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Sample records for putative basic leucine

  1. TCFL4: a gene at 17q21.1 encoding a putative basic helix-loop-helix leucine-zipper transcription factor.

    PubMed

    Bjerknes, M; Cheng, H

    1996-11-28

    TCFL4 (transcription factor like 4) is the HGMW-approved symbol for the gene of a widely expressed putative basic helix-loop-helix leucine-zipper (bHLH-zip) transcription factor which is located 3' to HSD17B1 (17-beta-hydroxysteroid dehydrogenase gene) at 17q21.1, centromeric to the BRCA1 (a gene implicated in familial breast cancer) locus. We report the human gene structure and the murine cDNA sequence of two variants, about 1.5 and 2.2 kb in size. The deduced protein is highly conserved between mouse and man. PMID:8973301

  2. Trp387 and the putative leucine zippers of PGH synthases-1 and -2.

    PubMed

    Hsi, L C; Tsai, A L; Kulmacz, R J; English, D G; Siefker, A O; Otto, J C; Smith, W L

    1993-01-01

    The active site sequence 385-YHWH-388 of ovine prostaglandin endoperoxide synthase-1 (PGHS-1) has residues critical for cyclooxygenase and peroxidase catalysis. Tyr385 is essential for cyclooxygenase activity, His386, for peroxidase activity, and His388, for both activities. To determine the importance of Trp387, we used site-directed mutagenesis to replace Trp387 of PGHS-1 with arginine, phenylalanine, and serine. W387R and W387S lacked significant activity. W387F retained both cyclooxygenase and peroxidase activities. Thus, we conclude that Trp387 is not essential for catalysis by PGHS-1. Purified PGHS-1 is a homodimer. There are two putative leucine zipper regions in ovine PGHS-1 involving residues 345-366 and 487-508. We tested for a role of these leucine zippers as determinants of dimer formation. Helix-breaking proline mutations were introduced at Leu359 or Leu501. Neither of these residues proved to be essential for peroxidase activity; but, mutations at each residue greatly reduced or eliminated cyclooxygenase activity. Both mutant proteins chromatographed as dimers on Sephacryl G-200. Thus, neither of these putative leucine zipper regions alone is responsible for PGHS-1 dimer formation. PMID:8357979

  3. Simulations of potentials of mean force for separating a leucine zipper dimer and the basic region of a basic region leucine zipper dimer.

    PubMed

    Cukier, Robert I

    2014-09-01

    Basic region leucine zipper (bZIP) transcription factors involved in DNA recognition are dimeric proteins. The monomers consist of two subdomains, a leucine zipper sequence responsible for dimerization and a highly basic DNA recognition sequence. Leucine zippers are strongly dimerized, and in a bZIP, the basic region can, in the absence of DNA, undergo extensive relative monomer-to-monomer fluctuations. In this work, LZ and bZIP potentials of mean force (PMFs), which provide free energies along reaction coordinates, are simulated with a distance replica exchange method. The method uses restraint potentials to provide sampling along a reaction coordinate and enhances configuration space exploration by exchanging information between neighboring restraint potential configurations. Restraint potentials that are constructed from sums over a number of atom distances are employed. Their use requires a modification of the Weighted Histogram Analysis Method (WHAM) procedure to combine and unbias the data from the different restraint-potential-biased window densities to provide a PMF. These methods are first used to obtain a PMF for separating a leucine zipper (GCN4-p1) of the yeast transcriptional activator GCN4. The PMF indicates a very strong binding free energy that only weakens when the monomers are separated by about 12 Å, which is about 6 Å beyond their bound, dimer equilibrium distance. PMFs are also obtained for separating the basic subdomain monomer parts of the GCN4 bZIP transcriptional factor, in the absence of DNA. In a monomer separation range spanning the open, crystal-based structure to closer configurations, the basic subdomain PMF is quite flat, implying essentially thermal sampling in this distance range. A PMF generated starting from a "collapsed" state, taken from a previous simulation ( J. Phys. Chem. B 2012 , 116 , 6071 ), where collapsed refers to the feature that the basic subdomain monomers are also effectively dimerized, shows that this state is

  4. The "putative" leucine zipper region of murine leukemia virus transmembrane protein (P15e) is essential for viral infectivity.

