porcine retinal explants were also treated with BMS-5 and analyzed 24 hours after detachment. Because Ca2+ influx contributes to axonal retraction, L-type channels were blocked in some experiments with nicardipine. Results Phosphorylated LIMK is present in rod terminals during retraction and in newly formed processes. Axonal retraction over 7 hours was significantly reduced by inhibition of LIMK or its regulators, ROCK and Pak. Process growth was reduced by LIMK or Pak inhibition especially at the basal (axon-bearing) region of the rod cells. Combining Ca2+ channel and LIMK inhibition had no additional effect on retraction but did further inhibit sprouting after 3 days. In detached porcine retina, LIMK inhibition reduced rod axonal retraction and improved retinal morphology. Conclusions Thus structural remodeling, in the form of either axonal retraction or neuritic growth, requires LIMK activity. LIM kinase inhibition may have therapeutic potential for reducing pathologic rod terminal plasticity after retinal injury. PMID:26658506

  3. Improvements in the profiles and distributions of nitric acid and nitrogen dioxide with the LIMS version 6 dataset

    NASA Astrophysics Data System (ADS)

    Remsberg, E.; Natarajan, M.; Marshall, B. T.; Gordley, L. L.; Thompson, R. E.; Lingenfelser, G.

    2010-05-01

    The quality of the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) nitric acid (HNO3) and nitrogen dioxide (NO2) profiles and distributions of 1978/1979 are described after their processing with an updated, Version 6 (V6) algorithm and subsequent archival in 2002. Estimates of the precision and accuracy of both of those species are developed and provided herein. The character of the V6 HNO3 profiles is relatively unchanged from that of the earlier LIMS Version 5 (V5) profiles, except in the upper stratosphere where the interfering effects of CO2 are accounted for better with V6. The accuracy of the retrieved V6 NO2 is also significantly better in the middle and upper stratosphere, due to improvements in its spectral line parameters and in the reduced biases for the accompanying V6 temperature and water vapor profiles. As a result of these important updates, there is better agreement with theoretical calculations for profiles of the HNO3/NO2 ratio, day-to-night NO2 ratio, and with estimates of the production of NO2 in the mesosphere and its descent to the upper stratosphere during polar night. In particular, the findings for middle and upper stratospheric NO2 should also be more compatible with those obtained from more recent satellite sensors because the effects of the spin-splitting of the NO2 lines are accounted for now with the LIMS V6 algorithm. The improved precisions and more frequent retrievals of the LIMS profiles along their orbit tracks provide for better continuity and detail in map analyses of these two species on pressure surfaces. It is judged that the chemical effects of the oxides of nitrogen on ozone can be studied quantitatively throughout the stratosphere with the LIMS V6 data.

  4. LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs.

    PubMed

    Po'uha, S T; Shum, M S Y; Goebel, A; Bernard, O; Kavallaris, M

    2010-01-28

    LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH-EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.

  5. Islamic Education Philosophy Development (Study Analysis on Ta'lim Al-Kitab Al-Zarnuji Muta'allim Works)

    ERIC Educational Resources Information Center

    Asrori, H. Achmad

    2016-01-01

    "Ta'lim Muta'allim" is one of the monumental works of Shaykh Tajuddin Nu'man ibn Ibrahim ibn al-Khalil al-Zarnuji, who lived in the 6th century H/13-14 M. The reason for writing this study ie: (1) it is very rich with the basic values of Islamic education, (2) the values are already widely practiced in the world of education, especially…

  6. Improvements in the profiles and distributions of nitric acid and nitrogen dioxide with the LIMS version 6 dataset

    NASA Astrophysics Data System (ADS)

    Remsberg, E.; Natarajan, M.; Marshall, T.; Gordley, L. L.; Thompson, R. E.; Lingenfelser, G.

    2010-02-01

    The quality of the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) nitric acid (HNO3) and nitrogen dioxide (NO2) profiles and distributions of 1978/1979 is described after their processing with an updated, Version 6 (V6) algorithm and subsequent archival in 2002. Estimates of the precision and accuracy of both of those species are developed and provided herein. The character of the V6 HNO3 profiles is relatively unchanged from that of the earlier LIMS Version 5 (V5) profiles, except in the upper stratosphere where the interfering effects of CO2 are accounted for better with V6. The accuracy of the retrieved V6 NO2 is also significantly better in the middle and upper stratosphere, due to improvements in its spectral line parameters and in the reduced biases for the accompanying V6 temperature and water vapor profiles. As a result of these important updates, there is better agreement with theoretical calculations for profiles of the HNO3/NO2 ratio, day-to-night NO2 ratio, and with estimates of the production of NO2 in the mesosphere and its descent to the upper stratosphere during polar night. The improved precisions and more frequent retrievals of the profiles along the LIMS orbit tracks provide for better continuity and detail in map analyses of these two species on pressure surfaces. It is judged that the chemical effects of the oxides of nitrogen on ozone can be examined quantitatively throughout the stratosphere with the LIMS V6 data, and that the findings will be more compatible with those obtained from measurements of the same species from subsequent satellite sensors.

  7. Speciation of surface gold in pressure oxidized carbonaceous gold ores by TOF-SIMS and TOF-LIMS

    NASA Astrophysics Data System (ADS)

    Dimov, S. S.; Chryssoulis, S. L.; Sodhi, R. N.

    2003-01-01

    To the best of our knowledge, this is the first attempt ever to speciate gold preg-robbed by carbonaceous matter using a surface sensitive microbeam technique. This approach enables the direct determination of gold species sorbed on carbonaceous particulates thus providing a new tool in understanding the chemistry of gold sorption on carbon. The reasoning behind this effort was to study the detrimental effect chloride ions have on gold recovery by pressure oxidation of carbonaceous sulfide ores, a technology largely used by the mining industry. The characterization of the sorbed gold species involved three surface sensitive microbeam analytical techniques (TOF-SIMS, TOF-LIMS and XPS) providing confirmatory results for better accuracy. Optimum conditions for detection of gold compounds with minimum fragmentation by TOF-SIMS and TOF-LIMS mass spectrometers have been determined. A reference library of 16 major gold complexes with halogen, thiosulfate, cyanide and thiocyanate groups relevant to the gold recovery processes has been established. The most suitable of the microbeam techniques tested was found to be negative (-ve) ion TOF-LIMS, offering best sensitivity and a small analytical spot size.

  8. Structure and chromosomal localization of the genomic locus encoding the Kiz1 LIM-kinase gene

    SciTech Connect

    Bernard, O.; Burkitt, V.; Webb, G.C.

    1996-08-01

    We have cloned and characterized the mouse gene encoding Kiz1/Limk1, a new member of the zinc-finger LIM family that also has a kinase domain. The gene encompasses 25 kb of the mouse genome, and the organization of its 16 exons does not correlate with its functional domains. The promoter region of Kiz1/Limk1 was identified by cloning a 1.06-kb genomic fragment upstream from the first ATG in a promotorless CAT vector. This construct was demonstrated to drive CAT expression in Jurkat cells. The promoter sequence lacks conventional TATA and CAAT motifs but contains consensus binding sequences for several transcriptional regulators implicated in control of transcription in many different cell types, including Sp1, Ets, and E2A. Analysis of the chromosomal localization of KIZ1/LIMK1 indicates that it lies on human chromosome 17 in the region 17q25 and on mouse Chromosome 5, band G2. 15 refs., 3 figs., 1 tab.

  9. ROCK1 via LIM kinase regulates growth, maturation and actin based functions in mast cells

    PubMed Central

    Kapur, Reuben; Shi, Jianjian; Ghosh, Joydeep; Munugalavadla, Veerendra; Sims, Emily; Martin, Holly; Wei, Lei; Mali, Raghuveer Singh

    2016-01-01

    Understanding mast cell development is essential due to their critical role in regulating immunity and autoimmune diseases. Here, we show how Rho kinases (ROCK) regulate mast cell development and can function as therapeutic targets for treating allergic diseases. Rock1 deficiency results in delayed maturation of bone marrow derived mast cells (BMMCs) in response to IL-3 stimulation and reduced growth in response to stem cell factor (SCF) stimulation. Further, integrin-mediated adhesion and migration, and IgE-mediated degranulation are all impaired in Rock1-deficient BMMCs. To understand the mechanism behind altered mast cell development in Rock1−/− BMMCs, we analyzed the activation of ROCK and its downstream targets including LIM kinase (LIMK). We observed reduced activation of ROCK, LIMK, AKT and ERK1/2 in Rock1-deficient BMMCs in response to SCF stimulation. Further, loss of either Limk1 or Limk2 also demonstrated altered BMMC maturation and growth; combined deletion of both Limk1 and Limk2 resulted in further reduction in BMMC maturation and growth. In passive cutaneous anaphylaxis model, deficiency of Rock1 or treatment with ROCK inhibitor Fasudil protected mice against IgE-mediated challenge. Our results identify ROCK/LIMK pathway as a novel therapeutic target for treating allergic diseases involving mast cells. PMID:26943578

  10. Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities

    PubMed Central

    2010-01-01

    Background Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups. Results We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects. Conclusions The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities. PMID:20482787

  11. A manual for a Laboratory Information Management System (LIMS) for light stable isotopes

    USGS Publications Warehouse

    Coplen, Tyler B.

    1998-01-01

    The reliability and accuracy of isotopic data can be improved by utilizing database software to (i) store information about samples, (ii) store the results of mass spectrometric isotope-ratio analyses of samples, (iii) calculate analytical results using standardized algorithms stored in a database, (iv) normalize stable isotopic data to international scales using isotopic reference materials, and (v) generate multi-sheet paper templates for convenient sample loading of automated mass-spectrometer sample preparation manifolds. Such a database program is presented herein. Major benefits of this system include (i) an increase in laboratory efficiency, (ii) reduction in the use of paper, (iii) reduction in workload due to the elimination or reduction of retyping of data by laboratory personnel, and (iv) decreased errors in data reported to sample submitters. Such a database provides a complete record of when and how often laboratory reference materials have been analyzed and provides a record of what correction factors have been used through time. It provides an audit trail for stable isotope laboratories. Since the original publication of the manual for LIMS for Light Stable Isotopes, the isotopes 3 H, 3 He, and 14 C, and the chlorofluorocarbons (CFCs), CFC-11, CFC-12, and CFC-113, have been added to this program.

  12. A manual for a laboratory information management system (LIMS) for light stable isotopes

    USGS Publications Warehouse

    Coplen, Tyler B.

    1997-01-01

    The reliability and accuracy of isotopic data can be improved by utilizing database software to (i) store information about samples, (ii) store the results of mass spectrometric isotope-ratio analyses of samples, (iii) calculate analytical results using standardized algorithms stored in a database, (iv) normalize stable isotopic data to international scales using isotopic reference materials, and (v) generate multi-sheet paper templates for convenient sample loading of automated mass-spectrometer sample preparation manifolds. Such a database program is presented herein. Major benefits of this system include (i) an increase in laboratory efficiency, (ii) reduction in the use of paper, (iii) reduction in workload due to the elimination or reduction of retyping of data by laboratory personnel, and (iv) decreased errors in data reported to sample submitters. Such a database provides a complete record of when and how often laboratory reference materials have been analyzed and provides a record of what correction factors have been used through time. It provides an audit trail for stable isotope laboratories. Since the original publication of the manual for LIMS for Light Stable Isotopes, the isotopes 3 H, 3 He, and 14 C, and the chlorofluorocarbons (CFCs), CFC-11, CFC-12, and CFC-113, have been added to this program.

  13. Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

    PubMed Central

    Morcillo, Patrick; Rosen, Christina; Baylies, Mary K.; Dorsett, Dale

    1997-01-01

    The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer–promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer–promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair–rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer–promoter interactions. PMID:9334334

  14. Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for β-Cell Regeneration

    PubMed Central

    Xu, Jin; Cui, Jiaxi; Del Campo, Aranzazu; Shin, Chong Hyun

    2016-01-01

    The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of β-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in β-cell regeneration. PMID:26845333

  15. Involvement of LIM kinase 1 in actin polarization in human CD4 T cells

    PubMed Central

    Xu, Xuehua; Guo, Jia; Vorster, Paul; Wu, Yuntao

    2012-01-01

    Chemokine binding to cognate receptors induces actin dynamics that are a major driving force for T cell migration and chemotactic motility. HIV-1 binding to the chemokine coreceptor CXCR4 initiates chemotactic signaling, mimicking chemokine-induced actin dynamics to facilitate infection processes such as entry, early DNA synthesis, and nuclear migration. Recently, we identified that HIV-triggered early actin polymerization is mediated through the Rac1-PAK1/2-LIMK1-cofilin pathway. Inhibition of LIMK1 (LIM domain kinase 1), a kinase phosphorylating cofilin, through shRNA knockdown decreases actin polymerization and T cell chemotaxis toward SDF-1. The LIMK1 knockdown T cells also supported lower viral entry, DNA synthesis and nuclear migration, suggesting a critical role of LIMK1-mediated actin dynamics in the initiation of HIV-1 infection. Surprisingly, LIMK1 knockdown in CEM-SS T cells did not lead to an overall change in the ratio of phospho-cofilin to total cofilin although there was a measurable decrease in the amount of actin filaments in cells. The decrease in filamentous actin in LIMK1 knockdown cells was found to mainly occur in polarized cap region rich in F-actin. These results suggest that LIMK1 may be involved in spontaneous actin polarization in transformed T cells. The inhibition of T cell chemotaxis by LIMK1 knockdown likely result from inhibition of localized LIMK1 activation and cofilin phosphorylation that are required for polarized actin polymerization for directional cell migration. The inhibition of HIV-1 infection by LIMK1 knockdown may also result from the decrease of actin-rich membrane protrusions that may be preferred viral entry sites in T cells. PMID:23060964

  16. LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B

    PubMed Central

    Poirault-Chassac, Sonia; Adam, Frédéric; Muczynski, Vincent; Aymé, Gabriel; Casari, Caterina; Bordet, Jean-Claude; Soukaseum, Christelle; Rothschild, Chantal; Proulle, Valérie; Pietrzyk-Nivau, Audrey; Berrou, Eliane; Christophe, Olivier D.; Rosa, Jean-Philippe; Lenting, Peter J.; Bryckaert, Marijke; Baruch, Dominique

    2016-01-01

    von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein’s multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics. PMID:27734030

  17. FluxCTTX: A LIMS-based tool for management and analysis of cytotoxicity assays data

    PubMed Central

    2015-01-01

    Background Cytotoxicity assays have been used by researchers to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds or screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical. These assays may be used as an alternative to animal experimentation and are becoming increasingly important in modern laboratories. However, the execution of these assays in large scale and different laboratories requires, among other things, the management of protocols, reagents, cell lines used as well as the data produced, which can be a challenge. The management of all this information is greatly improved by the utilization of computational tools to save time and guarantee quality. However, a tool that performs this task designed specifically for cytotoxicity assays is not yet available. Results In this work, we have used a workflow based LIMS -- the Flux system -- and the Together Workflow Editor as a framework to develop FluxCTTX, a tool for management of data from cytotoxicity assays performed at different laboratories. The main work is the development of a workflow, which represents all stages of the assay and has been developed and uploaded in Flux. This workflow models the activities of cytotoxicity assays performed as described in the OECD 129 Guidance Document. Conclusions FluxCTTX presents a solution for the management of the data produced by cytotoxicity assays performed at Interlaboratory comparisons. Its adoption will contribute to guarantee the quality of activities in the process of cytotoxicity tests and enforce the use of Good Laboratory Practices (GLP). Furthermore, the workflow developed is complete and can be adapted to other contexts and different tests for management of other types of data. PMID:26696462

  18. Shallow electrical resistivity imaging of the Limón fault, Chagres River Watershed, Panama Canal

    NASA Astrophysics Data System (ADS)

    Mojica, Alexis; Pérez, Tatiana; Toral, Jaime; Miranda, Roberto; Franceschi, Pastora; Calderón, Carlos; Vergara, Fidedigna

    2017-03-01

    The aim of this study was the use of electrical resistivity imaging to investigate the geometry of the southwest portion of one of the most important geologic fault zones of the Panama Canal Watershed: the Limón fault. This fault is characterized by its juxtaposition of pre-Tertiary andesitic basalt (Playa Venado Formation) against late Oligocene Tertiary sediments (Caimito Formation). In this zone, four 2D electrical resistivity tomography profiles were conducted perpendicular to the fault trace: T-1, T-2, T-3 and T-4. The T-1, T-3, and T-4 profiles were long profiles (235 m for the first two and 215 m for the last one), with a goal of determining the depth of the geologic boundary between the sedimentary and andesitic deposits. The T-2 profile was a short profile (23.5 m), with the objective of calibrating the results with data provided by the paleoseismic trenching previously developed in the area of interest. For these tests, two electrode arrays of types Wenner-Schlumberger and Dipole-Dipole, were used. For the inversion routine, two regularized least-squares methods were used: the smoothness-constrained method and robust inversion. The long electrical resistivity tomography profiles were able to identify a set of electrical anomalies associated with the andesitic basalt and the Tertiary sediments and with that, the contact geometry between these formations. In these profiles, fault angle measurements ranged from 60° to 80° with respect to the ground surface. In the T-2 profile, the electrical anomalies showed a good association with the results of the paleoseismic study. This allowed identification of the colluvium and alluvium covering the gravel and sand debris that mark the gradual transition to the soils of the Caimito Formation. Finally, a set of 2D synthetic models was developed for each of the T-1, T-3, and T-4 profiles with the objective of optimizing interpretation of the field results.

  19. Transcription of follicle-stimulating hormone subunit genes is modulated by porcine LIM homeobox transcription factors, LHX2 and LHX3

    PubMed Central

    YOSHIDA, Saishu; KATO, Takako; NISHIMURA, Naoto; KANNO, Naoko; CHEN, Mo; UEHARU, Hiroki; NISHIHARA, Hiroto; KATO, Yukio

    2016-01-01

    The LIM-homeobox transcription factors LHX2 and LHX3s (LHX3a and LHX3b) are thought to be involved in regulating the pituitary glycoprotein hormone subunit genes Cga and Fshβ. These two factors show considerable differences in their amino acid sequences for DNA binding and protein-protein interactions and in their vital function in pituitary development. Hence, we compared the DNA binding properties and transcriptional activities of Cga and Fshβ between LHX2 and LHX3s. A gel mobility shift assay for approximately 1.1 kb upstream of Cga and 2.0 kb upstream of Fshβ varied in binding profiles between LHX2 and LHX3s. DNase I footprinting revealed DNA binding sites in 8 regions of the Cga promoter for LHX2 and LHX3s with small differences in the binding range and strength. In the Fshβ promoter, 14 binding sites were identified for LHX2 and LHX3, respectively. There were alternative binding sites to either gene in addition to similar differences observed in the Cga promoter. The transcriptional activities of LHX2 and LHX3s according to a reporter assay showed cell-type dependent activity with repression in the pituitary gonadotrope lineage LβT2 cells and stimulation in Chinese hamster ovary lineage CHO cells. Reactivity of LHX2 and LHX3s was observed in all regions, and differences were observed in the 5'-upstream region of Fshβ. However, immunohistochemistry showed that LHX2 resides in a small number of gonadotropes in contrast to LHX3. Thus, LHX3 mainly controls Cga and Fshβ expression. PMID:26853788

  20. [Molecular variation of the shuttle craft and Lim3 genes, controlling the development of the nervous system, in a natural Drosophila melanogaster population].

    PubMed

    Simonenko, A V; Rybina, O Iu; Pasiukova, E G

    2008-09-01

    Comparative polymorphism of the first exon and first intron of the shuttle craft (stc) and Lim3 genes and their putative regulatory 5'-flanking sequences was analyzed using 20 sequenced natural alleles. A comparison of the stc and Lim3 genes showed that the extent of polymorphism was similar in their introns and corresponded to the variation level characteristic of Drosophila melanogaster, while the putative regulatory region and first intron of the stc gene proved to be more variable than the corresponding regions of the Lim3 gene. Since the genes under study occurred on the same chromosomes isolated from one population and were close together in a region having a high recombination rate, the difference in the extent of polymorphism between the regulatory and coding regions was explained by individual characteristics of each gene. The results made it possible to assume that the extent of polymorphism of the coding gene regions is maintained by balancing selection.

  1. Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899)

    SciTech Connect

    Andrew, S.M.; Teh, J.G.; Johnstone, R.W.; Russell, S.M.; Whitehead, R.H.; McKenzie, I.F.; Pietersz, G.A. )

    1990-09-01

    One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.

  2. Effect of the HITRAN 92 spectral data on the retrieval of NO2 mixing ratios from Nimbus 7 LIMS

    NASA Technical Reports Server (NTRS)

    Remsburg, Ellis E.; Bhatt, Praful P.; Eckman, Richard S.; Gordley, Larry L.; Russell, James M., III; Siskind, David E.