    PubMed

    Ramsdale, E E; Kingsman, S M; Kingsman, A J

    1996-06-01

    In order to determine the role of the putative leucine zipper region of murine leukemia virus (MLV) transmembrane protein p15E, nine mutations in this region were introduced by site-directed mutagenesis. None of these mutations affected the expression or transport of the envelope protein or incorporation into virions. The mutants were analyzed for their ability to infect NIH3T3 cells and to induce cell fusion in a rat XC cell fusion assay. Mutations removing the charge of the hydrophilic residues reduced infectivity in NIH3T3 cells but had either no effect or a minor effect on envelope-induced XC cell fusion. Six mutations of hydrophobic residues of the putative leucine zipper region were constructed; four completely abolished the ability to infect NIH3T3 cells and these mutant envelopes were also unable to induce cell fusion in the XC cell fusion assay. These data demonstrate the absolute requirement for the putative leucine zipper region for both fusion and infection of MLV. PMID:8659102

  5. Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola (endosymbiont of aphids).

    PubMed Central

    van Ham, R C; Moya, A; Latorre, A

    1997-01-01

    An 8.5-kb plasmid encoding genes (leuABCD) involved in leucine biosynthesis and a small plasmid of 1.74 kb of yet unknown function were found in the intracellular symbiont, Buchnera aphidicola, of two divergent aphid species, Thelaxes suberi and Tetraneura caerulescens, respectively. The leuABCD-carrying plasmid (pBTs1) was amplified from total aphid DNA by inverse long PCR, using outwardly oriented oligonucleotide primers specific to leuA. The resulting 8.2-kb PCR fragment as well as the 1.74-kb plasmid (pBTc1) were cloned and sequenced. pBTs1 differed from a previously described B. aphidicola plasmid (pRPE) of the aphid Rhopalosiphum padi by the presence of a small heat shock gene (ibp) and in the order of the leuABCD and repA genes. Comparison of both leucine plasmids to the small plasmid pBTc1 revealed extensive similarity with respect to putative replication functions as well as in the presence of a highly conserved open reading frame that was found to be homologous to Escherichia coli YqhA and Haemophilus influenzae HI0507 and which may encode an integral membrane protein. The three B. aphidicola plasmids most likely evolved from a common ancestral replicon, which in turn may be distantly related to IncFII plasmids. Phylogenetic affiliations of the B. aphidicola strains of the two aphid species were assessed by sequencing of their 16S rRNA genes. Evaluation of the distribution of the leuABCD-encoding plasmids within a phylogenetic framework suggests independent origins for pBTs1 and pRPE from an ancestral replicon resembling pBTc1. The implications for symbiotic essential amino acid biosynthesis and provisioning are discussed. PMID:9244264

  6. Evolutionary and Expression Analyses of the Apple Basic Leucine Zipper Transcription Factor Family

    PubMed Central

    Zhao, Jiao; Guo, Rongrong; Guo, Chunlei; Hou, Hongmin; Wang, Xiping; Gao, Hua

    2016-01-01

    Transcription factors (TFs) play essential roles in the regulatory networks controlling many developmental processes in plants. Members of the basic leucine (Leu) zipper (bZIP) TF family, which is unique to eukaryotes, are involved in regulating diverse processes, including flower and vascular development, seed maturation, stress signaling, and defense responses to pathogens. The bZIP proteins have a characteristic bZIP domain composed of a DNA-binding basic region and a Leu zipper dimerization region. In this study, we identified 112 apple (Malus domestica Borkh) bZIP TF-encoding genes, termed MdbZIP genes. Synteny analysis indicated that segmental and tandem duplication events, as well as whole genome duplication, have contributed to the expansion of the apple bZIP family. The family could be divided into 11 groups based on structural features of the encoded proteins, as well as on the phylogenetic relationship of the apple bZIP proteins to those of the model plant Arabidopsis thaliana (AtbZIP genes). Synteny analysis revealed that several paired MdbZIP genes and AtbZIP gene homologs were located in syntenic genomic regions. Furthermore, expression analyses of group A MdbZIP genes showed distinct expression levels in 10 different organs. Moreover, changes in these expression profiles in response to abiotic stress conditions and various hormone treatments identified MdbZIP genes that were responsive to high salinity and drought, as well as to different phytohormones. PMID:27066030