    1994-01-01

    To ensure spectral consistency when comparing Nimbus 7 Limb Infrared Monitor of the Stratosphere Experiment (LIMS) NO2 distributions with those from Atmosphere Trace Molecule Spectroscopy (ATMOS) and Upper Atmosphere Research Satellite Experiments (UARS), 1 day (May 5, 1979) of LIMS measurements were reprocessed using the NO2 line list on the HITRAN 92 tape compiled by the Airforce Geophysics Laboratory (AFGL). The revised NO2 mixing ratios are smaller by up to 20%. The decrease is not constant with height, latitude, or time of day but depends on the absolute amount of NO2 in the profile, as a result of a change in the degree of saturation for the strong NO2 spectral lines. The revised NO2 agrees better with correlative measurements and with NO2 distributions from the Stratospheric Aerosol and Gas Experiment (SAGE) and Halogen Occultation Experiment (HALOE) satellite experiments but not with those from ATMOS 85. Profiles of the day/night ratio of revised NO2 are now larger near 5 hPa. There is also some improvement between observed and modeled ozone in the upper stratosphere, when the revised nighttime NO2 profile is used as the estimate of NO(y) for the model calculations.

  3. Permian high pressure rocks—the basement of the Sierra de Limón Verde in Northern Chile

    NASA Astrophysics Data System (ADS)

    Lucassen, F.; Franz, G.; Laber, A.

    1999-03-01

    The gneisses and metabasites of the Sierra de Limón Verde were investigated by P-T-t determinations. The rocks are unique in the Central Andes because of their high pressure metamorphic conditions with P≈13±1 kbar at T≈660-720°C. Their age of metamorphism is ≈270 Ma, based on Sm-Nd mineral isochrons. Final uplift of the isolated basement block occurred in the Triassic with a K-Ar age of biotite at ca 235 Ma. In our interpretation, the protolith of the Permian metamorphic rocks is the crust that formed and stabilized during Early Paleozoic. The Sierra de Limón Verde rocks give insight into the lowermost part of the crust in Early Mesozoic. Its Sm-Nd isotopic composition is indistinguishable from the composition of the crust that formed in the Early Paleozoic metamorphic-magmatic cycle ( ca 500 Ma) in northern Chile and NW Argentina. The tectonic-geodynamic setting that triggered the high P (˜45 km depth) metamorphism and the locally restricted exhumation of the rocks remains speculative. Continental collision or a subduction related accretionary complex is unlikely considering the regional geological situation. Transpression-transtension in a strike slip system along the continental margin is suggested as a hypothesis for future investigations.

  4. The mean ozone profile and its temperature sensitivity in the upper stratosphere and lower mesosphere - An analysis of LIMS observations

    NASA Astrophysics Data System (ADS)

    Froidevaux, Lucien; Allen, Mark; Berman, Stanley; Daughton, Alan

    1989-05-01

    Multiple simultaneous LIMS (Limb Infrared Monitor of the Stratosphere) observations of both O3 and temperature T in the upper stratosphere and lower mesosphere are analyzed to derive O3-T correlations for comparisons with model calculations. The zonally averaged O3 profile is compared with results from a simplified photochemical model that assumes O3 to be in a photochemical steady state. The model profile is systematically lower than the observed profile. Key parameters are identified in which changes can systematically increase the O3 profile in both the stratosphere and the mesosphere. The LIMS-derived values for the sensitivity of O3 to changes in T are compared with equilibrium model calculations which include the T-driven opacity feedback effect on photodissociation rate constants. The theoretical O3 response to temperature perturbations is investigated. It is shown how the O3-T sensitivity coefficient is affected by zonal and vertical advection terms as well as by the photochemical coupling between O3 and T.

  5. Effect of the HITRAN 92 spectral data on the retrieval of NO2 mixing ratios from Nimbus 7 LIMS

    NASA Astrophysics Data System (ADS)

    Remsburg, Ellis E.; Bhatt, Praful P.; Eckman, Richard S.; Gordley, Larry L.; Russell, James M., III; Siskind, David E.

    1994-11-01

    To ensure spectral consistency when comparing Nimbus 7 Limb Infrared Monitor of the Stratosphere Experiment (LIMS) NO2 distributions with those from Atmosphere Trace Molecule Spectroscopy (ATMOS) and Upper Atmosphere Research Satellite Experiments (UARS), 1 day (May 5, 1979) of LIMS measurements were reprocessed using the NO2 line list on the HITRAN 92 tape compiled by the Airforce Geophysics Laboratory (AFGL). The revised NO2 mixing ratios are smaller by up to 20%. The decrease is not constant with height, latitude, or time of day but depends on the absolute amount of NO2 in the profile, as a result of a change in the degree of saturation for the strong NO2 spectral lines. The revised NO2 agrees better with correlative measurements and with NO2 distributions from the Stratospheric Aerosol and Gas Experiment (SAGE) and Halogen Occultation Experiment (HALOE) satellite experiments but not with those from ATMOS 85. Profiles of the day/night ratio of revised NO2 are now larger near 5 hPa. There is also some improvement between observed and modeled ozone in the upper stratosphere, when the revised nighttime NO2 profile is used as the estimate of NO(y) for the model calculations.

  6. A guide for the laboratory information management system (LIMS) for light stable isotopes--Versions 7 and 8

    USGS Publications Warehouse

    Coplen, Tyler B.

    2000-01-01

    The reliability and accuracy of isotopic data can be improved by utilizing database software to (i) store information about samples, (ii) store the results of mass spectrometric isotope-ratio analyses of samples, (iii) calculate analytical results using standardized algorithms stored in a database, (iv) normalize stable isotopic data to international scales using isotopic reference materials, and (v) generate multi-sheet paper templates for convenient sample loading of automated mass-spectrometer sample preparation manifolds. Such a database program, the Laboratory Information Management System (LIMS) for Light Stable Isotopes, is presented herein. Major benefits of this system include (i) a dramatic improvement in quality assurance, (ii) an increase in laboratory efficiency, (iii) a reduction in workload due to the elimination or reduction of retyping of data by laboratory personnel, and (iv) a decrease in errors in data reported to sample submitters. Such a database provides a complete record of when and how often laboratory reference materials have been analyzed and provides a record of what correction factors have been used through time. It provides an audit trail for laboratories. LIMS for Light Stable Isotopes is available for both Microsoft Office 97 Professional and Microsoft Office 2000 Professional as versions 7 and 8, respectively. Both source code (mdb file) and precompiled executable files (mde) are available. Numerous improvements have been made for continuous flow isotopic analysis in this version (specifically 7.13 for Microsoft Access 97 and 8.13 for Microsoft Access 2000). It is much easier to import isotopic results from Finnigan ISODAT worksheets, even worksheets on which corrections for amount of sample (linearity corrections) have been added. The capability to determine blank corrections using isotope mass balance from analyses of elemental analyzer samples has been added. It is now possible to calculate and apply drift corrections to isotopic

  7. Thrombin selectively engages LIM kinase 1 and slingshot-1L phosphatase to regulate NF-κB activation and endothelial cell inflammation.

    PubMed

    Leonard, Antony; Marando, Catherine; Rahman, Arshad; Fazal, Fabeha

    2013-11-01

    Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury and its more severe form acute respiratory distress syndrome. Alterations in actin cytoskeleton are shown to be crucial for NF-κB regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin in mediating cytoskeletal alterations essential for NF-κB activation and EC inflammation. The present study describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-κB activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion inhibited RelA/p65 phosphorylation at Ser(536), a critical event conferring transcriptional competency to the bound NF-κB. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 by blocking IKKβ-dependent phosphorylation/degradation of IκBα. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNF-α-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.

  8. Molecular cloning of LIM homeodomain transcription factor Lhx2 as a transcription factor of porcine follicle-stimulating hormone beta subunit (FSHβ) gene.

    PubMed

    Kato, Takako; Ishikawa, Akio; Yoshida, Saishu; Sano, Yoshiya; Kitahara, Kousuke; Nakayama, Michie; Susa, Takao; Kato, Yukio

    2012-01-01

    We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone β subunit gene (Fshβ) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LβT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LβT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshβ, it is possible that LHX2 controls the synthesis of FSH at the transcription level.

  9. [Diet of the capybara Hydrochaeris hydrochaeris (Rodentia: Hydrocharidae) in Caño Limón, Arauca, Colombia].

    PubMed

    Forero-Montaña, Jimena; Betancur, Julio; Cavelier, Jaime

    2003-06-01

    We studied the composition and seasonal variation of the diet of the capybara (Hydrochaeris hydrochaeris) in the flooded savannas of Caño Limón, Colombia. This was achieved by direct observation of the consumption patterns of these animals. The capybaras only consumed plants, and their diet included 89 species of 22 families. Sixty three percent of these plant species had not been reported before. The most commonly consumed plants (94% of the diet), belonged to the Poaceae, Cyperaceae, Leguminosae and Pontederiaceae. Only seven species represented 60% of the total diet: the grasses Hymenachne amplexicaulis (16.9%), Digitaria bicornis (4.5%) and Panicum maximum (4.4%) and the Cyperaceae Rynchospora corymbosa (4.4%). There was seasonal variation in the diet composition of capybaras.

  10. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  11. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  12. A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

    PubMed

    Robertson, Neil O; Shah, Manan; Matthews, Jacqueline M

    2016-10-10

    We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10(-9) -10(-5)  m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.

  13. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  14. LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori.

    PubMed

    Kimoto, Mai; Tsubota, Takuya; Uchino, Keiro; Sezutsu, Hideki; Takiya, Shigeharu

    2015-01-01

    In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.

  15. Complete genome sequence of Cellulophaga lytica type strain (LIM-21T)

    SciTech Connect

    Pati, Amrita; Abt, Birte; Teshima, Hazuki; Nolan, Matt; Lapidus, Alla L.; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxanne; Han, Cliff; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Pagani, Ioanna; Mavromatis, K; Ovchinnikova, Galina; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Detter, J. Chris; Brambilla, Evelyne-Marie; Kannan, K. Palani; Rohde, Manfred; Spring, Stefan; Goker, Markus; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Ivanova, N

    2011-01-01

    Cellulophaga lytica (Lewin 1969) Johansen et al. 1999 is the type species of the genus Cellulophaga, which belongs to the family Flavobacteriaceae within the phylum 'Bacteroidetes' and was isolated from marine beach mud in Limon, Costa Rica. The species is of biotechnological interest because its members produce a wide range of extracellular enzymes capable of degrading proteins and polysaccharides. After the genome sequence of Cellulophaga algicola this is the second completed genome sequence of a member of the genus Cellulophaga. The 3,765,936 bp long genome with its 3,303 protein-coding and 55 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  16. Complete genome sequence of Cellulophaga lytica type strain (LIM-21T)

    PubMed Central

    Pati, Amrita; Abt, Birte; Teshima, Hazuki; Nolan, Matt; Lapidus, Alla; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxane; Han, Cliff; Goodwin, Lynne; Pitluck, Sam; Liolios, Konstantinos; Pagani, Ioanna; Mavromatis, Konstantinos; Ovchinikova, Galina; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Jeffries, Cynthia D.; Detter, John C.; Brambilla, Evelyne-Marie; Kannan, K. Palani; Rohde, Manfred; Spring, Stefan; Göker, Markus; Woyke, Tanja; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter; Ivanova, Natalia

    2011-01-01

    Cellulophaga lytica (Lewin 1969) Johansen et al. 1999 is the type species of the genus Cellulophaga, which belongs to the family Flavobacteriaceae within the phylum 'Bacteroidetes' and was isolated from marine beach mud in Limon, Costa Rica. The species is of biotechnological interest because its members produce a wide range of extracellular enzymes capable of degrading proteins and polysaccharides. After the genome sequence of Cellulophaga algicola this is the second completed genome sequence of a member of the genus Cellulophaga. The 3,765,936 bp long genome with its 3,303 protein-coding and 55 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project. PMID:21677859

  17. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    PubMed

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants.

  18. Computational Prediction of Protein-Protein Interactions of Human Tyrosinase

    PubMed Central

    Wang, Su-Fang; Oh, Sangho; Si, Yue-Xiu; Wang, Zhi-Jiang; Han, Hong-Yan; Lee, Jinhyuk; Qian, Guo-Ying

    2012-01-01

    The various studies on tyrosinase have recently gained the attention of researchers due to their potential application values and the biological functions. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein-protein interactions between tyrosinase and three binding partners, four and half LIM domains 2 (FHL2), cytochrome b-245 alpha polypeptide (CYBA), and RNA-binding motif protein 9 (RBM9). Our interaction simulations showed significant binding energy scores of −595.3 kcal/mol for FHL2, −859.1 kcal/mol for CYBA, and −821.3 kcal/mol for RBM9. We also investigated the residues of each protein facing toward the predicted site of interaction with tyrosinase. Our computational predictions will be useful for elucidating the protein-protein interactions of tyrosinase and studying its binding mechanisms. PMID:22577521

  19. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  20. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  1. The LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain

    PubMed Central

    Zhao, Yangu; Marín, Oscar; Hermesz, Edit; Powell, Aaron; Flames, Nuria; Palkovits, Miklós; Rubenstein, John L. R.; Westphal, Heiner

    2003-01-01

    Forebrain cholinergic neurons play important roles as striatal local circuit neurons and basal telencephalic projection neurons. The genetic mechanisms that control development of these neurons suggest that most of them are derived from the basal telencephalon where Lhx8, a LIM-homeobox gene, is expressed. Here we report that mice with a null mutation of Lhx8 are deficient in the development of forebrain cholinergic neurons. Lhx8 mutants lack the nucleus basalis, a major source of the cholinergic input to the cerebral cortex. In addition, the number of cholinergic neurons is reduced in several other areas of the subcortical forebrain in Lhx8 mutants, including the caudate-putamen, medial septal nucleus, nucleus of the diagonal band, and magnocellular preoptic nucleus. Although cholinergic neurons are not formed, initial steps in their specification appear to be preserved, as indicated by a presence of cells expressing a truncated Lhx8 mRNA and mRNA of the homeobox gene Gbx1. These results provide genetic evidence supporting an important role for Lhx8 in development of cholinergic neurons in the forebrain. PMID:12855770

  2. Expression and function of the LIM-homeodomain transcription factor Islet-1 in the developing and mature vertebrate retina.

    PubMed

    Bejarano-Escobar, Ruth; Álvarez-Hernán, Guadalupe; Morona, Ruth; González, Agustín; Martín-Partido, Gervasio; Francisco-Morcillo, Javier

    2015-09-01

    The LIM-homeodomain transcription factor Islet-1 (Isl1) has been widely used as a marker of different subtypes of neurons in the developing and mature retina of vertebrates. During retinal neurogenesis, early Isl1 expression is detected in the nuclei of neuroblasts that give rise to ganglion, amacrine, bipolar, and horizontal cells. In the mature retina, Isl1 expression is restricted to the nuclei of ganglion cells, cholinergic amacrine cells, ON-bipolar cells, and subpopulations of horizontal cells. Recent studies have explored the functional mechanisms of Isl1 during specification and differentiation of these retinal cell types. Thus, conditional inactivation of Isl1 in the developing mouse retina disrupts retinal function, and also results in optic nerve hypoplasia, marked reductions in mature ganglion, amacrine, and bipolar cells, and a substantial increase in horizontal cells. Furthermore, conditional knockout shows delayed ganglion cell axon growth, ganglion cell axon guidance error, and ganglion cell nerve fiber defasciculation. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different vertebrate retinal cell types. This review examines whether the expression pattern of Isl1 during vertebrate retinal development is conserved across vertebrate species, and discusses current understanding of the developmental functions of Isl1 in retinogenesis.

  3. LIM-homeobox gene 2 promotes tumor growth and metastasis by inducing autocrine and paracrine PDGF-B signaling.

    PubMed

    Kuzmanov, Aleksandar; Hopfer, Ulrike; Marti, Patricia; Meyer-Schaller, Nathalie; Yilmaz, Mahmut; Christofori, Gerhard

    2014-03-01

    An epithelial-mesenchymal transition (EMT) is a critical process during embryonic development and the progression of epithelial tumors to metastatic cancers. Gene expression profiling has uncovered the transcription factor LIM homeobox gene 2 (Lhx2) with up-regulated expression during TGFβ-induced EMT in normal and cancerous breast epithelial cells. Loss and gain of function experiments in transgenic mouse models of breast cancer and of insulinoma in vivo and in breast cancer cells in vitro indicate that Lhx2 plays a critical role in primary tumor growth and metastasis. Notably, the transgenic expression of Lhx2 during breast carcinogenesis promotes vessel maturation, primary tumor growth, tumor cell intravasation and metastasis by directly inducing the expression of platelet-derived growth factor (PDGF)-B in tumor cells and by indirectly increasing the expression of PDGF receptor-β (PDGFRβ) on tumor cells and pericytes. Pharmacological inhibition of PDGF-B/PDGFRβ signaling reduces vessel functionality and tumor growth and Lhx2-induced cell migration and cell invasion. The data indicate a dual role of Lhx2 during EMT and tumor progression: by inducing the expression of PDGF-B, Lhx2 provokes an autocrine PDGF-B/PDGFRβ loop required for cell migration, invasion and metastatic dissemination and paracrine PDGF-B/PDGFRβ signaling to support blood vessel functionality and, thus, primary tumor growth.

  4. High resolution seismic imaging of faults beneath Limón Bay, northern Panama Canal, Republic of Panama

    NASA Astrophysics Data System (ADS)

    Pratt, Thomas L.; Holmes, Mark; Schweig, Eugene S.; Gomberg, Joan; Cowan, Hugh A.

    2003-06-01

    High-resolution seismic reflection profiles from Limón Bay, Republic of Panama, were acquired as part of a seismic hazard investigation of the northern Panama Canal region. The seismic profiles image gently west and northwest dipping strata of upper Miocene Gatún Formation, unconformably overlain by a thin (<20 m) sequence of Holocene muds. Numerous faults, which have northeast trends where they can be correlated between seismic profiles, break the upper Miocene strata. Some of the faults have normal displacement, but on many faults, the amount and type of displacement cannot be determined. The age of displacement is constrained to be Late Miocene or younger, and regional geologic considerations suggest Pliocene movement. The faults may be part of a more extensive set of north- to northeast-trending faults and fractures in the canal region of central Panama. Low topography and the faults in the canal area may be the result of the modern regional stress field, bending of the Isthmus of Panama, shearing in eastern Panama, or minor deformation of the Panama Block above the Caribbean subduction zone. For seismic hazard analysis of the northern canal area, these faults led us to include a source zone of shallow faults proximal to northern canal facilities.

  5. Interactions between Arctic sea ice drift, concentration and thickness modeled by NEMO-LIM3 at different resolutions

    NASA Astrophysics Data System (ADS)

    Docquier, David; Massonnet, François; Raulier, Jonathan; Lecomte, Olivier; Fichefet, Thierry

    2016-04-01

    Sea ice concentration and thickness have substantially decreased in the Arctic since the beginning of the satellite era. As a result, mechanical strength has decreased allowing more fracturing and leading to increased sea ice drift. However, recent studies have highlighted that the interplay between sea ice thermodynamics and dynamics is poorly represented in contemporary global climate model (GCM) simulations. Thus, the considerable inter-model spread in terms of future sea ice extent projections could be reduced by better understanding the interactions between drift, concentration and thickness. This study focuses on the results coming from the global coupled ocean-sea ice model NEMO-LIM3 between 1979 and 2012. Three different simulations are forced by the Drakkar Forcing Set (DFS) 5.2 and run on the global tripolar ORCA grid at spatial resolutions of 0.25, 1° and 2°. The relation between modeled sea ice drift, concentration and thickness is further analyzed, compared to observations and discussed in the framework of the above-mentioned poor representation. It is proposed as a process-based metric for evaluating model performance. This study forms part of the EU Horizon 2020 PRIMAVERA project aiming at developing a new generation of advanced and well-evaluated high-resolution GCMs.

  6. LIM kinase inhibition reduces breast cancer growth and invasiveness but systemic inhibition does not reduce metastasis in mice.

    PubMed

    Li, Rong; Doherty, Judy; Antonipillai, Juliana; Chen, Sheng; Devlin, Mark; Visser, Kathryn; Baell, Jonathan; Street, Ian; Anderson, Robin L; Bernard, Ora

    2013-04-01

    Metastasis is the major cause of morbidity and mortality in cancer patients. An understanding of the genes that regulate metastasis and development of therapies to target these genes is needed urgently. Since members of the LIM kinase (LIMK) family are key regulators of the actin cytoskeleton and are involved in cell motility and invasion, LIMK is considered to be a good therapeutic target for metastatic disease. Here we investigated the consequences of LIMK inhibition on growth and metastasis of human and mouse mammary tumors. LIMK activity was reduced in tumor cells by expression of dominant-negative LIMK1, by RNA interference or with a selective LIMK inhibitor. The extent of phosphorylation of the LIMK substrate, cofilin, of proliferation and invasion in 2D and 3D culture and of tumor growth and metastasis in mice were assessed. Inhibition of LIMK activity efficiently reduced the pro-invasive properties of tumor cells in vitro. Tumors expressing dominant-negative LIMK1 grew more slowly and were less metastatic in mice. However, systemic administration of a LIMK inhibitor did not reduce either primary tumor growth or spontaneous metastasis. Surprisingly, metastasis to the liver was increased after administration of the inhibitor. These data raise a concern about the use of systemic LIMK inhibitors for the treatment of metastatic breast cancer.

  7. Effects of different agricultural systems on soil quality in Northern Limón province, Costa Rica.

    PubMed

    Cornwell, Emma

    2014-09-01

    Conversion of native rainforest ecosystems in Limón Province of Costa Rica to banana and pineapple monoculture has led to reductions in biodiversity and soil quality. Agroforestry management of cacao (Theobroma cacao) is an alternative system that may maintain the agricultural livelihood of the region while more closely mimicking native ecosystems. This study compared physical, biological and chemical soil quality indicators of a cacao plantation under organic agroforestry management with banana, pineapple, and pasture systems; a native forest nearby served as a control. For bulk density and earthworm analysis, 18 samples were collected between March and April 2012 from each ecosystem paired with 18 samples from the cacao. Cacao had a lower bulk density than banana and pineapple monocultures, but greater than the forest (p < 0.05). Cacao also hosted a greater number and mass of earthworms than banana and pineapple (p < 0.05), but similar to forest and pasture. For soil chemical characteristics, three composite samples were collected in March 2012 from each agroecosystem paired with three samples from the cacao plantation. Forest and pineapple ecosystems had the lowest pH, cation exchange capacity, and exchangeable nutrient cations, while cacao had the greatest (p < 0.05). Total nutrient levels of P and N were slightly greater in banana, pineapple and pasture than in cacao; probably related to addition of chemical fertilizer and manure from cattle grazing. Forest and cacao also had greater %C, than other ecosystems, which is directly related to soil organic matter content (p < 0.0001). Overall, cacao had more favorable physical, biological and chemical soil characteristics than banana and pineapple monocultures, while trends were less conclusive compared to the pastureland. While organic cacao was inferior to native forest in some soil characteristics such as bulk density and organic carbon, its soil quality did best mimic that of the native forest. This supports

  8. Context-specific function of the LIM homeobox 1 transcription factor in head formation of the mouse embryo.

    PubMed

    Fossat, Nicolas; Ip, Chi Kin; Jones, Vanessa J; Studdert, Joshua B; Khoo, Poh-Lynn; Lewis, Samara L; Power, Melinda; Tourle, Karin; Loebel, David A F; Kwan, Kin Ming; Behringer, Richard R; Tam, Patrick P L

    2015-06-01

    Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.

  9. LIMS (Limb Infrared Monitor of the Stratosphere) observation of traveling planetary waves and potential vorticity advection in the stratosphere and mesosphere

    SciTech Connect

    Dunkerton, T.J. )

    1991-02-20

    Eastward and westward traveling waves were observed by the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) during the northern winter 1978-1979. Eastward waves were prevalent in early winter and were involved in a minor Canadian warming in December 1978. A large westward traveling wave, as described by previous authors, was observed in January 1979 during a series of minor warmings. By comparing these two events, it is shown that in both cases the superposition of traveling and quasi-stationary waves led to constructive interference that was responsible for the warmings. However, there was significant asymmetry between eastward and westward traveling components. Eastward disturbances were confined mostly within the polar vortex, whereas quasi-stationary and westward traveling components propagated to the vortex periphery and beyond, into the tropics and mid-latitude mesosphere. This behavior is consistent with Rossby wave propagation and indicates that the location and magnitude of planetary wave breaking is sensitive to the frequency spectrum entering the middle atmosphere. However, this asymmetry is also a signature of the nonlinear critical layer as it projects onto the frequency spectrum. Both interpretations are shown to be valid during wave events observed by LIMS. A local Eulerian analysis of potential vorticity (PV) transport indicates that adiabatic, geostrophic advection by the resolvable scales of motion explains qualitatively (but not quantitatively) the observed potential vorticity tendencies in the LIMS northern hemisphere winter. In particular, calculated advection explains the eastward rotation of the main vortex, intrusion of low PV air into the polar cap, and formation of high PV filaments at the vortex periphery.

  10. Neotectonics and paleoseismology of the Limón and pedro miguel faults in Panamá: earthquake hazard to the Panamá canal

    USGS Publications Warehouse

    Rockwell, Thomas; Gath, Edon; Gonzalez, Tania; Madden, Chris; Verdugo, Danielle; Lippincott, Caitlin; Dawson, Tim; Owen, Lewis A.; Fuchs, Markus; Cadena, Ana; Williams, Pat; Weldon, Elise; Franceschi, Pastora

    2010-01-01

    We present new geologic, tectonic geomorphic, and geochronologic data on the slip rate, timing, and size of past surface ruptures for the right-lateral Lim??n and Pedro Miguel faults in central Panam??. These faults are part of a system of conjugate faults that accommodate the internal deformation of Panam?? resulting from the ongoing collision of Central and South America. There have been at least three surface ruptures on the Lim??n fault in the past 950-1400 years, with the most recent during the past 365 years. Displacement in this young event is at least 1.2 m (based on trenching) and may be 1.6-2 m (based on small channel offsets). Awell-preserved 4.2 m offset suggests that the penultimate event also sustained significant displacement. The Holocene slip rate has averaged about 6 mm=yr, based on a 30-m offset terrace riser incised into a 5-ka abandoned channel. The Pedro Miguel fault has sustained three surface ruptures in the past 1600 years, the most recent being the 2 May 1621 earthquake that partially destroyed Panam?? Viejo. At least 2.1 m of slip occurred in this event near the Canal, with geomorphic offsets suggesting 2.5-3 m. The historic Camino de Cruces is offset 2.8 m, indicating multimeter displacement over at least 20 km of fault length. Channel offsets of 100-400 m, together with a climate-induced incision model, suggest a Late Quaternary slip rate of about 5 mm=yr, which is consistent with the paleoseismic results. Comparison of the timing of surface ruptures between the Lim??n and Pedro Miguel faults suggests that large earthquakes may rupture both faults with 2-3 m of displacement for over 40 km, such as is likely in earthquakes in the M 7 range. Altogether, our observations indicate that the Lim??n and Pedro Miguel faults represent a significant seismic hazard to central Panam?? and, specifically, to the Canal and Panam?? City.

  11. Characteristics of wintertime and autumn nitric acid chemistry as defined by Limb Infrared Monitor of the Stratosphere (LIMS) data

    NASA Astrophysics Data System (ADS)

    Rood, Richard B.; Douglass, Anne R.; Kaye, Jack A.; Considine, David B.

    1993-10-01

    Earlier two-dimensional (2-D) and three-dimensional (3-D) model experiments have shown that the Limb Infrared Monitor of the Stratosphere (LIMS) nitric acid data do not behave as expected from conventional gas phase chemical reactions. As contrasted to two-dimensional (2-D) model results, the 3-D model suggests that the discrepancies are at middle latitudes outside of the polar vortex, rather than at polar latitudes. Using only the data record, the characteristics of the nitric acid behavior arc further examined. The data inside the Aleutian anticyclone are examined during the January wave 1 warming. The anticyclone provides a large isolated region of air that moves from about 40°N to 60°N during the warming. Ozone remains approximately constant during this transit, while nitric acid increases more than 1 parts per billion by volume (ppbv). Both ozone and water vapor fields develop a wave 1 structure during the warming, as expected. Nitric acid, which is also expected to develop a wave 1 signal, develops a prominent wave 2 structure. This structure is observed between 50 and 5 mbar. A prominent feature of the nitric acid field is the persistent "bending" of contours due to strong meridional flow. Since these contours persist, instead of aligning with the flow, there must be chemical processes maintaining the nitric acid with timescales shorter than the advective timescale. The time constant for this chemical process ranges from approximately 1 day at 70°N to about 4 days at 30°N. When the time constant is used in the 3-D model, all of the basic characteristics of the observations are simulated. It is not clear what chemical mechanisms are responsible for this behavior. There is a strong relationship between the insolation and the shortcomings of the nitric acid simulations. Heterogeneous reactions on background aerosols are considered, but their spatial, temporal, and chemical characteristics are not clearly consistent with the needed changes in the chemistry

  12. LIMS (Limb Infrared Monitor of the Stratosphere) observation of traveling planetary waves and potential vorticity advection in the stratosphere and mesosphere

    NASA Astrophysics Data System (ADS)

    Dunkerton, Timothy J.

    1991-02-01

    Eastward and westward traveling waves were observed by the Nimbus 7 Limb Infrared Monitor of the Stratosphere (LIMS) during the northern winter 1978-1979. Eastward waves were prevalent in early winter and were involved in a minor Canadian warming in December 1978. A large westward traveling wave, as described by previous authors, was observed in January 1979 during a series of minor warmings. By comparing these two events, it is shown that in both cases the superposition of traveling and quasi-stationary waves led to constructive interference that was responsible for the warmings. However, there was significant asymmetry between eastward and westward traveling components. A local Eulerian analysis of potential vorticity (PV) transport indicates that adiabatic, geostrophic advection by the resolvable scales of motion explains qualitatively (but not quantitatively) the observed potential vorticity tendencies in the LIMS Northern Hemisphere winter. In particular, calculated advection explains the eastward rotation of the main vortex, intrusion of low PV air into the polar cap, and formation of high PV filaments at the vortex periphery.

  13. Laboratory information management system for membrane protein structure initiative--from gene to crystal.

    PubMed

    Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

    2008-12-01

    Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

  14. Do Deregulated Cas Proteins Induce Genomic Instability in Early-Stage Ovarian Cancer

    DTIC Science & Technology

    2006-12-01

    5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Erica A. Golemis, Ph.D. 5d. PROJECT NUMBER...separable. REPORTABLE OUTCOMES: To date, this project has resulted in two manuscripts that have been published in Nature Cell Biology and in...Cell Cycle. A third manuscript, describing HEF1-Aurora A- Ajuba interactions, is currently in preparation. Data from this project has been presented

  15. The Limón, Costa Rica earthquake of April 22, 1991: Back arc thrusting and collisional tectonics in a subduction environment

    NASA Astrophysics Data System (ADS)

    SuáRez, Gerardo; Pardo, Mario; DomíNguez, Jaime; Ponce, Lautaro; Montero, Walter; Boschini, Ileana; Rojas, Wilfredo

    1995-04-01

    On April 22, 1991, a large earthquake (Mw = 7.7) occurred along the Caribbean coast of Costa Rica and western Panama. The rupture area of the fault mapped from the aftershocks is 45×85 km2;. The distribution of aftershocks and the local geological record suggest that faulting occurred on a blind thrust sheet that shallows toward the northeast. Uplift of the Caribbean coast ranging from 1.5 m near Puerto Limón and decreasing gradually toward the southeast was observed along the Caribbean. Northwest of Puerto Limón no significant coastal uplift was observed. This observation agrees with the aftershock data suggesting the rupture did not extend to the northwest of this location. The Limón earthquake also triggered aftershocks on secondary faults in the crust. These events are apparently associated with a family of imbricate thrust and strike-slip faults that lie in the eastern piedmont of the Talamanca Cordillera. The historical seismicity indicates that the Caribbean coast has been the site of several historical earthquakes with magnitudes greater than 7.0. On April 26, 1916, another earthquake (Ms = 6.9) took place in the same region. Summing the scalar seismic moment release along the Caribbean coast, the average rate of slip is approximately 0.8 cm/yr, compared with a value of 0.4 to 0.8 cm/yr along the Pacific subduction zone, depending on the estimated width of the seismogenic zone. Therefore a large fraction of the relative plate motion between the Cocos and Caribbean plates (9.8 cm/yr) appears to be taken up by crustal deformation in the back arc. The tectonic regime in the area appears to be dominated by the collision of the buoyant Cocos ridge with the subduction zone. The absence of a Wadati-Benioff zone where the Cocos ridge collides with the trench suggests the slab does not subduct beneath the Osa Peninsula; this is supported by the Pliocene gap of volcanism present in Costa Rica. Thus the predicted relative motion between the Cocos and Caribbean

  16. Gsh-4 encodes a LIM-type homeodomain, is expressed in the developing central nervous system and is required for early postnatal survival.

    PubMed Central

    Li, H; Witte, D P; Branford, W W; Aronow, B J; Weinstein, M; Kaur, S; Wert, S; Singh, G; Schreiner, C M; Whitsett, J A

    1994-01-01

    We present an initial characterization of the murine Gsh-4 gene which is shown to encode a LIM-type homeodomain. Genes in this category are known to control late developmental cell-type specification events in simpler organisms. Whole mount and serial section in situ hybridizations show transient Gsh-4 expression in ventrolateral regions of the developing neural tube and hindbrain. Mice homozygous for a targeted mutation in Gsh-4 suffer early postnatal death resulting from immature lungs which do not inflate. Prenatal administration of progesterone and glucocorticoid, to extend gestational term and accelerate maturation, resulted in lung inflation at birth. Nevertheless, the hormonally treated mutants generally failed to survive beyond an hour after birth, due to ineffective breathing efforts. It is concluded that Gsh-4 plays a critical role in the development of respiratory control mechanisms and in the normal growth and maturation of the lung. Images PMID:7913017

  17. Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins*

    PubMed Central

    Yi, Chae-ryun; Allen, John E.; Russo, Brian; Lee, Soo Young; Heindl, Jason E.; Baxt, Leigh A.; Herrera, Bobby Brooke; Kahoud, Emily; MacBeath, Gavin; Goldberg, Marcia B.

    2014-01-01

    Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain. PMID:25124035

  18. The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory.

    PubMed

    Morris, Chris; Pajon, Anne; Griffiths, Susanne L; Daniel, Ed; Savitsky, Marc; Lin, Bill; Diprose, Jonathan M; da Silva, Alan Wilter; Pilicheva, Katya; Troshin, Peter; van Niekerk, Johannes; Isaacs, Neil; Naismith, James; Nave, Colin; Blake, Richard; Wilson, Keith S; Stuart, David I; Henrick, Kim; Esnouf, Robert M

    2011-04-01

    The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service.

  19. The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory

    PubMed Central

    Morris, Chris; Pajon, Anne; Griffiths, Susanne L.; Daniel, Ed; Savitsky, Marc; Lin, Bill; Diprose, Jonathan M.; Wilter da Silva, Alan; Pilicheva, Katya; Troshin, Peter; van Niekerk, Johannes; Isaacs, Neil; Naismith, James; Nave, Colin; Blake, Richard; Wilson, Keith S.; Stuart, David I.; Henrick, Kim; Esnouf, Robert M.

    2011-01-01

    The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service. PMID:21460443

  20. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

    PubMed Central

    Barba, Marta; Pirozzi, Filomena; Saulnier, Nathalie; Vitali, Tiziana; Natale, Maria Teresa; Logroscino, Giandomenico; Robbins, Paul D.; Gambotto, Andrea; Neri, Giovanni; Michetti, Fabrizio; Pola, Enrico; Lattanzi, Wanda

    2012-01-01

    Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs), can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC. PMID:23097599

  1. Epigenetic repression of PDZ-LIM domain-containing protein 2 promotes ovarian cancer via NOS2-derived nitric oxide signaling.

    PubMed

    Zhao, Linjie; Yu, Chuan; Zhou, Shengtao; Lau, Wayne Bond; Lau, Bonnie; Luo, Zhongyue; Lin, Qiao; Yang, Huiliang; Xuan, Yu; Yi, Tao; Zhao, Xia; Wei, Yuquan

    2016-01-12

    Ovarian cancer constitutes one of the most lethal gynaecological malignancies worldwide and currently no satisfactory therapeutic approaches have been established. Therefore, elucidation of molecular mechanisms to develop targeted therapy of ovarian cancer is crucial. PDLIM2 is critical to promote ubiquitination of nuclear p65 and thus its role in inflammation has been highlighted recently. We demonstrate that PDLIM2 is decreased in both ovarian high-grade serous carcinoma and in various human ovarian cancer cell lines compared with normal ovary tissues and human ovarian surface epithelial cells (HOSE). Further functional analysis revealed that PDLIM2 is epigenetically repressed in ovarian cancer development and inhibition of PDLIM2 promoted ovarian cancer growth both in vivo and in vitro via NOS2-derived nitric oxide signaling, leading to recruitment of M2 type macrophages. These results suggest that PDLIM2 might be involved in ovarian cancer pathogenesis, which could serve as a promising therapeutic target for ovarian cancer patients.

  2. Neogene-quaternary Ostracoda and paleoenvironments, of the Limón basin, Costa Rica, and Bocas del Toro basin, Panama

    USGS Publications Warehouse

    Borne, P.F.; Cronin, T. M.; Hazel, J.E.

    1999-01-01

    Tropical marine ostracodes from Neogene and Quaternary sediments of the Central American Caribbean region have been the subject of biostratigraphic, ecological, taxonomic, and evolutionary studies. As part of the Panama Paleontology Project (PPP), Neogene and Quaternary ostracodes are being studied from the Central American region. The overall goal of this research is to evaluate the impact of the emergence of the Central American Isthmus as a land barrier between the Caribbean/tropical Atlantic and the Pacific oceans on marine ostracode biodiversity and the oceanic environments in which extant ostracodes evolved. Due to the ecological specificity of many living tropical ostracode species, they are ideally suited for reconstructing paleoenvironments on the basis of their occurrence in fossil assemblages, which in turn can lead to a better understanding of the tropical climatic and tectonic history of Central America. The principal aims of this chapter are: (a) to document the composition of the ostracode assemblages from the Limón Basin of Costa Rica and the Bocas del Toro Basin of Panama, two areas yielding extensive ma rine ostracode assemblages; (b) to describe the environments of deposition within these basins; and (c) to document the stratigraphic distribution of potentially agediagnostic ostracode species in the Limón and Bocas del Toro basins in order to enhance their use in Central American biostratigraphy. A secondary, but none-the-less important goal is to assemble a database on the distribution of modem ostracode species in the Caribbean and adjacent areas as a basis for comparison with fossil assemblages. Although the ecological, biostratigraphic and paleoenvironmental conclusions presented here will improve as additional material is studied, these fossil and modem ostracode databases constitute the foundation for future evolutionary and geochernical studies of tropical Caribbean and eastern Pacific Ocean ostracodes. Moreover, we present here evidence

  3. ProteinTracker: an application for managing protein production and purification

    PubMed Central

    2012-01-01

    Background Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks), or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS). Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. Findings This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes), cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. Conclusions ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org. PMID:22574679

  4. Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain

    PubMed Central

    Shimada, Naoto; Inami, Show; Sato, Shoma; Kitamoto, Toshihiro; Sakai, Takaomi

    2016-01-01

    Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies. PMID:27853240

  5. Indentured migration and differential gender gene flow: the origin and evolution of the East-Indian community of Limón, Costa Rica.

    PubMed

    Castrì, Loredana; Otárola, Flory; Blell, Mwenza; Ruiz, Ernesto; Barrantes, Ramiro; Luiselli, Donata; Pettener, Davide; Madrigal, Lorena

    2007-10-01

    After the emancipation of African slaves in the Caribbean, the labor void left by out-migrating former slaves was filled by in-migrating indentured servants from prepartition India and China. In some areas of the Caribbean such as Trinidad, Suriname, and Guyana, the East-Indian migrants formed large communities. In this article, we report a study based on mtDNA and Y-chromosomal markers of a small East-Indian community from Limón, Costa Rica. The purpose of the project is to determine the place of origin in the Indian subcontinent of the ancestors of our group and the contributions to its gene pool through gene flow by members of other ethnic groups. Both Y-chromosome and mtDNA suggest that the Indo-Costa Ricans descend from migrants primarily from Central India. While both paternal and maternal markers indicate that this group is overwhelmingly of Indian origin, they also indicate that males and females of African, European, and Amerindian origin contributed to it differently. We discuss our results in the historical context of the virtual extinction of Amerindian Caribbean groups, the forced migration of African slaves to the Caribbean, and the gene flow between Amerindians, Europeans, East-Indians, and Africans that eventually produced the Caribbean's currently diverse gene pool.

  6. The expression of LIM-homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation.

    PubMed

    Inoue, Junji; Ueda, Yuuki; Bando, Tetsuya; Mito, Taro; Noji, Sumihare; Ohuchi, Hideyo

    2013-09-01

    Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye-related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM-homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle-like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.

  7. The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types.

    PubMed

    Zhang, Feifan; Bhattacharya, Abhishek; Nelson, Jessica C; Abe, Namiko; Gordon, Patricia; Lloret-Fernandez, Carla; Maicas, Miren; Flames, Nuria; Mann, Richard S; Colón-Ramos, Daniel A; Hobert, Oliver

    2014-01-01

    Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.

  8. The co-factor of LIM domains (CLIM/LDB/NLI) maintains basal mammary epithelial stem cells and promotes breast tumorigenesis.