  7. The Importance of Being Flexible: The Case of Basic Region Leucine Zipper Transcriptional Regulators

    PubMed Central

    Miller, Maria

    2009-01-01

    Large volumes of protein sequence and structure data acquired by proteomic studies led to the development of computational bioinformatic techniques that made possible the functional annotation and structural characterization of proteins based on their primary structure. It has become evident from genome-wide analyses that many proteins in eukaryotic cells are either completely disordered or contain long unstructured regions that are crucial for their biological functions. The content of disorder increases with evolution indicating a possibly important role of disorder in the regulation of cellular systems. Transcription factors are no exception and several proteins of this class have recently been characterized as premolten/molten globules. Yet, mammalian cells rely on these proteins to control expression of their 30,000 or so genes. Basic region:leucine zipper (bZIP) DNA-binding proteins constitute a major class of eukaryotic transcriptional regulators. This review discusses how conformational flexibility “built” into the amino acid sequence allows bZIP proteins to interact with a large number of diverse molecular partners and to accomplish their manifold cellular tasks in a strictly regulated and coordinated manner. PMID:19519454

  8. Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene

    PubMed Central

    Manna, Pulak R.; Dyson, Matthew T.; Stocco, Douglas M.

    2016-01-01

    The regulation of steroidogenic acute regulatory protein (StAR) gene transcription by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP response element (CRE, TGACGTGA). This regulation is coordinated by multiple transcription factors that bind to sequence-specific elements located approximately 150 bp upstream of the transcription start site. Among the proteins that bind within this region, the basic leucine zipper (bZIP) family of transcription factors, i.e. CRE binding protein (CREB)/CRE modulator (CREM)/activating transcription factor (ATF), activator protein 1 (AP-1; Fos/Jun), and CCAAT enhancer binding protein β (C/EBPβ), interact with an overlapping region (−81/−72 bp) in the StAR promoter, mediate stimulus-transcription coupling of cAMP signaling and play integral roles in regulating StAR gene expression. These bZIP proteins are structurally similar and bind to DNA sequences as dimers; however, they exhibit discrete transcriptional activities, interact with several transcription factors and other properties that contribute in their regulatory functions. The 5′-flanking −81/−72 bp region of the StAR gene appears to function as a key element within a complex cAMP response unit by binding to different bZIP members, and the StAR promoter displays variable states of cAMP responsivity contingent upon the occupancy of these cis-elements with these transcription factors. The expression and activities of CREB/CREM/ATF, Fos/Jun and C/EBPβ have been demonstrated to be mediated by a plethora of extracellular signals, and the phosphorylation of these proteins at several Ser and Thr residues allows recruitment of the transcriptional coactivator CREB binding protein (CBP) or its functional homolog p300 to the StAR promoter. This review will focus on the current level of understanding of the roles of selective bZIP family proteins within the complex series of processes involved in regulating StAR gene transcription. PMID:19150388

  9. Basic Leucine Zipper (bZIP) Domain Transcription Factor MBZ1 Regulates Cell Wall Integrity, Spore Adherence, and Virulence in Metarhizium robertsii *

    PubMed Central

    Huang, Wei; Shang, Yanfang; Chen, Peilin; Cen, Kai; Wang, Chengshu

    2015-01-01

    Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels. PMID:25673695

  10. Neuroprotective role of the basic leucine zipper transcription factor NFIL3 in models of amyotrophic lateral sclerosis.