    PubMed

    Salmans, Michael L; Yu, Zhengquan; Watanabe, Kazuhide; Cam, Eric; Sun, Peng; Smyth, Padhraic; Dai, Xing; Andersen, Bogi

    2014-07-01

    Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

  9. A core transcriptional network composed of Pax2/8, Gata3 and Lim1 regulates key players of pro/mesonephros morphogenesis.

    PubMed

    Boualia, Sami Kamel; Gaitan, Yaned; Tremblay, Mathieu; Sharma, Richa; Cardin, Julie; Kania, Artur; Bouchard, Maxime

    2013-10-15

    Translating the developmental program encoded in the genome into cellular and morphogenetic functions requires the deployment of elaborate gene regulatory networks (GRNs). GRNs are especially crucial at the onset of organ development where a few regulatory signals establish the different programs required for tissue organization. In the renal system primordium (the pro/mesonephros), important regulators have been identified but their hierarchical and regulatory organization is still elusive. Here, we have performed a detailed analysis of the GRN underlying mouse pro/mesonephros development. We find that a core regulatory subcircuit composed of Pax2/8, Gata3 and Lim1 turns on a deeper layer of transcriptional regulators while activating effector genes responsible for cell signaling and tissue organization. Among the genes directly affected by the core components are the key developmental molecules Nephronectin (Npnt) and Plac8. Hence, the pro/mesonephros GRN links together several essential genes regulating tissue morphogenesis. This renal GRN sheds new light on the disease group Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) in that gene mutations are expected to generate different phenotypic outcomes as a consequence of regulatory network deficiencies rather than threshold effects from single genes.

  10. HalX: an open-source LIMS (Laboratory Information Management System) for small- to large-scale laboratories.

    PubMed

    Prilusky, Jaime; Oueillet, Eric; Ulryck, Nathalie; Pajon, Anne; Bernauer, Julie; Krimm, Isabelle; Quevillon-Cheruel, Sophie; Leulliot, Nicolas; Graille, Marc; Liger, Dominique; Trésaugues, Lionel; Sussman, Joel L; Janin, Joël; van Tilbeurgh, Herman; Poupon, Anne

    2005-06-01

    Structural genomics aims at the establishment of a universal protein-fold dictionary through systematic structure determination either by NMR or X-ray crystallography. In order to catch up with the explosive amount of protein sequence data, the structural biology laboratories are spurred to increase the speed of the structure-determination process. To achieve this goal, high-throughput robotic approaches are increasingly used in all the steps leading from cloning to data collection and even structure interpretation is becoming more and more automatic. The progress made in these areas has begun to have a significant impact on the more 'classical' structural biology laboratories, dramatically increasing the number of individual experiments. This automation creates the need for efficient data management. Here, a new piece of software, HalX, designed as an 'electronic lab book' that aims at (i) storage and (ii) easy access and use of all experimental data is presented. This should lead to much improved management and tracking of structural genomics experimental data.

  11. Stabilization of a binary protein complex by intein-mediated cyclization.

    PubMed

    Jeffries, Cy M; Graham, Stephen C; Stokes, Philippa H; Collyer, Charles A; Guss, J Mitchell; Matthews, Jacqueline M

    2006-11-01

    The study of protein-protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein-mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM-only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X-ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.

  12. The four and a half LIM domains 2 (FHL2) regulates ovarian granulosa cell tumor progression via controlling AKT1 transcription

    PubMed Central

    Hua, G; He, C; Lv, X; Fan, L; Wang, C; Remmenga, S W; Rodabaugh, K J; Yang, L; Lele, S M; Yang, P; Karpf, A R; Davis, J S; Wang, C

    2016-01-01

    The four and a half LIM domains 2 (FHL2) has been shown to play important roles in the regulation of cell proliferation, survival, adhesion, motility and signal transduction in a cell type and tissue-dependent manner. However, the function of FHL2 in ovarian physiology and pathology is unclear. The aim of this study was to determine the role and functional mechanism of FHL2 in the progression of ovarian granulosa cell tumors (GCTs). Immunohistochemical analysis indicated that FHL2 was overexpressed in GCT tissues. Cellular localization of FHL2 in GCT cells was cell cycle dependent. Knockdown of FHL2 suppressed GCT cell growth, reduced cell viability and inhibited cell migration. Consistently, ectopic expression of FHL2 in GCT cells with very low endogenous FHL2 promoted cell growth, improved cell viability and enhance cell migration. Importantly, overexpression of FHL2 promoted GCT progression in vivo. Mechanistic studies indicated that FHL2 regulates AKT1 gene expression in vitro and in vivo. Knockdown of FHL2 or AKT1 in GCT cell lines induced very similar phenotypes. Ectopic expression of constitutively active AKT1 rescued FHL2 knockdown-induced arrest of GCT cell growth and reduction of GCT cell viability, suggesting that FHL2 regulates GCT cell growth and viability through controlling AKT1 expression. Finally, co-immunoprecipitation and chromatin immunoprecipitation analyses indicated that FHL2 functions as a co-activator of NFκB and AP-1 to regulate AKT1 gene transcription. In conclusion, results from the present study indicate that FHL2 exerts its oncogenic action in GCT cells via controlling AKT1 gene expression. FHL2 is a promising target for the development of novel drugs against ovarian granulosa cell tumor. PMID:27415427

  13. Olfactory and amygdalar structures of the chicken ventral pallium based on the combinatorial expression patterns of LIM and other developmental regulatory genes.

    PubMed

    Abellán, Antonio; Legaz, Isabel; Vernier, Baptiste; Rétaux, Sylvie; Medina, Loreta

    2009-09-20

    We compared the combinatorial expression patterns of several LIM domain-containing regulatory genes in the ventrolateral pallium of mouse and chicken, in order to identify the homologues of the ventral pallial amygdala and other olfactory structures in birds. Lmo3, Lmo4, Lhx2, and Lhx9 showed comparable expression patterns in the telencephalon of mouse and chicken, which allowed distinction of the ventrolateral pallium and, particularly, the ventral pallial amygdala and entorhinal cortex. Lmo3 was expressed in most of the ventrolateral pallium in both species, including, in chicken, the piriform cortex and dorsal ventricular ridge (mesopallium, nidopallium, and arcopallium) and, in mouse, the piriform cortex, most of the claustral complex, and the pallial amygdala. Lhx9 was differentially expressed in the ventral pallium, where it was restricted to its rostral (olfactory bulb) and caudal (amygdalar and entorhinal) poles. In the caudal pole, expression of Lhx9 overlapped that of its paralog Lhx2. According to these expression patterns, the chicken ventral pallial amygdala appears to include the caudal dorsolateral pallium, the caudal nidopallium, and the whole arcopallium, and each one relates to a distinct ventricular sector. Finally, the combinatorial expression patterns of Lmo3, Lhx9, and Lmo4 distinguished four distinct subdivisions in the superficial, olfactorecipient area of the chicken ventral pallium, which appear comparable to the piriform, entorhinal, amygdalopiriform, and amygdalar cortices of mammals. The results are discussed in the context of the two existing, opposite views on the homology of the dorsal ventricular ridge of sauropsids and in terms of the evolution of pallial derivatives.

  14. A LIM-homeobox gene is required for differentiation of Wnt-expressing cells at the posterior end of the planarian body.

    PubMed

    Hayashi, Tetsutaro; Motoishi, Minako; Yazawa, Shigenobu; Itomi, Kazu; Tanegashima, Chiharu; Nishimura, Osamu; Agata, Kiyokazu; Tarui, Hiroshi

    2011-09-01

    Planarians have high regenerative ability, which is dependent on pluripotent adult somatic stem cells called neoblasts. Recently, canonical Wnt/β-catenin signaling was shown to be required for posterior specification, and Hedgehog signaling was shown to control anterior-posterior polarity via activation of the Djwnt1/P-1 gene at the posterior end of planarians. Thus, various signaling molecules play an important role in planarian stem cell regulation. However, the molecular mechanisms directly involved in stem cell differentiation have remained unclear. Here, we demonstrate that one of the planarian LIM-homeobox genes, Djislet, is required for the differentiation of Djwnt1/P-1-expressing cells from stem cells at the posterior end. RNA interference (RNAi)-treated planarians of Djislet [Djislet(RNAi)] show a tail-less phenotype. Thus, we speculated that Djislet might be involved in activation of the Wnt signaling pathway in the posterior blastema. When we carefully examined the expression pattern of Djwnt1/P-1 by quantitative real-time PCR during posterior regeneration, we found two phases of Djwnt1/P-1 expression: the first phase was detected in the differentiated cells in the old tissue in the early stage of regeneration and then a second phase was observed in the cells derived from stem cells in the posterior blastema. Interestingly, Djislet is expressed in stem cell-derived DjPiwiA- and Djwnt1/P-1-expressing cells, and Djislet(RNAi) only perturbed the second phase. Thus, we propose that Djislet might act to trigger the differentiation of cells expressing Djwnt1/P-1 from stem cells.

  15. [Intestinal parasites in white-faced capuchin monkeys Cebus capucinus (Primates: Cebidae) inhabiting a protected area in the Limón province of Northeastern Costa Rica].

    PubMed

    Chinchilla, Misael; Urbani, Bernardo; Valerio, Idalia; Vanegas, Juan Carlos

    2010-12-01

    Deforestation of tropical forests is threatening monkey biodiversity and their health status, dependent of an ecologically undisturbed area. To asses this relationship, we analyzed parasite occurrence in their intestines. The study was conducted at the Estación Biológica La Suerte (EBLS), Limón, Costa Rica. The group of white-faced capuchin monkeys (Cebus capucinus) was observed between March and December of 2006. A total of 75 feces samples were obtained. Once a sample was collected, the eaten plant type was identified to family and species level, and feces were processed in the laboratory to determine parasite incidence. Results showed that Moraceae was the most represented family in the samples. Among parasites, Strongyloides spp. and Acanthocephala were the most common. Positive prevalence of parasites was found similar and independent of sex and age of capuchin individuals. Microsporids were mainly reported in feces associated with Piperaceae. A low presence of these parasites was found in samples associated with Myrtaceae, with possible anti-parasite active components. The occurrence of parasites was relatively high in EBLS, when compared to other regions in Costa Rica. The higher occurrence of parasites observed in capuchins at EBLS may be due to the fact that this rain forest is surrounded by areas affected by human activities. We suggest the promotion of research in neotropical primates parasitology, for a better comprehension of the parasite-host relationship, and in a long term, being able to understand the ecosystems where they coexist, and consequently, preserve the biodiversity of the whole region.

  16. The interaction between the adaptor protein APS and Enigma is involved in actin organisation.

    PubMed

    Barrès, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2005-08-15

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

  17. Chemical analysis of solid materials by a LIMS instrument designed for space research: 2D elemental imaging, sub-nm depth profiling and molecular surface analysis

    NASA Astrophysics Data System (ADS)

    Moreno-García, Pavel; Grimaudo, Valentine; Riedo, Andreas; Neuland, Maike B.; Tulej, Marek; Broekmann, Peter; Wurz, Peter

    2016-04-01

    Direct quantitative chemical analysis with high lateral and vertical resolution of solid materials is of prime importance for the development of a wide variety of research fields, including e.g., astrobiology, archeology, mineralogy, electronics, among many others. Nowadays, studies carried out by complementary state-of-the-art analytical techniques such as Auger Electron Spectroscopy (AES), X-ray Photoelectron Spectroscopy (XPS), Secondary Ion Mass Spectrometry (SIMS), Glow Discharge Time-of-Flight Mass Spectrometry (GD-TOF-MS) or Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) provide extensive insight into the chemical composition and allow for a deep understanding of processes that might have fashioned the outmost layers of an analyte due to its interaction with the surrounding environment. Nonetheless, these investigations typically employ equipment that is not suitable for implementation on spacecraft, where requirements concerning weight, size and power consumption are very strict. In recent years Laser Ablation/Ionization Mass Spectrometry (LIMS) has re-emerged as a powerful analytical technique suitable not only for laboratory but also for space applications.[1-3] Its improved performance and measurement capabilities result from the use of cutting edge ultra-short femtosecond laser sources, improved vacuum technology and fast electronics. Because of its ultimate compactness, simplicity and robustness it has already proven to be a very suitable analytical tool for elemental and isotope investigations in space research.[4] In this contribution we demonstrate extended capabilities of our LMS instrument by means of three case studies: i) 2D chemical imaging performed on an Allende meteorite sample,[5] ii) depth profiling with unprecedented sub-nm vertical resolution on Cu electrodeposited interconnects[6,7] and iii) preliminary molecular desorption of polymers without assistance of matrix or functionalized substrates.[8] On the whole

  18. From information management to protein annotation: preparing protein structures for drug discovery.

    PubMed

    Peat, Tom; de La Fortelle, Eric; Culpepper, Janice; Newman, Janet

    2002-11-01

    In contrast to academic pursuits of structural genomics, Structural GenomiX (SGX) solves protein structures at high throughput for the main purpose of enhancing drug-discovery projects, either internally or in partnership with pharmaceutical/biotechnology companies. This involves a radical redesign of the pipeline of methods that turn a gene sequence into a three-dimensional protein structure. The various processes all report electronically to a Laboratory Information Management System (LIMS) to make sure all the parameters of the experiment are recorded in an accessible and 'mineable' form, helping guarantee reproducibility of results. Quality control at several key points keeps the process from branching out on a wrong hypothesis. Protein annotation, in a broad sense, takes care of the interpretation of a protein crystal structure or the crystal structure of one or several protein-ligand complexes. This interpretation both gathers all necessary biological information (protein function, mechanism, specific features within a protein family etc.) and hands over this information in a form accessible to medicinal chemistry teams designing specific small-molecule agonists or antagonists.

  19. LIMS for Lasers 2015 for achieving long-term accuracy and precision of δ2H, δ17O, and δ18O of waters using laser absorption spectrometry

    USGS Publications Warehouse

    Coplen, Tyler B.; Wassenaar, Leonard I

    2015-01-01

    Although laser absorption spectrometry (LAS) instrumentation is easy to use, its incorporation into laboratory operations is not easy, owing to extensive offline manipulation of comma-separated-values files for outlier detection, between-sample memory correction, nonlinearity (δ-variation with water amount) correction, drift correction, normalization to VSMOW-SLAP scales, and difficulty in performing long-term QA/QC audits. METHODS: A Microsoft Access relational-database application, LIMS (Laboratory Information Management System) for Lasers 2015, was developed. It automates LAS data corrections and manages clients, projects, samples, instrument-sample lists, and triple-isotope (δ(17) O, δ(18) O, and δ(2) H values) instrumental data for liquid-water samples. It enables users to (1) graphically evaluate sample injections for variable water yields and high isotope-delta variance; (2) correct for between-sample carryover, instrumental drift, and δ nonlinearity; and (3) normalize final results to VSMOW-SLAP scales. RESULTS: Cost-free LIMS for Lasers 2015 enables users to obtain improved δ(17) O, δ(18) O, and δ(2) H values with liquid-water LAS instruments, even those with under-performing syringes. For example, LAS δ(2) HVSMOW measurements of USGS50 Lake Kyoga (Uganda) water using an under-performing syringe having ±10 % variation in water concentration gave +31.7 ± 1.6 ‰ (2-σ standard deviation), compared with the reference value of +32.8 ± 0.4 ‰, after correction for variation in δ value with water concentration, between-sample memory, and normalization to the VSMOW-SLAP scale. CONCLUSIONS: LIMS for Lasers 2015 enables users to create systematic, well-founded instrument templates, import δ(2) H, δ(17) O, and δ(18) O results, evaluate performance with automatic graphical plots, correct for δ nonlinearity due to variable water concentration, correct for between-sample memory, adjust for drift, perform VSMOW-SLAP normalization, and

  20. LIMS for Lasers 2015 for achieving long-term accuracy and precision of δ2H, δ17O, and δ18O of waters using laser absorption spectrometry

    USGS Publications Warehouse

    Coplen, Tyler B.; Wassenaar, Leonard I

    2015-01-01

    RationaleAlthough laser absorption spectrometry (LAS) instrumentation is easy to use, its incorporation into laboratory operations is not easy, owing to extensive offline manipulation of comma-separated-values files for outlier detection, between-sample memory correction, nonlinearity (δ-variation with water amount) correction, drift correction, normalization to VSMOW-SLAP scales, and difficulty in performing long-term QA/QC audits.MethodsA Microsoft Access relational-database application, LIMS (Laboratory Information Management System) for Lasers 2015, was developed. It automates LAS data corrections and manages clients, projects, samples, instrument-sample lists, and triple-isotope (δ17O, δ18O, and δ2H values) instrumental data for liquid-water samples. It enables users to (1) graphically evaluate sample injections for variable water yields and high isotope-delta variance; (2) correct for between-sample carryover, instrumental drift, and δ nonlinearity; and (3) normalize final results to VSMOW-SLAP scales.ResultsCost-free LIMS for Lasers 2015 enables users to obtain improved δ17O, δ18O, and δ2H values with liquid-water LAS instruments, even those with under-performing syringes. For example, LAS δ2HVSMOW measurements of USGS50 Lake Kyoga (Uganda) water using an under-performing syringe having ±10 % variation in water concentration gave +31.7 ± 1.6 ‰ (2-σ standard deviation), compared with the reference value of +32.8 ± 0.4 ‰, after correction for variation in δ value with water concentration, between-sample memory, and normalization to the VSMOW-SLAP scale.ConclusionsLIMS for Lasers 2015 enables users to create systematic, well-founded instrument templates, import δ2H, δ17O, and δ18O results, evaluate performance with automatic graphical plots, correct for δ nonlinearity due to variable water concentration, correct for between-sample memory, adjust for drift, perform VSMOW-SLAP normalization, and perform long-term QA/QC audits

  1. Assessing water salinity along River Limón and Caño San Miguel irrigation paleochannel (Maracaibo, Venezuela) as affected by the balance of soluble salts in alluvium soils

    NASA Astrophysics Data System (ADS)

    Rodriguez, Raquel; Moreno, Juan; Hermosilla, Daphne; Gascó, Antonio

    2013-04-01

    The saline degradation of soils that are irrigated with brackish water is worrisome increasing worldwide, and it may further affect the salinity of fresh water in those streams flowing across. The problem that is caused by an increasing concentration of salts that are more soluble than gypsum depends on the quality of irrigation water, climatic aridity, and drainage limitations. All these conditions meet in the alluvium soils of River Limón basin that are crossed by Caño San Miguel irrigation paleochannel. River Limón's regulation by closing Manuelote and Tulé artificial reservoirs has diminished the input of water and sediments from flooding events, which exerted dilutive effects in the past. In addition, the balance of soluble salts in these soils has also registered further net accumulation during those extremely dry years happened before 2006, because the great dilution contribution of ombrogenic dammed water coming from rain has not been enough to compensate salts concentration generated by water evapotranspiration in those irrigated soils of the middle basin, particularly in the absence of superficial runoff and deep drainage. Considering those semi-arid climate conditions prevailing in the area (annual precipitation = 710 mm; potential evapotransporation = 2361 mm), it resulted that water analyses in River Limón showed a ten-fold increased maximum annual salinity concentration (March) along the stream; that is, an electric conductivity (Ce) of 0.37 dS•m-1 (at 25 °C) at Puente Carrasquero pumping station, where water for crop irrigation is subtracted, turns to 34.60 dS•m-1 (at 25 °C) at its base level in Puerto Mara, where it discharges to Lake Maracaibo. In addition, the quality of irrigation water from Caño San Miguel, which aggregates to those coming from River Limón at the pumping station located in Carrasquero just before running through the alluvium of this water stream, resulted pretty irregular. In short, it spanned form C1 to C4 soil

  2. Kinematic 3-D Retro-Modeling of an Orogenic Bend in the South Limón Fold-and-Thrust Belt, Eastern Costa Rica: Prediction of the Incremental Internal Strain Distribution

    NASA Astrophysics Data System (ADS)

    Brandes, Christian; Tanner, David C.; Winsemann, Jutta

    2016-10-01

    The South Limón fold-and-thrust belt, in the back-arc area of southern Costa Rica, is characterized by a 90° curvature of the strike of the thrust planes and is therefore a natural laboratory for the analysis of curved orogens. The analysis of curved fold-and-thrust belts is a challenge because of the varying structural orientations within the belt. Based on seismic reflection lines, we created a 3-D subsurface model containing three major thrust faults and three stratigraphic horizons. 3-D kinematic retro-deformation modeling was carried out to analyze the spatial evolution of the fold-and-thrust belt. The maximum amount of displacement on each of the faults is (from hinterland to foreland); thrust 1: 800 m; thrust 2: 600 m; thrust 3: 250 m. The model was restored sequentially to its pre-deformational state. The strain history of the stratigraphic horizons in the model was calculated at every step. This shows that the internal strain pattern has an abrupt change at the orogenic bend. Contractional strain occurs in the forelimbs of the hanging-wall anticlines, while a zone of dilative strain spreads from the anticline crests to the backlimbs. The modeling shows that a NNE-directed transport direction best explains the structural evolution of the bend. This would require a left-lateral strike-slip zone in the North to compensate for the movement and thereby decoupling the South Limón fold-and-thrust belt from northern Costa Rica. Therefore, our modeling supports the presence of the Trans-Isthmic fault system, at least during the Plio-Pleistocene.

  3. A multidimensional proteomic approach to identify hypertrophy-associated proteins.

    PubMed

    Lindsey, Merry L; Goshorn, Danielle K; Comte-Walters, Susana; Hendrick, Jennifer W; Hapke, Elizabeth; Zile, Michael R; Schey, Kevin

    2006-04-01

    Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.

  4. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    SciTech Connect

    Cambier, Linda; Pomies, Pascal

    2011-06-17

    Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  5. The Origin and Evolution of the Plant Cell Surface: Algal Integrin-Associated Proteins and a New Family of Integrin-Like Cytoskeleton-ECM Linker Proteins

    PubMed Central

    Becker, Burkhard; Doan, Jean Michel; Wustman, Brandon; Carpenter, Eric J.; Chen, Li; Zhang, Yong; Wong, Gane K.-S.; Melkonian, Michael

    2015-01-01

    The extracellular matrix of scaly green flagellates consists of small organic scales consisting of polysaccharides and scale-associated proteins (SAPs). Molecular phylogenies have shown that these organisms represent the ancestral stock of flagellates from which all green plants (Viridiplantae) evolved. The molecular characterization of four different SAPs is presented. Three SAPs are type-2 membrane proteins with an arginine/alanine-rich short cytoplasmic tail and an extracellular domain that is most likely of bacterial origin. The fourth protein is a filamin-like protein. In addition, we report the presence of proteins similar to the integrin-associated proteins α-actinin (in transcriptomes of glaucophytes and some viridiplants), LIM-domain proteins, and integrin-associated kinase in transcriptomes of viridiplants, glaucophytes, and rhodophytes. We propose that the membrane proteins identified are the predicted linkers between scales and the cytoskeleton. These proteins are present in many green algae but are apparently absent from embryophytes. These proteins represent a new protein family we have termed gralins for green algal integrins. Gralins are absent from embryophytes. A model for the evolution of the cell surface proteins in Plantae is discussed. PMID:25977459

  6. Singlet CH domain containing human multidomain proteins: an inventory.

    PubMed

    Friedberg, Felix

    2010-03-01

    The actin cytoskeleton presents the basic force in processes such as cytokinesis, endocytosis, vesicular trafficking and cell migration. Here, we list 30 human singlet CH (calpononin homology/actin binding) containing multidomain molecules, each encoded by one gene. We show the domain distributions as given by the SMART program. These mosaic proteins organize geographically the placement of selected proteins in proximity within the cell. In most instances, their precise location, their actin binding capacity by way of the singlet CH (or by other domains?) and their physiological functions need further elucidation. A dendrogram based solely on the relationship for the human singlet CH domains (in terms of AA sequences) for the various molecules that possess the domain, implies that the singlet descended from a common ancestor which in turn sprouted three main branches of protein products. Each branch bifurcated multiple times thus accounting for a cornucopia of products. Wherever, additional (unassigned), highly homologous regions exist in related proteins (e.g., in LIM and LMO7 or in Tangerin and EH/BP1), these unrecognized domain regions await assignment as specific functional domains. Frequently genes coding multidomain proteins duplicated. The varying modular nature within multidomain proteins should have accelerated evolutionary changes to a degree not feasible to achieve by means of mere post-duplication mutational changes.

  7. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

    PubMed

    Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2006-11-01

    APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

  8. Comparative expression analysis of cysteine-rich intestinal protein family members crip1, 2 and 3 during Xenopus laevis embryogenesis.

    PubMed

    Hempel, Annemarie; Kühl, Susanne J

    2014-01-01

    Members of the cysteine-rich intestinal protein (Crip) family belong to the group 2 LIM proteins. Crip proteins are widely expressed in adult mammals but their expression profile and function during embryonic development are still mostly unknown. In this study, we have described for the first time the spatio-temporal expression pattern of the three family members crip1, crip2 and crip3 during Xenopus laevis embryogenesis by RT-PCR and whole mount in situ hybridization approaches. We observed that all three genes are expressed in the pronephros, branchial arches and the eye. Furthermore, crip1 transcripts could be visualized in the developing cranial ganglia and neural tube. In contrast, crip2 could be detected in the cardiovascular system, the brain and the neural tube while crip3 was expressed in the cranial ganglions and the heart. Based on these findings, we suggest that each crip family member may play an important role during embryonic development.

  9. Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

    PubMed Central

    2013-01-01

    Background Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well. Results To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co

  10. The p53 co-activator Zac1 neither induces cell cycle arrest nor apoptosis in chicken Lim1 horizontal progenitor cells.

    PubMed

    Fard, S Shirazi; Blixt, Mke; Hallböök, F

    2015-01-01

    Chicken horizontal progenitor cells are able to enter their final mitosis even in the presence of DNA damage despite having a functional p53-p21 system. This suggests that they are resistant to DNA damage and that the regulation of the final cell cycle of horizontal progenitor cells is independent of the p53-p21 system. The activity of p53 is regulated by positive and negative modulators, including the zinc finger containing transcription factor Zac1 (zinc finger protein that regulates apoptosis and cell cycle arrest). Zac1 interacts with and enhances the activity of p53, thereby inducing cell cycle arrest and apoptosis. In this work, we use a gain-of-function assay in which mouse Zac1 (mZac1) is overexpressed in chicken retinal progenitor cells to study the effect on the final cell cycle of horizontal progenitor cells. The results showed that overexpression of mZac1 induced expression of p21 in a p53-dependent way and arrested the cell cycle as well as triggered apoptosis in chicken non-horizontal retinal progenitor cells. The negative regulation of the cell cycle by mZac1 is consistent with its proposed role as a tumour-suppressor gene. However, the horizontal cells were not affected by mZac1 overexpression. They progressed into S- and late G2/M-phase despite overexpression of mZac1. The inability of mZac1 to arrest the cell cycle in horizontal progenitor cells support the notion that the horizontal cells are less sensitive to events that triggers the p53 system during their terminal and neurogenic cell cycle, compared with other retinal cells. These properties are associated with a cell that has a propensity to become neoplastic and thus with a cell that may develop retinoblastoma.

  11. SPINS: a laboratory information management system for organizing and archiving intermediate and final results from NMR protein structure determinations.

    PubMed

    Baran, Michael C; Moseley, Hunter N B; Aramini, James M; Bayro, Marvin J; Monleon, Daniel; Locke, Jessica Y; Montelione, Gaetano T

    2006-03-01

    Recent technological advances and experimental techniques have contributed to an increasing number and size of NMR datasets. In order to scale up productivity, laboratory information management systems for handling these extensive data need to be designed and implemented. The SPINS (Standardized ProteIn Nmr Storage) Laboratory Information Management System (LIMS) addresses these needs by providing an interface for archival of complete protein NMR structure determinations, together with functionality for depositing these data to the public BioMagResBank (BMRB). The software tracks intermediate files during each step of an NMR structure-determination process, including: data collection, data processing, resonance assignments, resonance assignment validation, structure calculation, and structure validation. The underlying SPINS data dictionary allows for the integration of various third party NMR data processing and analysis software, enabling users to launch programs they are accustomed to using for each step of the structure determination process directly out of the SPINS user interface.

  12. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  13. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  14. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  15. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  16. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  17. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  18. Cypher/ZASP Is a Novel A-kinase Anchoring Protein*

    PubMed Central

    Lin, Changsong; Guo, Xiaogang; Lange, Stephan; Liu, Jie; Ouyang, Kunfu; Yin, Xiang; Jiang, Liujun; Cai, Yibo; Mu, Yongxin; Sheikh, Farah; Ye, Sheng; Chen, Ju; Ke, Yuehai; Cheng, Hongqiang

    2013-01-01

    PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser265 and Ser296 by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser1928 induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins. PMID:23996002

  19. Whey Protein

    MedlinePlus

    ... inflammation (polymyalgia rheumatica). Taking whey protein in a dairy product twice daily for 8 weeks does not improve muscle function, walking speed, or other movement tests in people with polymyalgia rheumatica. Other conditions. More evidence is needed to rate whey protein for these uses.

  20. Single and multiple CH (calponin homology) domain containing multidomain proteins in Arabidopsis and Saccharomyces: an inventory.

    PubMed

    Friedberg, Felix

    2011-01-01

    Genes for individual domains such as CH, lim, ankyrin, PH and RhoGAP, IQ motif, Ig_FLMN, spectrin, and EF hand probably existed in early evolution before there were plants, fungi or animals so that when we examine multidomain proteins in Arabidopsis, Saccharomyces, Dictyostelium or Homo Sapiens we encounter various combinations of such domains. While all of these four species express Fimbrin and EB1, the lists of CH containing multidomain proteins, however, differ in number and in type for each of them. There was no further great increase in the number of new single domain proteins. Still many new multidomain genes evolved--but far more so in metazoans--than in plants or fungi. In both plants and fungi only singlet CH domains but no doublets (other than those forming the Fimbrin quadruplet) were incorporated. That is in these two branches one finds no alpha actinin, dystrophin or filamin even though the individual building blocks (i.e. domains such as spectrin or IG-FLMN) were available in Arabidopsis. Possibly transposons create new chimeric multidomain genes by mixing and matching genes or gene fragments.

  1. Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment

    PubMed Central

    1995-01-01

    The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus- host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle. PMID:7540613

  2. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  3. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples

    PubMed Central

    Belov, Larissa; Matic, Kieran J.; Hallal, Susannah; Best, O. Giles; Mulligan, Stephen P.; Christopherson, Richard I.

    2016-01-01

    Extracellular vesicles (EV) are membranous particles (30–1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19+ EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals. PMID:27086589

  4. Bioinformatics analysis of the proteins interacting with LASP-1 and their association with HBV-related hepatocellular carcinoma

    PubMed Central

    Kong, Fan-Yun; Zhu, Ting; Li, Nan; Cai, Yun-Fei; Zhou, Kai; Wei, Xiao; Kou, Yan-Bo; You, Hong-Juan; Zheng, Kui-Yang; Tang, Ren-Xian

    2017-01-01

    LIM and SH3 domain protein (LASP-1) is responsible for the development of several types of human cancers via the interaction with other proteins; however, the precise biological functions of proteins interacting with LASP-1 are not fully clarified. Although the role of LASP-1 in hepatocarcinogenesis has been reported, the implication of LASP-1 interactors in HBV-related hepatocellular carcinoma (HCC) is not clearly evaluated. We obtained information regarding LASP-1 interactors from public databases and published studies. Via bioinformatics analysis, we found that LASP-1 interactors were related to distinct molecular functions and associated with various biological processes. Through an integrated network analysis of the interaction and pathways of LASP-1 interactors, cross-talk between different proteins and associated pathways was found. In addition, LASP-1 and several its interactors are significantly altered in HBV-related HCC through microarray analysis and could form a complex co-expression network. In the disease, LASP-1 and its interactors were further predicted to be regulated by a complex interaction network composed of different transcription factors. Besides, numerous LASP-1 interactors were associated with various clinical factors and related to the survival and recurrence of HBV-related HCC. Taken together, these results could help enrich our understanding of LASP-1 interactors and their relationships with HBV-related HCC. PMID:28266596

  5. ORFer – retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files

    PubMed Central

    2002-01-01

    Background Functional genomics involves the parallel experimentation with large sets of proteins. This requires management of large sets of open reading frames as a prerequisite of the cloning and recombinant expression of these proteins. Results A Java program was developed for retrieval of protein and nucleic acid sequences and annotations from NCBI GenBank, using the XML sequence format. Annotations retrieved by ORFer include sequence name, organism and also the completeness of the sequence. The program has a graphical user interface, although it can be used in a non-interactive mode. For protein sequences, the program also extracts the open reading frame sequence, if available, and checks its correct translation. ORFer accepts user input in the form of single or lists of GenBank GI identifiers or accession numbers. It can be used to extract complete sets of open reading frames and protein sequences from any kind of GenBank sequence entry, including complete genomes or chromosomes. Sequences are either stored with their features in a relational database or can be exported as text files in Fasta or tabulator delimited format. The ORFer program is freely available at http://www.proteinstrukturfabrik.de/orfer. Conclusion The ORFer program allows for fast retrieval of DNA sequences, protein sequences and their open reading frames and sequence annotations from GenBank. Furthermore, storage of sequences and features in a relational database is supported. Such a database can supplement a laboratory information system (LIMS) with appropriate sequence information. PMID:12493080

  6. ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle

    PubMed Central

    Martinelli, Valentina C.; Kyle, W. Buck; Kojic, Snezana; Vitulo, Nicola; Li, Zhaohui; Belgrano, Anna; Maiuri, Paolo; Banks, Lawrence; Vatta, Matteo; Valle, Giorgio; Faulkner, Georgine

    2014-01-01

    ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein. PMID:24647531

  7. Age-dependent decline of nogo-a protein in the mouse cerebrum.

    PubMed

    Kumari, Anita; Thakur, M K

    2014-11-01

    Nogo-A, a myelin-associated neurite growth inhibitory protein, is implicated in synaptic plasticity. It binds to its receptor namely the Nogo-66 receptor1 (NgR1) and regulates filamentous (F) actin dynamics via small GTPases of the Rho family, RhoA kinase (ROCK), LimK and cofilin. These proteins are associated with the structural plasticity, one of the components of synaptic plasticity, which is known to decline with normal aging. So, the level of Nogo-A and its receptor NgR1 are likely to vary during normal brain aging. However, it is not clearly understood how the levels of Nogo-A and its receptor NgR1 change in the cerebrum during aging. Several studies show an age- and gender-dependent decline in synaptic plasticity. Therefore, the present study was planned to analyze the relative changes in the mRNA and protein levels of Nogo-A and NgR1 in both male and female mice cerebrum during normal aging. Western blot analysis has shown decrease in Nogo-A protein level during aging in both male and female mice cerebrum. This was further confirmed by immunofluorescence analysis. RT-PCR analysis of Nogo-A mRNA showed no significant difference in the above-mentioned groups. This was also supported by in situ hybridization. NgR1 protein and its mRNA expression levels showed no significant alteration with aging in the cerebrum of both male and female mice. Taken together, we speculate that the downregulation of Nogo-A protein might have a role in the altered synaptic plasticity during aging.

  8. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  9. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

    PubMed

    Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS.

  10. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  11. Heat Shock 70-kDa Protein 5 (Hspa5) Is Essential for Pronephros Formation by Mediating Retinoic Acid Signaling*

    PubMed Central

    Shi, Weili; Xu, Gang; Wang, Chengdong; Sperber, Steven M.; Chen, Yonglong; Zhou, Qin; Deng, Yi; Zhao, Hui

    2015-01-01

    Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and β1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development. PMID:25398881

  12. Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization.

    PubMed

    Miehe, Ulrich; Neumaier-Wagner, Peruka; Kadyrov, Mamed; Goyal, Pankaj; Alfer, Joachim; Rath, Werner; Huppertz, Berthold

    2005-01-01

    The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.

  13. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  14. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  15. Interaction of TACC proteins with the FHL family: implications for ERK signaling

    PubMed Central

    Lauffart, Brenda; Sondarva, Gautam V.; Gangisetty, Omkaram; Cincotta, Melissa

    2007-01-01

    The Transforming acidic coiled coil (TACC) proteins play a conserved role in normal development and tumorigenesis through interactions with multiple complexes involved in transcription, translation, and centrosomal dynamics. However, despite significant work on the function of TACC3 in the control of centrosomal mechanics, relatively little functional data is known about the family’s founding member, TACC1. From a continued analysis of clones isolated by an unbiased yeast two-hybrid assay, we now show direct physical interactions between the TACC1 and the FHL (Four and a Half LIM-only) family of proteins. The authenticity of these interactions was validated both in vitro and in cellular systems. The FHLs exhibit diverse biological roles such as the regulation of the actin cytoskeleton and are promiscuous coregulators for several transcription factors. The interaction of the endogenous TACC-FHL proteins is primarily localized to the nucleus. However, similar to FHL2, overexpression of TACC1A in HEK293 is able to sequester serum activated ERK to the cytoplasm. This has the effect of reducing the serum induced transcriptional response of the c-fos and c-jun genes. The observation that TACCs can interact with the FHLs and alter their serum induced activities raises the possibility that the TACCs participate in crosstalk between cell signaling pathways important for cancer development and tumor progression. The transforming acidic coiled coil genes are known to be important prognostic indicators for breast, ovarian and lung cancer. In this manuscript, we identify a novel interaction between the TACCs and the FHL protein family. This interaction has an affect on ERK and may in part explain the variable associations and changes in subcellular locations of each family with specific subtypes of malignancy. PMID:18481206

  16. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  17. ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration

    PubMed Central

    Lecroisey, Claire; Brouilly, Nicolas; Qadota, Hiroshi; Mariol, Marie-Christine; Rochette, Nicolas C.; Martin, Edwige; Benian, Guy M.; Ségalat, Laurent; Mounier, Nicole; Gieseler, Kathrin

    2013-01-01

    In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy. PMID:23427270

  18. Loss of the Cytoskeletal Protein Pdlim7 Predisposes Mice to Heart Defects and Hemostatic Dysfunction

    PubMed Central

    Krcmery, Jennifer; Gupta, Rajesh; Sadleir, Rudyard W.; Ahrens, Molly J.; Misener, Sol; Kamide, Christine; Fitchev, Philip; Losordo, Douglas W.; Crawford, Susan E.; Simon, Hans-Georg

    2013-01-01

    The actin-associated protein Pdlim7 is essential for heart and fin development in zebrafish; however, the expression and function of this PDZ-LIM family member in the mammal has remained unclear. Here, we show that Pdlim7 predominantly localizes to actin-rich structures in mice including the heart, vascular smooth muscle, and platelets. To test the requirement for Pdlim7 in mammalian development and function, we analyzed a mouse strain with global genetic inactivation of Pdlim7. We demonstrate that Pdlim7 loss-of-function leads to significant postnatal mortality. Inactivation of Pdlim7 does not disrupt cardiac development, but causes mild cardiac dysfunction in adult mice. Adult Pdlim7-/- mice displayed increased mitral and tricuspid valve annulus to body weight ratios. These structural aberrations in Pdlim7-/- mice were supported by three-dimensional reconstructions of adult cardiac valves, which revealed increased surface area to volume ratios for the mitral and tricuspid valve leaflets. Unexpectedly, we found that loss of Pdlim7 triggers systemic venous and arterial thrombosis, leading to significant mortality shortly after birth in Pdlim7+/- (11/60) and Pdlim7-/- (19/35) mice. In line with a prothrombotic phenotype, adult Pdlim7-/- mice exhibit dramatically decreased tail bleed times compared to controls. These findings reveal a novel and unexpected function for Pdlim7 in maintaining proper hemostasis in neonatal and adult mice. PMID:24278323

  19. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  20. The involvement of cysteine-rich intestinal protein in early development and innate immunity of Asiatic hard clam, Meretrix meretrix.

    PubMed

    Chen, Hongjian; Yang, Xue; Tang, Ting; Li, Juan; Liu, Baozhong; Liu, Fengsong; Xie, Song

    2014-10-01

    Cysteine-rich intestinal protein (CRIP), a Zn(2+)-binding protein, contains a single copy of the highly conserved double-zinc-finger structure known as the LIM (lin-11-isl-1-mec-3) motif. In this paper, a cDNA encoding MmCRIP was isolated from the Asiatic hard clam Meretrix meretrix. The full-length cDNA of MmCRIP consists of a 237-bp open reading frame that encodes a polypeptide of 78 amino acids with a predicted molecular weight (MW) of 8635.8 Da and theoretical isoelectric point (pI) of 9.01. Bioinformatics analysis showed that it belonged to a new member of the CRIP subfamily. Relationship analysis revealed that MmCRIP has high-levels of sequence similarity to many CRIPs reported in other animals, particularly in invertebrates. Real-time PCR analysis showed that the highest level of MmCRIP expression was in hemocyte tissue and at pediveligers stage. To investigate immune function, mature clams were challenged with Aeromonas hydrophila. During A. hydrophila infection, up-regulation of MmCRIP transcript in clam's hemocyte, gill and hepatopancreas was detected. DsRNAi (double-strand RNA interference) approach was employed to study the function of MmCRIP and the data showed that inactivation of the MmCRIP gene blocked larvae development and caused mass mortalities. The probable roles of MmCRIP in clam early development and innate immunity are presented for the first time.

  1. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  2. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  3. Kinetic characterization of the protein Z-dependent protease inhibitor reaction with blood coagulation factor Xa.

    PubMed

    Huang, Xin; Swanson, Richard; Broze, George J; Olson, Steven T

    2008-10-31

    Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.

  4. Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice.

    PubMed

    Kolpe, Annasaheb B; Kiener, Tanja K; Grotenbreg, Gijsbert M; Kwang, Jimmy

    2012-09-01

    Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215-219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

  5. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  6. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  7. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  8. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  9. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  10. Modeling and Simulation of Mold Filling with LIMS

    DTIC Science & Technology

    2007-11-02

    University of Delaware All rights reservedSimacek ONR Workshop - 4 RTM and VARTM Processes RTM VARTM 2 July 2003© 2003 University of Delaware All...Tools? Ø Can We See Into the Mold? F In RTM Manufacturing NO! F In VARTM or Laboratory A little Ø Can We Measure What is Happening? F Yes, but at High...Pressure Transducers Flow meter Inlet 2 July 2003© 2003 University of Delaware All rights reservedSimacek ONR Workshop - 22 VARTM Specific

  11. Miniature LIMS System for In Situ Detection of Biosignatures

    NASA Astrophysics Data System (ADS)

    Riedo, A.; Tulej, M.; Neuland, M. B.; Wurz, P.

    2016-05-01

    The current measurement capabilities of our miniature Laser Ablation Ionization Mass Spectrometer for sensitive and quantitative in situ chemical analyses (element, isotope and molecular) of solids on planetary surfaces will be presented.

  12. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  13. Activation of a bacterial lipase by its chaperone.

    PubMed Central

    Hobson, A H; Buckley, C M; Aamand, J L; Jørgensen, S T; Diderichsen, B; McConnell, D J

    1993-01-01

    The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase. Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans. Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA. Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA. However, no lipase activity is observed in the absence of limA. limA has been overexpressed and encodes a 33-kDa protein, Lim. If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation. Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody. The Lim protein has therefore the properties of a chaperone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685908

  14. Intraflagellar Transport Protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain

    PubMed Central

    Gorivodsky, Marat; Mukhopadhyay, Mahua; Wilsch-Braeuninger, Michaela; Phillips, Matthew; Teufel, Andreas; Kim, Changmee; Malik, Nasir; Huttner, Wieland; Westphal, Heiner

    2008-01-01

    IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the Intraflagellar Transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5–13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling. PMID:18930042

  15. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  16. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  17. Whey protein fractionation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  18. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  19. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

  20. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  1. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    PubMed

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  2. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity‡

    PubMed Central

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B.; Titus, Louisa; Boden, Scott D.

    2010-01-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1ΔSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  3. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  4. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  5. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  6. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  7. CSRP2 — EDRN Public Portal

    Cancer.gov

    CSRP2 encodes a LIM domain protein. LIM domain proteins may be involved in regulatory processes important for development and cellular differentiation. CSRP2 has two copies of the cysteine-rich amino acid sequence motif (LIM) with putative zinc-binding activity, and may be involved in regulating ordered cell growth. CSRP2 is thought to play a role in the development of the embryonic vascular system.

  8. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  9. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  10. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  11. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  12. Texturized dairy proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  13. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  14. The E5 Proteins

    PubMed Central

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 is the PDGF β receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis. PMID:23731971

  15. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  16. Protein-protein interactions in multienzyme megasynthetases.

    PubMed

    Weissman, Kira J; Müller, Rolf

    2008-04-14

    The multienzyme polyketide synthases (PKSs), nonribosomal polypeptide synthetases (NRPSs), and their hybrids are responsible for the construction in bacteria of numerous natural products of clinical value. These systems generate high structural complexity by using a simple biosynthetic logic--that of the assembly line. Each of the individual steps in building the metabolites is designated to an independently folded domain within gigantic polypeptides. The domains are clustered into functional modules, and the modules are strung out along the proteins in the order in which they act. Every metabolite results, therefore, from the successive action of up to 100 individual catalysts. Despite the conceptual simplicity of this division-of-labor organization, we are only beginning to decipher the molecular details of the numerous protein-protein interactions that support assembly-line biosynthesis, and which are critical to attempts to re-engineer these systems as a tool in drug discovery. This review aims to summarize the state of knowledge about several aspects of protein-protein interactions, including current architectural models for PKS and NRPS systems, the central role of carrier proteins, and the structural basis for intersubunit recognition.

  17. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  18. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  19. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  20. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  1. [Atypical ubiquitination of proteins].

    PubMed

    Buneeva, O A; Medvedev, A E

    2016-07-01

    Ubiquitination is a type of posttranslational modification of intracellular proteins characterized by covalent attachment of one (monoubiquitination) or several (polyubiquitination) of ubiquitin molecules to target proteins. In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins not necessarily targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes and altered polyubiquitination of certain proteins is crucial for development of serious diseases.

  2. Protein and vegetarian diets.

    PubMed

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  3. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  4. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  5. Conversion of the LIMA1 tumour suppressor into an oncogenic LMO-like protein by API2-MALT1 in MALT lymphoma.

    PubMed

    Nie, Zilin; Du, Ming-Qing; McAllister-Lucas, Linda M; Lucas, Peter C; Bailey, Nathanael G; Hogaboam, Cory M; Lim, Megan S; Elenitoba-Johnson, Kojo S J

    2015-01-08

    MALT1 is the only known paracaspase and is a critical mediator of B- and T-cell receptor signalling. The function of the MALT1 gene is subverted by oncogenic chimeric fusions arising from the recurrent t(11;18)(q21;q21) aberration, which is the most frequent translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. API2-MALT1-positive MALT lymphomas manifest antibiotic resistance and aggressive clinical behaviour with poor clinical outcome. However, the mechanisms underlying API2-MALT1-induced MALT lymphomagenesis are not fully understood. Here we show that API2-MALT1 induces paracaspase-mediated cleavage of the tumour suppressor protein LIMA1. LIMA1 binding by API2-MALT1 is API2 dependent and proteolytic cleavage is dependent on MALT1 paracaspase activity. Intriguingly, API2-MALT1-mediated proteolysis generates a LIM domain-only (LMO)-containing fragment with oncogenic properties in vitro and in vivo. Importantly, primary MALT lymphomas harbouring the API2-MALT1 fusion uniquely demonstrate LIMA1 cleavage fragments. Our studies reveal a novel paracaspase-mediated oncogenic gain-of-function mechanism in the pathogenesis of MALT lymphoma.

  6. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  7. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  8. Protein flexibility as a biosignal.

    PubMed

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  9. Antimicrobial proteins: From old proteins, new tricks.

    PubMed

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis.

  10. Protein-protein Interactions using Radiolytic Footprinting

    SciTech Connect

    Takamoto,K.; Chance, M.

    2006-01-01

    Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.

  11. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation.

  12. Mayaro virus proteins.

    PubMed

    Mezencio, J M; Rebello, M A

    1993-01-01

    Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

  13. TRIM proteins and diseases.

    PubMed

    Watanabe, Masashi; Hatakeyama, Shigetsugu

    2017-01-07

    Ubiquitination is one of the posttranslational modifications that regulates a number of intracellular events including signal transduction, protein quality control, transcription, cell cycle, apoptosis and development. The ubiquitin system functions as a garbage machine to degrade target proteins and as a regulator for several signalling pathways. Biochemical reaction of ubiquitination requires several enzymes including E1, E2 and E3, and E3 ubiquitin ligases play roles as receptors for recognizing target proteins. Most of the tripartite motif (TRIM) proteins are E3 ubiquitin ligases. Recent studies have shown that some TRIM proteins function as important regulators for a variety of diseases including cancer, inflammatory diseases, infectious diseases, neuropsychiatric disorders, chromosomal abnormalities and developmental diseases. In this review, we summarize the involvement of TRIM proteins in the aetiology of various diseases.

  14. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  15. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  16. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  17. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  18. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  19. Chemical Synthesis of Proteins

    PubMed Central

    Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

    2010-01-01

    Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

  20. PIC: Protein Interactions Calculator

    PubMed Central

    Tina, K. G.; Bhadra, R.; Srinivasan, N.

    2007-01-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic–aromatic interactions, aromatic–sulphur interactions and cation–π interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar–apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  1. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  2. Dietary proteins and angiogenesis.

    PubMed

    Medina, Miguel Ángel; Quesada, Ana R

    2014-01-17

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

  3. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  4. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  5. TRIM proteins in development.

    PubMed

    Petrera, Francesca; Meroni, Germana

    2012-01-01

    TRIM proteins play important roles in several patho-physiological processes. Their common activity within the ubiquitylation pathway makes them amenable to a number of diverse biological roles. Many of the TRIM genes are highly and sometimes specifically expressed during embryogenesis, it is therefore not surprising that several of them might be involved in developmental processes. Here, we primarily discuss the developmental implications of two subgroups of TRIM proteins that conserved domain composition and functions from their invertebrate ancestors. The two groups are: the TRIM-NHL proteins implicated in miRNA processing regulation and the TRIM-FN3 proteins involved in ventral midline development.

  6. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  7. Acanthamoeba castellanii STAT Protein

    PubMed Central

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  8. Protein intakes in India.

    PubMed

    Swaminathan, Sumathi; Vaz, Mario; Kurpad, Anura V

    2012-08-01

    Indian diets derive almost 60 % of their protein from cereals with relatively low digestibility and quality. There have been several surveys of diets and protein intakes in India by the National Nutrition Monitoring Board (NNMB) over the last 25 years, in urban and rural, as well as in slum dwellers and tribal populations. Data of disadvantaged populations from slums, tribals and sedentary rural Indian populations show that the protein intake (mainly from cereals) is about 1 gm/kg/day. However, the protein intake looks less promising in terms of the protein digestibility corrected amino acid score (PDCAAS), using lysine as the first limiting amino acid, where all populations, particularly rural and tribal, appear to have an inadequate quality to their protein intake. The protein: energy (PE) ratio is a measure of dietary quality, and has been used in the 2007 WHO/FAO/UNU report to define reference requirement values with which the adequacy of diets can be evaluated in terms of a protein quality corrected PE ratio. It is likely that about one third of this sedentary rural population is at risk of not meeting their requirements. These levels of risk of deficiency are in a population with relatively low BMI populations, whose diets are also inadequate in fruits and vegetables. Therefore, while the burden of enhancing the quality of protein intake in rural India exists, the quality of the diet, in general, represents a challenge that must be met.

  9. Self assembling proteins

    DOEpatents

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  10. Ultrafiltration of pegylated proteins

    NASA Astrophysics Data System (ADS)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  11. Regulation of protein secretion by ... protein secretion?

    PubMed

    Atmakuri, Krishnamohan; Fortune, Sarah M

    2008-09-11

    Mycobacterium tuberculosis (Mtb) requires an alternative protein secretion system, ESX1, for virulence. Recently, Raghavan et al. (2008) reported a new regulatory circuit that may explain how ESX1 activity is controlled during infection. Mtb appears to regulate ESX1 by modulating transcription of associated genes rather than structural components of the secretion system itself.

  12. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  13. The Caenorhabditis elegans Ldb/NLI/Clim orthologue ldb-1 is required for neuronal function.

    PubMed

    Cassata, G; Röhrig, S; Kuhn, F; Hauri, H P; Baumeister, R; Bürglin, T R

    2000-10-01

    LIM homeodomain (LIM-HD) and nuclear LIM-only proteins play important roles in a variety of developmental processes in animals. In some cases their activities are modulated by a nuclear LIM binding protein family called Ldb/NLI/Clim. Here we characterize the Ldb/NLI/Clim orthologue ldb-1 of the nematode Caenorhabditis elegans. Two alternatively spliced variants exist, which differ in their amino-termini. The ldb-1 orthologue of Caenorhabditis briggsae has the same structure as that of C. elegans and is highly conserved throughout the open reading frame, while conservation to fly and vertebrate proteins is restricted to specific domains: the dimerization domain, the nuclear localization sequence, and the LIM interaction domain. C. elegans ldb-1 is expressed in neurogenic tissues in embryos, in all neurons in larval and adult stages, and in vulval cells, gonadal sheath cells, and some body muscle cells. C. elegans LDB-1 is able to specifically bind LIM domains in yeast two-hybrid assays. RNA inactivation studies suggest that C. elegans ldb-1 is not required for the differentiation of neurons that express the respective LIM-HD genes or for LIM-HD gene autoregulation. In contrast, ldb-1 is necessary for several neuronal functions mediated by LIM-HD proteins, including the transcriptional activation of mec-2, the mechanosensory neuron-specific stomatin.

  14. Engineered Protein Polymers

    DTIC Science & Technology

    2010-05-31

    of each pure polymer, we plan to combine the various polymer solutions in different ratios to tune the composition and physico-chemical properties...protein materials as vehicles for storage and delivery of small molecules. Each protein polymer under concentrations for particle formation ( vida

  15. Multidomain proteins under force.

    PubMed

    Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel

    2017-04-28

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  16. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  17. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  18. Poxviral Ankyrin Proteins

    PubMed Central

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  19. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  20. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  1. Proteins in unexpected locations.

    PubMed Central

    Smalheiser, N R

    1996-01-01

    Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer. PMID:8862516

  2. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  3. Transdermal delivery of proteins.

    PubMed

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  4. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  5. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  6. Protein Unfolding and Alzheimer's

    NASA Astrophysics Data System (ADS)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  7. Junin virus structural proteins.

    PubMed Central

    De Martínez Segovia, Z M; De Mitri, M I

    1977-01-01

    Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined. PMID:189088

  8. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so

  9. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  10. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  11. [Controversies around diet proteins].

    PubMed

    Cichosz, Grazyna; Czeczot, Hanna

    2013-12-01

    Critical theories regarding proteins of anima origin are still and still popularized, though they are ungrounded from scientific point of view. Predominance of soya proteins over the animal ones in relation to their influence on calcium metabolism, bone break risk or risk of osteoporosis morbidity has not been confirmed in any honest, reliable research experiment. Statement, that sulphur amino acids influence disadvantageously on calcium metabolism of human organism and bone status, is completely groundless, the more so as presence of sulphur amino acids in diet (animal proteins are their best source) is the condition of endogenic synthesis of glutathione, the key antioxidant of the organism, and taurine stimulating brain functioning. Deficiency of proteins in the diet produce weakness of intellectual effectiveness and immune response. There is no doubt that limitation of consumption of animal proteins of standard value is not good for health.

  12. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  13. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  14. Regulation of protein turnover by heat shock proteins.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.

  15. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  16. Prediction of protein-protein interactions: unifying evolution and structure at protein interfaces.

    PubMed

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-06-01

    The vast majority of the chores in the living cell involve protein-protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein-protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations.

  17. NMCP/LINC proteins

    PubMed Central

    Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

    2013-01-01

    Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins. PMID:24128696

  18. TRIM proteins in cancer.

    PubMed

    Cambiaghi, Valeria; Giuliani, Virginia; Lombardi, Sara; Marinelli, Cristiano; Toffalorio, Francesca; Pelicci, Pier Giuseppe

    2012-01-01

    Some members of the tripartite motif (TRIM/RBCC) protein family are thought to be important regulators of carcinogenesis. This is not surprising as the TRIM proteins are involved in several biological processes, such as cell growth, development and cellular differentiation and alteration of these proteins can affect transcriptional regulation, cell proliferation and apoptosis. In particular, four TRIM family genes are frequently translocated to other genes, generating fusion proteins implicated in cancer initiation and progression. Among these the most famous is the promyelocytic leukaemia gene PML, which encodes the protein TRIM19. PML is involved in the t(15;17) translocation that specifically occurs in Acute Promyelocytic Leukaemia (APL), resulting in a PML-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. Other members of the TRIM family are linked to cancer development without being involved in chromosomal re-arrangements, possibly through ubiquitination or loss of tumour suppression functions. This chapter discusses the biological functions of TRIM proteins in cancer.

  19. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  20. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  1. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  2. Piezoelectric allostery of protein

    NASA Astrophysics Data System (ADS)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

  3. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  4. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  5. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  6. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  7. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  8. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression.

  9. Evolution of proteins.

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1971-01-01

    The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

  10. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  11. Teaching resources. Protein phosphatases.

    PubMed

    Salton, Stephen R

    2005-03-01

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

  12. Electrochromatographic separation of proteins

    NASA Technical Reports Server (NTRS)

    Basak, S. K.; Velayudhan, A.; Kohlmann, K.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

    1995-01-01

    We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

  13. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  14. Plant protein glycosylation

    PubMed Central

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  15. Protein Model Database

    SciTech Connect

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  16. Protein Colloidal Aggregation Project

    NASA Technical Reports Server (NTRS)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  17. Fully automated protein purification

    PubMed Central

    Camper, DeMarco V.; Viola, Ronald E.

    2009-01-01

    Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol. PMID:19595984

  18. Protein fabrication automation

    PubMed Central

    Cox, J. Colin; Lape, Janel; Sayed, Mahmood A.; Hellinga, Homme W.

    2007-01-01

    Facile “writing” of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable. PMID:17242375

  19. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  20. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  1. Occupational protein contact dermatitis.

    PubMed

    Barbaud, Annick; Poreaux, Claire; Penven, Emmanuelle; Waton, Julie

    2015-01-01

    Occupational contact dermatitis is generally caused by haptens but can also be induced by proteins causing mainly immunological contact urticaria (ICU); chronic hand eczema in the context of protein contact dermatitis (PCD). In a monocentric retrospective study, from our database, only 31 (0.41%) of patients with contact dermatitis had positive skin tests with proteins: 22 had occupational PCD, 3 had non-occupational PCD, 5 occupational ICU and 1 cook had a neutrophilic fixed food eruption (NFFE) due to fish. From these results and analysis of literature, the characteristics of PCD can be summarized as follows. It is a chronic eczematous dermatitis, possibly exacerbated by work, suggestive if associated with inflammatory perionyxix and immediate erythema with pruritis, to be investigated when the patient resumes work after a period of interruption. Prick tests with the suspected protein-containing material are essential, as patch tests have negative results. In case of multisensitisation revealed by prick tests, it is advisable to analyse IgE against recombinant allergens. A history of atopy, found in 56 to 68% of the patients, has to be checked for. Most of the cases are observed among food-handlers but PCD can also be due to non-edible plants, latex, hydrolysed proteins or animal proteins. Occupational exposure to proteins can thus lead to the development of ICU. Reflecting hypersensitivity to very low concentrations of allergens, investigating ICU therefore requires caution and prick tests should be performed with a diluted form of the causative protein-containing product. Causes are food, especially fruit peel, non-edible plants, cosmetic products, latex, animals.

  2. Chirality and protein biosynthesis.

    PubMed

    Banik, Sindrila Dutta; Nandi, Nilashis

    2013-01-01

    Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

  3. Protein Nitrogen Determination

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  4. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  5. Protein conducting nanopores

    NASA Astrophysics Data System (ADS)

    Harsman, Anke; Krüger, Vivien; Bartsch, Philipp; Honigmann, Alf; Schmidt, Oliver; Rao, Sanjana; Meisinger, Christof; Wagner, Richard

    2010-11-01

    About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40SC as well as a mutant Tom40SC (\\mathrm {S}_{54} \\to E ) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40SC corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40SC S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with \\bar {t}_{\\mathrm {off}} \\cong 1.1 ms for the wildtype, whereas the mutant Tom40SC S54E displayed a biphasic dwelltime distribution (\\bar {t}_{\\mathrm {off}}^1 \\cong 0.4 ms \\bar {t}_{\\mathrm {off}}^2 \\cong 4.6 ms).

  6. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  7. Motor proteins 1: kinesins.

    PubMed

    Bloom, G S; Endow, S A

    1995-01-01

    Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles [17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292] or books [49, 227, 293] that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.

  8. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  9. Disease specific protein corona

    NASA Astrophysics Data System (ADS)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  10. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  11. Food protein sources.

    PubMed

    Pirie, N W

    1976-07-01

    Work on food, planned by the U.M. (Use and Management) Section of the U.K. committe, was limited to sources of protein because we agreed that more problems calling for research were likely to arise in getting adequate supplies of protein than of other types of food. Deer meat can be produced on land too rough and exposed for sheep; parts of the work on their metabolism and food requirements necessitated building a mobile laboratory. The manner in which the nutritive value of maize is affected by changes in the ratios in which the component proteins are present, stimulated similar studies on barley and groundnut. There is good quality protein in coconuts and leaves but its use in human food is restricted by the presence of fibre. Methods for separating protein from fibre and other deleterious components were improved. In cooperation with scientists in India and Nigeria, the potential yield of protein-deficient foods. e.g. cassava, were 'ennobled' by growing micro-organisms on them with the addition of a cheap source of nitrogen.

  12. Protein-Protein Interfaces in Viral Capsids Are Structurally Unique.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2015-11-06

    Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein-protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein-protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein-protein interfaces that share similar physicochemical patterns with some protein-protein interfaces in the capsid set, using the program PCalign we developed for comparing protein-protein interfaces. This overlap in the structural space of protein-protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein-protein interfaces. Furthermore, the generic protein-protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein-protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy.

  13. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  14. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane

    PubMed Central

    Park, Jeong Soon; Lee, Woo Cheol; Yeo, Kwon Joo; Ryu, Kyoung-Seok; Kumarasiri, Malika; Hesek, Dusan; Lee, Mijoon; Mobashery, Shahriar; Song, Jung Hyun; Kim, Seung Il; Lee, Je Chul; Cheong, Chaejoon; Jeon, Young Ho; Kim, Hye-Yeon

    2012-01-01

    The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.—Park, J. S., Lee, W. C., Yeo, K. J., Ryu, K.-S., Kumarasiri, M., Hesek, D., Lee, M., Mobashery, S., Song, J. H., Lim, S. I., Lee, J. C., Cheong, C., Jeon, Y. H., Kim, H.-Y. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane. PMID:21965596

  15. Parallel Computational Protein Design

    PubMed Central

    Zhou, Yichao; Donald, Bruce R.; Zeng, Jianyang

    2016-01-01

    Computational structure-based protein design (CSPD) is an important problem in computational biology, which aims to design or improve a prescribed protein function based on a protein structure template. It provides a practical tool for real-world protein engineering applications. A popular CSPD method that guarantees to find the global minimum energy solution (GMEC) is to combine both dead-end elimination (DEE) and A* tree search algorithms. However, in this framework, the A* search algorithm can run in exponential time in the worst case, which may become the computation bottleneck of large-scale computational protein design process. To address this issue, we extend and add a new module to the OSPREY program that was previously developed in the Donald lab [1] to implement a GPU-based massively parallel A* algorithm for improving protein design pipeline. By exploiting the modern GPU computational framework and optimizing the computation of the heuristic function for A* search, our new program, called gOSPREY, can provide up to four orders of magnitude speedups in large protein design cases with a small memory overhead comparing to the traditional A* search algorithm implementation, while still guaranteeing the optimality. In addition, gOSPREY can be configured to run in a bounded-memory mode to tackle the problems in which the conformation space is too large and the global optimal solution cannot be computed previously. Furthermore, the GPU-based A* algorithm implemented in the gOSPREY program can be combined with the state-of-the-art rotamer pruning algorithms such as iMinDEE [2] and DEEPer [3] to also consider continuous backbone and side-chain flexibility. PMID:27914056

  16. Modeling Mercury in Proteins

    SciTech Connect

    Smith, Jeremy C; Parks, Jerry M

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  17. Benchtop Detection of Proteins

    NASA Technical Reports Server (NTRS)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  18. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  19. Heat Capacity in Proteins

    NASA Astrophysics Data System (ADS)

    Prabhu, Ninad V.; Sharp, Kim A.

    2005-05-01

    Heat capacity (Cp) is one of several major thermodynamic quantities commonly measured in proteins. With more than half a dozen definitions, it is the hardest of these quantities to understand in physical terms, but the richest in insight. There are many ramifications of observed Cp changes: The sign distinguishes apolar from polar solvation. It imparts a temperature (T) dependence to entropy and enthalpy that may change their signs and which of them dominate. Protein unfolding usually has a positive ΔCp, producing a maximum in stability and sometimes cold denaturation. There are two heat capacity contributions, from hydration and protein-protein interactions; which dominates in folding and binding is an open question. Theoretical work to date has dealt mostly with the hydration term and can account, at least semiquantitatively, for the major Cp-related features: the positive and negative Cp of hydration for apolar and polar groups, respectively; the convergence of apolar group hydration entropy at T ≈ 112°C; the decrease in apolar hydration Cp with increasing T; and the T-maximum in protein stability and cold denaturation.

  20. The ras superfamily proteins.

    PubMed

    Chardin, P

    1988-07-01

    Several recent discoveries indicate that the ras genes, frequently activated to a transforming potential in some human tumours, belong to a large family that can be divided into three main branches: the first branch represented by the ras, ral and rap genes; the second branch, by the rho genes; and the third branch, by the rab genes. The C-terminal end of the encoded proteins always includes a cystein, which may become fatty-acylated, suggesting a sub-membrane localization. The ras superfamily proteins share four regions of high homology corresponding to the GTP binding site; however, even in these regions, significant differences are found, suggesting that the various proteins may possess slightly different biochemical properties. Recent reports show that some of these proteins play an essential role in the control of physical processes such as cell motility, membrane ruffling, endocytosis and exocytosis. Nevertheless, the characterization of the proteins directly interacting with the ras or ras-related gene-products will be required to precisely understand their function.

  1. [Protein metabolism in vegans].

    PubMed

    Okuda, T; Miyoshi-Nishimura, H; Makita, T; Sugawa-Katayama, Y; Hazama, T; Simizu, T; Yamaguchi, Y

    1994-11-01

    To elucidate the mechanisms of adaptation to a low-energy and low-protein vegan diet, we carried out dietary surveys and nitrogen balance studies five times during one year on two women and a man who ate raw brown rice, raw green vegetables, three kinds of raw roots, fruit and salt daily. Individual subjects modified this vegan diet slightly. The mean daily energy intake of the subjects was 18, 14, and 32 kcal/kg, of body weight. The loss of body weight was about 10% of the initial level. The daily nitrogen balance was -32, -33, and -11 mg N/kg of body weight. In spite of the negative nitrogen balance, the results of routine clinical tests, initially normal, did not change with the vegan diet. Ten months after the start of the vegan diet, the subjects were given 15N urea orally. The incorporation of 15N into serum proteins suggested that these subjects could utilize urea nitrogen for body protein synthesis. The level of 15N in serum proteins was close to the level in other normal adult men on a low-protein diet with adequate energy for 2 weeks.

  2. Protein Dynamics in Enzymology

    NASA Astrophysics Data System (ADS)

    Brooks, , III

    2001-03-01

    Enzymes carry-out the chemical activity essential for living processes by providing particular structural arrangements of chemically functional moieties through the structure of their constituent proteins. They are suggested to be optimized through evolution to specifically bind the transition state for the chemical processes they participate in, thereby enhancing the rate of these chemical events by 6-12 orders of magnitude. However, proteins are malleable and fluctuating many-body systems and may also utilize coupling between motional processes with catalysis to regulate or promote these processes. Our studies are aimed at exploring the hypothesis that motions of the protein couple distant regions of the molecule to assist catalytic processes. We demonstrate, through the use of molecular simulations, that strongly coupled motions occur in regions of protein molecules distant in sequence and space from each other, and the enzyme’s active site, when the protein is in a reactant state. Further, we find that the presence of this coupling disappears in complexes no longer reactive-competent, i.e., for product configurations and mutant sequences. The implications of these findings and aspects of evolutionary relationships and mutational studies which support the coupling hypothesis will be discussed in the context of our work on dihydrofolate reductase.

  3. Protein folding and de novo protein design for biotechnological applications

    PubMed Central

    Khoury, George A.; Smadbeck, James; Kieslich, Chris A.; Floudas, Christodoulos A.

    2014-01-01

    In the post-genomic era, the medical/biological fields are advancing faster than ever. However, before the power of full-genome sequencing can be fully realized, the connection between amino acid sequence and protein structure, known as the protein folding problem, needs to be elucidated. The protein folding problem remains elusive, with significant difficulties still arising when modeling amino acid sequences lacking an identifiable template. Understanding protein folding will allow for unforeseen advances in protein design, often referred as the inverse protein folding problem. Despite challenges in protein folding, de novo protein design has recently demonstrated significant success via computational techniques. We review advances and challenges in protein structure prediction and de novo protein design, and highlight their interplay in successful biotechnological applications. PMID:24268901

  4. Matricellular proteins and biomaterials.

    PubMed

    Morris, Aaron H; Kyriakides, Themis R

    2014-07-01

    Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials.

  5. Advanced protein formulations

    PubMed Central

    Wang, Wei

    2015-01-01

    It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution. PMID:25858529

  6. Thermal hysteresis proteins.

    PubMed

    Barrett, J

    2001-02-01

    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  7. Thermodynamics of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  8. Protein crystallization studies

    NASA Technical Reports Server (NTRS)

    Lyne, James Evans

    1996-01-01

    The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

  9. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  10. Tracking protein aggregate interactions

    PubMed Central

    Bartz, Jason C; Nilsson, K Peter R

    2011-01-01

    Amyloid fibrils share a structural motif consisting of highly ordered β-sheets aligned perpendicular to the fibril axis.1, 2 At each fibril end, β-sheets provide a template for recruiting and converting monomers.3 Different amyloid fibrils often co-occur in the same individual, yet whether a protein aggregate aids or inhibits the assembly of a heterologous protein is unclear. In prion disease, diverse prion aggregate structures, known as strains, are thought to be the basis of disparate disease phenotypes in the same species expressing identical prion protein sequences.4–7 Here we explore the interactions reported to occur when two distinct prion strains occur together in the central nervous system. PMID:21597336

  11. Bioinformatics and Moonlighting Proteins

    PubMed Central

    Hernández, Sergio; Franco, Luís; Calvo, Alejandra; Ferragut, Gabriela; Hermoso, Antoni; Amela, Isaac; Gómez, Antonio; Querol, Enrique; Cedano, Juan

    2015-01-01

    Multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. Usually, moonlighting proteins are experimentally revealed by serendipity. For this reason, it would be helpful that Bioinformatics could predict this multifunctionality, especially because of the large amounts of sequences from genome projects. In the present work, we analyze and describe several approaches that use sequences, structures, interactomics, and current bioinformatics algorithms and programs to try to overcome this problem. Among these approaches are (a) remote homology searches using Psi-Blast, (b) detection of functional motifs and domains, (c) analysis of data from protein–protein interaction databases (PPIs), (d) match the query protein sequence to 3D databases (i.e., algorithms as PISITE), and (e) mutation correlation analysis between amino acids by algorithms as MISTIC. Programs designed to identify functional motif/domains detect mainly the canonical function but usually fail in the detection of the moonlighting one, Pfam and ProDom being the best methods. Remote homology search by Psi-Blast combined with data from interactomics databases (PPIs) has the best performance. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can only be used in very specific situations – it requires the existence of multialigned family protein sequences – but can suggest how the evolutionary process of second function acquisition took place. The multitasking protein database MultitaskProtDB (http://wallace.uab.es/multitask/), previously published by our group, has been used as a benchmark for the all of the analyses. PMID:26157797

  12. Antioxidants and protein oxidation.

    PubMed

    Griffiths, H R

    2000-11-01

    Proteins are susceptible to oxidation by reactive oxygen species, where the type of damage induced is characteristic of the denaturing species. The induction of protein carbonyls is a widely applied biomarker, arising from primary oxidative insult. However, when applied to complex biological and pathological conditions it can be subject to interference from lipid, carbohydrate and DNA oxidation products. More recently, interest has focused on the analysis of specific protein bound oxidised amino acids. Of the 22 amino acids, aromatic and sulphydryl containing residues have been regarded as being particularly susceptible to oxidative modification, with L-DOPA from tyrosine, ortho-tyrosine from phenylalanine; sulphoxides and disulphides from methionine and cysteine respectively; and kynurenines from tryptophan. Latterly, the identification of valine and leucine hydroxides, reduced from hydroperoxide intermediates, has been described and applied. In order to examine the nature of oxidative damage and protective efficacy of antioxidants the markers must be thoroughly evaluated for dosimetry in vitro following damage by specific radical species. Antioxidant protection against formation of the biomarker should be demonstrated in vitro. Quantification of biomarkers in proteins from normal subjects should be within the limits of detection of any analytical procedure. Further to this, the techniques for isolation and hydrolysis of specific proteins should demonstrate that in vitro oxidation is minimised. There is a need for the development of standards for quality assurance material to standardise procedures between laboratories. At present, antioxidant effects on protein oxidation in vivo are limited to animal studies, where dietary antioxidants have been reported to reduce dityrosine formation during rat exercise training. Two studies on humans have been reported last year. The further application of these methods to human studies is indicated, where the quality of the

  13. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets.

  14. SAP family proteins.

    PubMed

    Fujita, A; Kurachi, Y

    2000-03-05

    Thus far, five members including Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102, and PSD-93/chapsyn110 which belong to SAP family have been identified. Recent studies have revealed that these proteins play important roles in the localization and function of glutamate receptors and K(+) channels. Although most of them have been reported to be localized to the synapse, only one member, SAP97, is expressed also in the epithelial cells. In this review, we have summarized structural characters of SAP family proteins and discuss their functions in neurons and epithelial cells.

  15. Protein Biosynthesis in Mitochondria

    PubMed Central

    Kuzmenko, A. V.; Levitskii, S. A.; Vinogradova, E. N.; Atkinson, G. C.; Hauryliuk, V.; Zenkin, N.; Kamenski, P. A.

    2013-01-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis. PMID:24228873

  16. Congenital protein hypoglycosylation diseases

    PubMed Central

    Sparks, Susan E

    2012-01-01

    Glycosylation is an essential process by which sugars are attached to proteins and lipids. Complete lack of glycosylation is not compatible with life. Because of the widespread function of glycosylation, inherited disorders of glycosylation are multisystemic. Since the identification of the first defect on N-linked glycosylation in the 1980s, there are over 40 different congenital protein hypoglycosylation diseases. This review will include defects of N-linked glycosylation, O-linked glycosylation and disorders of combined N- and O-linked glycosylation. PMID:23776380

  17. [Protein-losing enteropathy].

    PubMed

    Parfenov, A I; Krums, L M

    2017-01-01

    Protein-losing enteropathy (PLE) is a rare complication of intestinal diseases. Its main manifestation is hypoproteinemic edema. The diagnosis of PLE is based on the verification of protein loss into the intestinal lumen, by determining fecal α1-antitrypsin concentration and clearance. The localization of the affected colonic segment is clarified using radiologic and endoscopic techniques. The mainstay of treatment for PLE is a fat-free diet enriched with medium-chain triglycerides. Surgical resection of the affected segment of the colon may be the treatment of choice for severe hypoproteinemia resistant to drug therapy.

  18. DELIVERY OF THERAPEUTIC PROTEINS

    PubMed Central

    Pisal, Dipak S.; Kosloski, Matthew P.; Balu-Iyer, Sathy V.

    2009-01-01

    The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g. liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches. PMID:20049941

  19. Protein biosynthesis in mitochondria.

    PubMed

    Kuzmenko, A V; Levitskii, S A; Vinogradova, E N; Atkinson, G C; Hauryliuk, V; Zenkin, N; Kamenski, P A

    2013-08-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

  20. Protein energy malnutrition.

    PubMed

    Grover, Zubin; Ee, Looi C

    2009-10-01

    Protein energy malnutrition (PEM) is a common problem worldwide and occurs in both developing and industrialized nations. In the developing world, it is frequently a result of socioeconomic, political, or environmental factors. In contrast, protein energy malnutrition in the developed world usually occurs in the context of chronic disease. There remains much variation in the criteria used to define malnutrition, with each method having its own limitations. Early recognition, prompt management, and robust follow up are critical for best outcomes in preventing and treating PEM.

  1. An introduction to protein moonlighting.

    PubMed

    Jeffery, Constance J

    2014-12-01

    Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple physiologically relevant biochemical or biophysical functions. Almost 300 proteins have been found to moonlight. The known examples of moonlighting proteins include diverse types of proteins, including receptors, enzymes, transcription factors, adhesins and scaffolds, and different combinations of functions are observed. Moonlighting proteins are expressed throughout the evolutionary tree and function in many different biochemical pathways. Some moonlighting proteins can perform both functions simultaneously, but for others, the protein's function changes in response to changes in the environment. The diverse examples of moonlighting proteins already identified, and the potential benefits moonlighting proteins might provide to the organism, such as through coordinating cellular activities, suggest that many more moonlighting proteins are likely to be found. Continuing studies of the structures and functions of moonlighting proteins will aid in predicting the functions of proteins identified through genome sequencing projects, in interpreting results from proteomics experiments, in understanding how different biochemical pathways interact in systems biology, in annotating protein sequence and structure databases, in studies of protein evolution and in the design of proteins with novel functions.

  2. Protein domain connectivity and essentiality

    NASA Astrophysics Data System (ADS)

    da F. Costa, L.; Rodrigues, F. A.; Travieso, G.

    2006-10-01

    Protein-protein interactions can be properly modeled as scale-free complex networks, while the lethality of proteins has been correlated with the node degrees, therefore defining a lethality-centrality rule. In this work the authors revisit this relevant problem by focusing attention not on proteins as a whole, but on their functional domains, which are ultimately responsible for their binding potential. Four networks are considered: the original protein-protein interaction network, its randomized version, and two domain networks assuming different lethality hypotheses. By using formal statistical analysis, they show that the correlation between connectivity and essentiality is higher for domains than for proteins.

  3. Conformation Distributions in Adsorbed Proteins.

    NASA Astrophysics Data System (ADS)

    Meuse, Curtis W.; Hubbard, Joseph B.; Vrettos, John S.; Smith, Jackson R.; Cicerone, Marcus T.

    2007-03-01

    While the structural basis of protein function is well understood in the biopharmaceutical and biotechnology industries, few methods for the characterization and comparison of protein conformation distributions are available. New methods capable of measuring the stability of protein conformations and the integrity of protein-protein, protein-ligand and protein-surface interactions both in solution and on surfaces are needed to help the development of protein-based products. We are developing infrared spectroscopy methods for the characterization and comparison of molecular conformation distributions in monolayers and in solutions. We have extracted an order parameter describing the orientational and conformational variations of protein functional groups around the average molecular values from a single polarized spectrum. We will discuss the development of these methods and compare them to amide hydrogen/deuterium exchange methods for albumin in solution and on different polymer surfaces to show that our order parameter is related to protein stability.

  4. NextGen protein design

    PubMed Central

    Regan, Lynne

    2014-01-01

    Protein engineering is at an exciting stage because designed protein–protein interactions are being used in many applications. For instance, three designed proteins are now in clinical trials. Although there have been many successes over the last decade, protein engineering still faces numerous challenges. Often, designs do not work as anticipated and they still require substantial redesign. The present review focuses on the successes, the challenges and the limitations of rational protein design today. PMID:24059497

  5. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  6. Protein states and proteinquakes.

    PubMed Central

    Ansari, A; Berendzen, J; Bowne, S F; Frauenfelder, H; Iben, I E; Sauke, T B; Shyamsunder, E; Young, R D

    1985-01-01

    After photodissociation of carbon monoxide bound to myoglobin, the protein relaxes to the deoxy equilibrium structure in a quake-like motion. Investigation of the proteinquake and of related intramolecular equilibrium motions shows that states and motions have a hierarchical glass-like structure. PMID:3860839

  7. Dynamics of protein conformations

    NASA Astrophysics Data System (ADS)

    Stepanova, Maria

    2010-10-01

    A novel theoretical methodology is introduced to identify dynamic structural domains and analyze local flexibility in proteins. The methodology employs a multiscale approach combining identification of essential collective coordinates based on the covariance analysis of molecular dynamics trajectories, construction of the Mori projection operator with these essential coordinates, and analysis of the corresponding generalized Langevin equations [M.Stepanova, Phys.Rev.E 76(2007)051918]. Because the approach employs a rigorous theory, the outcomes are physically transparent: the dynamic domains are associated with regions of relative rigidity in the protein, whereas off-domain regions are relatively soft. This also allows scoring the flexibility in the macromolecule with atomic-level resolution [N.Blinov, M.Berjanskii, D.S.Wishart, and M.Stepanova, Biochemistry, 48(2009)1488]. The applications include the domain coarse-graining and characterization of conformational stability in protein G and prion proteins. The results are compared with published NMR experiments. Potential applications for structural biology, bioinformatics, and drug design are discussed.

  8. Proteins of Excitable Membranes

    PubMed Central

    Nachmansohn, David

    1969-01-01

    Excitable membranes have the special ability of changing rapidly and reversibly their permeability to ions, thereby controlling the ion movements that carry the electric currents propagating nerve impulses. Acetylcholine (ACh) is the specific signal which is released by excitation and is recognized by a specific protein, the ACh-receptor; it induces a conformational change, triggering off a sequence of reactions resulting in increased permeability. The hydrolysis of ACh by ACh-esterase restores the barrier to ions. The enzymes hydrolyzing and forming ACh and the receptor protein are present in the various types of excitable membranes. Properties of the two proteins directly associated with electrical activity, receptor and esterase, will be described in this and subsequent lectures. ACh-esterase has been shown to be located within the excitable membranes. Potent enzyme inhibitors block electrical activity demonstrating the essential role in this function. The enzyme has been recently crystallized and some protein properties will be described. The monocellular electroplax preparation offers a uniquely favorable material for analyzing the properties of the ACh-receptor and its relation to function. The essential role of the receptor in electrical activity has been demonstrated with specific receptor inhibitors. Recent data show the basically similar role of ACh in the axonal and junctional membranes; the differences of electrical events and pharmacological actions are due to variations of shape, structural organization, and environment. PMID:19873642

  9. Cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  10. Chaos in protein dynamics.

    PubMed

    Braxenthaler, M; Unger, R; Auerbach, D; Given, J A; Moult, J

    1997-12-01

    MD simulations, currently the most detailed description of the dynamic evolution of proteins, are based on the repeated solution of a set of differential equations implementing Newton's second law. Many such systems are known to exhibit chaotic behavior, i.e., very small changes in initial conditions are amplified exponentially and lead to vastly different, inherently unpredictable behavior. We have investigated the response of a protein fragment in an explicit solvent environment to very small perturbations of the atomic positions (10(-3)-10(-9) A). Independent of the starting conformation (native-like, compact, extended), perturbed dynamics trajectories deviated rapidly, leading to conformations that differ by approximately 1 A RMSD within 1-2 ps. Furthermore, introducing the perturbation more than 1-2 ps before a significant conformational transition leads to a loss of the transition in the perturbed trajectories. We present evidence that the observed chaotic behavior reflects physical properties of the system rather than numerical instabilities of the calculation and discuss the implications for models of protein folding and the use of MD as a tool to analyze protein folding pathways.

  11. Tuber Storage Proteins

    PubMed Central

    SHEWRY, PETER R.

    2003-01-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose‐binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers. PMID:12730067

  12. Tuber storage proteins.

    PubMed

    Shewry, Peter R

    2003-06-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

  13. Protein thin film machines.

    PubMed

    Federici, Stefania; Oliviero, Giulio; Hamad-Schifferli, Kimberly; Bergese, Paolo

    2010-12-01

    We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fueled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model.

  14. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  15. 24-hour urine protein

    MedlinePlus

    ... your doctor may be able to order a test that is done on just one urine sample (protein-to-creatinine ratio). Normal Results The normal value is less than 100 milligrams per day or less than 10 milligrams per deciliter ... of these tests. Normal value ranges may vary slightly among different ...

  16. Protein Requirements during Aging

    PubMed Central

    Courtney-Martin, Glenda; Ball, Ronald O.; Pencharz, Paul B.; Elango, Rajavel

    2016-01-01

    Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals. PMID:27529275

  17. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  18. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  19. Protein specific polymeric immunomicrospheres

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping S. (Inventor); Dreyer, William J. (Inventor)

    1980-01-01

    Small, round, bio-compatible microspheres capable of covalently bonding proteins and having a uniform diameter below about 3500 A are prepared by substantially instantaneously initiating polymerization of an aqueous emulsion containing no more than 35% total monomer including an acrylic monomer substituted with a covalently bondable group such as hydroxyl, amino or carboxyl and a minor amount of a cross-linking agent.

  20. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  1. Monobodies and other synthetic binding proteins for expanding protein science.

    PubMed

    Sha, Fern; Salzman, Gabriel; Gupta, Ankit; Koide, Shohei

    2017-03-01

    Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

  2. Production of specific antibodies against protein A fusion proteins.

    PubMed Central

    Löwenadler, B; Nilsson, B; Abrahmsén, L; Moks, T; Ljungqvist, L; Holmgren, E; Paleus, S; Josephson, S; Philipson, L; Uhlén, M

    1986-01-01

    The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:3096719

  3. Accessory proteins for heterotrimeric G-proteins in the kidney

    PubMed Central

    Park, Frank

    2015-01-01

    Heterotrimeric G-proteins play a fundamentally important role in regulating signal transduction pathways in the kidney. Accessory proteins are being identified as direct binding partners for heterotrimeric G-protein α or βγ subunits to promote more diverse mechanisms by which G-protein signaling is controlled. In some instances, accessory proteins can modulate the signaling magnitude, localization, and duration following the activation of cell membrane-associated receptors. Alternatively, accessory proteins complexed with their G-protein α or βγ subunits can promote non-canonical models of signaling activity within the cell. In this review, we will highlight the expression profile, localization and functional importance of these newly identified accessory proteins to control the function of select G-protein subunits under normal and various disease conditions observed in the kidney. PMID:26300785

  4. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    PubMed Central

    2011-01-01

    Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions. PMID:21569443

  5. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  6. An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins.

    PubMed

    Howell, Jenika M; Winstone, Tara L; Coorssen, Jens R; Turner, Raymond J

    2006-04-01

    Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.

  7. Gastroprotective mechanisms of Citrus lemon (Rutaceae) essential oil and its majority compounds limonene and β-pinene: involvement of heat-shock protein-70, vasoactive intestinal peptide, glutathione, sulfhydryl compounds, nitric oxide and prostaglandin E₂.

    PubMed

    Rozza, Ariane Leite; Moraes, Thiago de Mello; Kushima, Hélio; Tanimoto, Alexandre; Marques, Márcia Ortiz Mayo; Bauab, Taís Maria; Hiruma-Lima, Clélia Akiko; Pellizzon, Cláudia Helena

    2011-01-15

    Citrus lemon (CL) belongs to Rutaceae family and is popularly known in Brazil as limão siciliano. The phytochemical analysis of CL fruit bark essential oil showed two majority components, limonene (LIM) and β-pinene (PIN). This study aimed to evaluate the gastroprotective mechanism of action from CL, LIM and PIN in ethanol- and indomethacin-induced gastric ulcers and its in vitro anti-Helicobacter pylori activity. After ethanol-induced gastric ulcer, the ulcer area was measured and the stomachs were destined to histology (HE and PAS), immunohistochemistry for HSP-70 and VIP and glutathione (GSH) measurement. The involvement of nitric oxide (NO) and sulfhydryl (SH) compounds was determined. The ulcer area for indomethacin-induced gastric ulcers was measured. PGE₂ concentration was biochemically measured. The minimum inhibitory concentration (MIC) against H. pylori was determined in vitro. In ethanol model, CL and LIM demonstrated 100% of gastroprotection, while PIN did not exert effective gastroprotection (53.26%). In the indomethacin model, CL and LIM offered effective gastroprotection but PIN did not show gastroprotective effect. The gastric ulcer area of rats pretreated with NO-synthase inhibitor or SH-blocker was decreased in comparison to the control group. The MIC obtained for CL was 125 μg/mL, for LIM was 75 μg/mL and for PIN was 500 μg/mL. The gastroprotective effect of CL and LIM was involved with increasing in mucus secretion, HSP-70 and VIP, but not with GSH, NO or SH compounds. CL gastroprotective mechanism is involved with PGE₂. PIN did not present gastroprotective activity.

  8. Direct Probing of Protein-Protein Interactions

    SciTech Connect

    Noy, A; Sulchek, T A; Friddle, R W

    2005-03-10

    This project aimed to establish feasibility of using experimental techniques based on direct measurements of interaction forces on the single molecule scale to characterize equilibrium interaction potentials between individual biological molecules. Such capability will impact several research areas, ranging from rapid interaction screening capabilities to providing verifiable inputs for computational models. It should be one of the enabling technologies for modern proteomics research. This study used a combination of Monte-Carlo simulations, theoretical considerations, and direct experimental measurements to investigate two model systems that represented typical experimental situations: force-induced melting of DNA rigidly attached to the tip, and force-induced unbinding of a protein-antibody pair connected to flexible tethers. Our results establish that for both systems researchers can use force spectroscopy measurements to extract reliable information about equilibrium interaction potentials. However, the approaches necessary to extract these potentials in each case--Jarzynski reconstruction and Dynamic Force Spectroscopy--are very different. We also show how the thermodynamics and kinetics of unbinding process dictates the choice between in each case.

  9. Septins: Regulators of Protein Stability

    PubMed Central

    Vagin, Olga; Beenhouwer, David O.

    2016-01-01

    Septins are small GTPases that play a role in several important cellular processes. In this review, we focus on the roles of septins in protein stabilization. Septins may regulate protein stability by: (1) interacting with proteins involved in degradation pathways, (2) regulating the interaction between transmembrane proteins and cytoskeletal proteins, (3) affecting the mobility of transmembrane proteins in lipid bilayers, and (4) modulating the interaction of proteins with their adaptor or signaling proteins. In this context, we discuss the role of septins in protecting four different proteins from degradation. First we consider botulinum neurotoxin serotype A (BoNT/A) and the contribution of septins to its extraordinarily long intracellular persistence. Next, we discuss the role of septins in stabilizing the receptor tyrosine kinases EGFR and ErbB2. Finally, we consider the contribution of septins in protecting hypoxia-inducible factor 1α (HIF-1α) from degradation. PMID:28066764

  10. The quality of microparticulated protein.

    PubMed

    Erdman, J W

    1990-08-01

    The purpose of this paper is to describe the effects of microparticulation upon the quality of microparticulated protein products and to confirm that microparticulation does not result in changes in protein structure or quality different from those that occur with cooking. Two products were tested: microparticulated egg white and skim milk proteins and microparticulated whey protein concentrate. Three approaches were used to monitor for changes in amino acid and protein value: amino acid analysis, protein efficiency ratio (PER) bioassay, and both one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Evaluation of the results of these tests indicates that no significant differences were found when comparing the premix before and after microparticulation. Significant differences also did not occur when the premix was cooked using conventional methods. Collectively, the data provide strong evidence that the protein microparticulation process used to prepare microparticulated protein products (e.g., Simplesse) does not alter the quality or nutritional value of protein in the final products.

  11. Dissecting protein-protein interactions using directed evolution.

    PubMed

    Bonsor, Daniel A; Sundberg, Eric J

    2011-04-05

    Protein-protein interactions are essential for life. They are responsible for most cellular functions and when they go awry often lead to disease. Proteins are inherently complex. They are flexible macromolecules whose constituent amino acid components act in combinatorial and networked ways when they engage one another in binding interactions. It is just this complexity that allows them to conduct such a broad array of biological functions. Despite decades of intense study of the molecular basis of protein-protein interactions, key gaps in our understanding remain, hindering our ability to accurately predict the specificities and affinities of their interactions. Until recently, most protein-protein investigations have been probed experimentally at the single-amino acid level, making them, by definition, incapable of capturing the combinatorial nature of, and networked communications between, the numerous residues within and outside of the protein-protein interface. This aspect of protein-protein interactions, however, is emerging as a major driving force for protein affinity and specificity. Understanding a combinatorial process necessarily requires a combinatorial experimental tool. Much like the organisms in which they reside, proteins naturally evolve over time, through a combinatorial process of mutagenesis and selection, to functionally associate. Elucidating the process by which proteins have evolved may be one of the keys to deciphering the molecular rules that govern their interactions with one another. Directed evolution is a technique performed in the laboratory that mimics natural evolution on a tractable time scale that has been utilized widely to engineer proteins with novel capabilities, including altered binding properties. In this review, we discuss directed evolution as an emerging tool for dissecting protein-protein interactions.

  12. 14-3-3 proteins: regulators of numerous eukaryotic proteins.

    PubMed

    van Heusden, G Paul H

    2005-09-01

    14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

  13. Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

    PubMed Central

    Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

    2013-01-01

    While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface

  14. Hydrogels Constructed from Engineered Proteins.

    PubMed

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed.

  15. Protein misfolding disorders and macroautophagy

    PubMed Central

    Menzies, Fiona M; Moreau, Kevin; Rubinsztein, David C

    2011-01-01

    A large group of diseases, termed protein misfolding disorders, share the common feature of the accumulation of misfolded proteins. The possibility of a common mechanism underlying either the pathogenesis or therapy for these diseases is appealing. Thus, there is great interest in the role of protein degradation via autophagy in such conditions where the protein is found in the cytoplasm. Here we review the growing evidence supporting a role for autophagic dysregulation as a contributing factor to protein accumulation and cellular toxicity in certain protein misfolding disorders and discuss the available evidence that upregulation of autophagy may be a valuable therapeutic strategy. PMID:21087849

  16. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  17. Biological Applications of Protein Splicing

    PubMed Central

    Vila-Perelló, Miquel; Muir, Tom W.

    2010-01-01

    Protein splicing is a naturally-occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer. PMID:20946979

  18. Misfolded Proteins and Retinal Dystrophies

    PubMed Central

    Lin, Jonathan H.; LaVail, Matthew M.

    2010-01-01

    Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death by activating the Unfolded Protein Response, a set of conserved intracellular signaling pathways that detect protein misfolding within the endoplasmic reticulum and control protective and proapoptotic signal transduction pathways. Here, we review the misfolded proteins associated with select types of retinitis pigmentosa, Stargadt-like macular degeneration, and Doyne Honeycomb Retinal Dystrophy and discuss the role that endoplasmic reticulum stress and UPR signaling play in their pathogenesis. Last, we review new therapies for these diseases based on preventing protein misfolding in the retina. PMID:20238009

  19. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  20. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  1. Statistical analysis and prediction of protein-protein interfaces.

    PubMed

    Bordner, Andrew J; Abagyan, Ruben

    2005-08-15

    Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously difficult to predict. We generated a large nonredundant data set of 1494 true protein-protein interfaces using biological symmetry annotation where necessary. The data set was carefully analyzed and a Support Vector Machine was trained on a combination of a new robust evolutionary conservation signal with the local surface properties to predict protein-protein interfaces. Fivefold cross validation verifies the high sensitivity and selectivity of the model. As much as 97% of the predicted patches had an overlap with the true interface patch while only 22% of the surface residues were included in an average predicted patch. The model allowed the identification of potential new interfaces and the correction of mislabeled oligomeric states.

  2. Mx proteins: antiviral proteins by chance or by necessity?

    PubMed

    Arnheiter, H; Meier, E

    1990-10-01

    The interferon-inducible Mx1 protein is responsible for inborn resistance of mice to influenza. It is now recognized that this protein is a member of a family of interferon-inducible, putative GTP-binding proteins found in many organisms. Thus, these proteins, called the Mx proteins, are found in species that are naturally infected with influenza virus, and also in species that are not. Some Mx proteins display a broader antiviral profile than the one observed for Mx1 in mice. Others, however, may not be antiviral. Two recently discovered GTP-binding proteins, Vps1p in yeast and dynamin in rat, are also related to Mx1. These proteins are synthesized constitutively and serve basic cellular functions.

  3. Collaborative protein filaments.

    PubMed

    Ghosal, Debnath; Löwe, Jan

    2015-09-14

    It is now well established that prokaryotic cells assemble diverse proteins into dynamic cytoskeletal filaments that perform essential cellular functions. Although most of the filaments assemble on their own to form higher order structures, growing evidence suggests that there are a number of prokaryotic proteins that polymerise only in the presence of a matrix such as DNA, lipid membrane or even another filament. Matrix-assisted filament systems are frequently nucleotide dependent and cytomotive but rarely considered as part of the bacterial cytoskeleton. Here, we categorise this family of filament-forming systems as collaborative filaments and introduce a simple nomenclature. Collaborative filaments are frequent in both eukaryotes and prokaryotes and are involved in vital cellular processes including chromosome segregation, DNA repair and maintenance, gene silencing and cytokinesis to mention a few. In this review, we highlight common principles underlying collaborative filaments and correlate these with known functions.

  4. Protein engineering of subtilisin.

    PubMed

    Bryan, P N

    2000-12-29

    The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.

  5. A magnetic protein biocompass

    NASA Astrophysics Data System (ADS)

    Qin, Siying; Yin, Hang; Yang, Celi; Dou, Yunfeng; Liu, Zhongmin; Zhang, Peng; Yu, He; Huang, Yulong; Feng, Jing; Hao, Junfeng; Hao, Jia; Deng, Lizong; Yan, Xiyun; Dong, Xiaoli; Zhao, Zhongxian; Jiang, Taijiao; Wang, Hong-Wei; Luo, Shu-Jin; Xie, Can

    2016-02-01

    The notion that animals can detect the Earth’s magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

  6. A magnetic protein biocompass.

    PubMed

    Qin, Siying; Yin, Hang; Yang, Celi; Dou, Yunfeng; Liu, Zhongmin; Zhang, Peng; Yu, He; Huang, Yulong; Feng, Jing; Hao, Junfeng; Hao, Jia; Deng, Lizong; Yan, Xiyun; Dong, Xiaoli; Zhao, Zhongxian; Jiang, Taijiao; Wang, Hong-Wei; Luo, Shu-Jin; Xie, Can

    2016-02-01

    The notion that animals can detect the Earth's magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

  7. Microdosing of protein drugs.

    PubMed

    Rowland, M

    2016-02-01

    Poor pharmacokinetics (PK) can seriously limit clinical utility. Knowing early whether a new compound is likely to have the desired PK profile at therapeutic doses is therefore important. One approach, microdosing, has shown high success with small molecular weight compounds, despite early skepticism. Vlaming et al. report the first, and successful, clinical application of a microdose of a humanized recombinant protein. But what is the likely success for this class of drugs more generally?

  8. Prion protein and aging

    PubMed Central

    Gasperini, Lisa; Legname, Giuseppe

    2014-01-01

    The cellular prion protein (PrPC) has been widely investigated ever since its conformational isoform, the prion (or PrPSc), was identified as the etiological agent of prion disorders. The high homology shared by the PrPC-encoding gene among mammals, its high turnover rate and expression in every tissue strongly suggest that PrPC may possess key physiological functions. Therefore, defining PrPC roles, properties and fate in the physiology of mammalian cells would be fundamental to understand its pathological involvement in prion diseases. Since the incidence of these neurodegenerative disorders is enhanced in aging, understanding PrPC functions in this life phase may be of crucial importance. Indeed, a large body of evidence suggests that PrPC plays a neuroprotective and antioxidant role. Moreover, it has been suggested that PrPC is involved in Alzheimer disease, another neurodegenerative pathology that develops predominantly in the aging population. In prion diseases, PrPC function is likely lost upon protein aggregation occurring in the course of the disease. Additionally, the aging process may alter PrPC biochemical properties, thus influencing its propensity to convert into PrPSc. Both phenomena may contribute to the disease development and progression. In Alzheimer disease, PrPC has a controversial role because its presence seems to mediate β-amyloid toxicity, while its down-regulation correlates with neuronal death. The role of PrPC in aging has been investigated from different perspectives, often leading to contrasting results. The putative protein functions in aging have been studied in relation to memory, behavior and myelin maintenance. In aging mice, PrPC changes in subcellular localization and post-translational modifications have been explored in an attempt to relate them to different protein roles and propensity to convert into PrPSc. Here we provide an overview of the most relevant studies attempting to delineate PrPC functions and fate in aging

  9. Dissecting Amelogenin Protein Nanospheres

    PubMed Central

    Bromley, Keith M.; Kiss, Andrew S.; Lokappa, Sowmya Bekshe; Lakshminarayanan, Rajamani; Fan, Daming; Ndao, Moise; Evans, John Spencer; Moradian-Oldak, Janet

    2011-01-01

    Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp161, Trp45, and Trp25) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (RH) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg·ml−1. We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp161) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp25 and Trp45 is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite. PMID:21840988

  10. Bone morphogenetic protein

    SciTech Connect

    Xiao Yongtao; Xiang Lixin; Shao Jianzhong

    2007-10-26

    Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor-beta superfamily. It has been demonstrated that BMPs had been involved in the regulation of cell proliferation, survival, differentiation and apoptosis. However, their hallmark ability is that play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. In this review, we mainly concentrate on BMP structure, function, molecular signaling and potential medical application.

  11. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  12. Protein-Protein Fusion Catalyzed by Sortase A

    PubMed Central

    Levary, David A.; Parthasarathy, Ranganath; Boder, Eric T.; Ackerman, Margaret E.

    2011-01-01

    Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality — demonstrating the robust and facile nature of this reaction. PMID:21494692

  13. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  14. Process for protein PEGylation.

    PubMed

    Pfister, David; Morbidelli, Massimo

    2014-04-28

    PEGylation is a versatile drug delivery technique that presents a particularly wide range of conjugation chemistry and polymer structure. The conjugated protein can be tuned to specifically meet the needs of the desired application. In the area of drug delivery this typically means to increase the persistency in the human body without affecting the activity profile of the original protein. On the other hand, because of the high costs associated with the production of therapeutic proteins, subsequent operations imposed by PEGylation must be optimized to minimize the costs inherent to the additional steps. The closest attention has to be given to the PEGylation reaction engineering and to the subsequent purification processes. This review article focuses on these two aspects and critically reviews the current state of the art with a clear focus on the development of industrial scale processes which can meet the market requirements in terms of quality and costs. The possibility of using continuous processes, with integration between the reaction and the separation steps is also illustrated.

  15. Papillomavirus E6 proteins

    SciTech Connect

    Howie, Heather L.; Katzenellenbogen, Rachel A.; Galloway, Denise A.

    2009-02-20

    The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition.

  16. Papillomavirus E6 proteins

    PubMed Central

    Howie, Heather L; Katzenellenbogen, Rachel A; Galloway, Denise A

    2009-01-01

    The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition. PMID:19081593

  17. Analysis of secreted proteins.

    PubMed

    Severino, Valeria; Farina, Annarita; Chambery, Angela

    2013-01-01

    Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.

  18. Infrared Protein Crystallography

    SciTech Connect

    J Sage; Y Zhang; J McGeehan; R Ravelli; M Weik; J van Thor

    2011-12-31

    We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through pola