    PubMed

    Tamai, So-ichi; Imaizumi, Keisuke; Kurabayashi, Nobuhiro; Nguyen, Minh Dang; Abe, Takaya; Inoue, Masatoshi; Fukada, Yoshitaka; Sanada, Kamon

    2014-01-17

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Here we show that the basic leucine zipper transcription factor NFIL3 (also called E4BP4) confers neuroprotection in models of ALS. NFIL3 is up-regulated in primary neurons challenged with neurotoxic insults and in a mouse model of ALS. Overexpression of NFIL3 attenuates excitotoxic neuronal damage and protects neurons against neurodegeneration in a cell-based ALS model. Conversely, reduction of NFIL3 exacerbates neuronal demise in adverse conditions. Transgenic neuronal expression of NFIL3 in ALS mice delays disease onset and attenuates motor axon and neuron degeneration. These results suggest that NFIL3 plays a neuroprotective role in neurons and constitutes a potential therapeutic target for neurodegeneration. PMID:24280221

  11. A leucine-to-proline mutation in the putative first transmembrane domain of the 22-kDa peripheral myelin protein in the trembler-J mouse.

    PubMed

    Suter, U; Moskow, J J; Welcher, A A; Snipes, G J; Kosaras, B; Sidman, R L; Buchberg, A M; Shooter, E M

    1992-05-15

    Peripheral myelin protein PMP-22 is a potential growth-regulating myelin protein that is expressed by Schwann cells and predominantly localized in compact peripheral myelin. A point mutation in the Pmp-22 gene of inbred trembler (Tr) mice was identified and proposed to be responsible for the Tr phenotype, which is characterized by paralysis of the limbs as well as tremors and transient seizures. In support of this hypothesis, we now report the fine mapping of the Pmp-22 gene to the immediate vicinity of the Tr locus on mouse chromosome 11. Furthermore, we have found a second point mutation in the Pmp-22 gene of trembler-J (TrJ) mice, which results in the substitution of a leucine residue by a proline residue in the putative first transmembrane region of the PMP-22 polypeptide. Tr and TrJ were previously mapped genetically as possible allelic mutations giving rise to similar, but not identical, phenotypes. This finding is consistent with the discovery of two different mutations in physicochemically similar domains of the PMP-22 protein. Our results strengthen the hypothesis that mutations in the Pmp-22 gene can lead to heterogeneous forms of peripheral neuropathies and offer clues toward possible explanations for the dominant inheritance of these disorders. PMID:1374899

  12. LMOh7858_0369, a gene encoding a putative leucine-rich repeat-containing protein, is required for virulence of Listeria monocytogenes.

    PubMed

    Zhang, Ting; Bae, Dongryeoul; Wang, Chinling

    2016-05-01

    Listeria monocytogenes possesses the highest number of leucine-rich repeat (LRR)-containing proteins among all Gram-positive bacteria; these LRR-containing molecules are known as the 'internalin' family. To understand the functions of largely uncharacterized LRR-containing molecules, we constructed seven deletion mutants in the L. monocytogenes H7858 strain targeting genes in this family and tested their virulence. Among the seven mutants, the ΔLMOh7858_0369 strain and the ΔLMOh7858_2546 strain showed significantly impaired invasiveness of HepG2 cells. We further tested the virulence of these two strains in the intravascular sepsis model using BALB/c mice. Interestingly, the ΔLMOh7858_0369 strain showed significant reduction in organ colonization, bacteremia and invasion of the brain compared with the parental wild-type strain. Host immune responses to listerial intravascular infection were measured at 24 and 72 h post-infection. Transcript levels of several proinflammatory cytokines and chemokines were significantly lower when induced by the ΔlmOh7858_0369 strain than when induced by the wild type. These results suggest that the putative LRR-containing protein encoded by LMOh7858_0369 might be a novel virulence factor of the L. monocytogenes H7858 strain. PMID:26976852

  13. Analysis of basic leucine zipper genes and their expression during bud dormancy in peach (Prunus persica).

    PubMed

    Sun, Ming-Yue; Fu, Xi-Ling; Tan, Qiu-Ping; Liu, Li; Chen, Min; Zhu, Cui-Ying; Li, Ling; Chen, Xiu-De; Gao, Dong-Sheng

    2016-07-01

    Dormancy is a biological characteristic developed to resist the cold conditions in winter. The bZIP transcription factors are present exclusively in eukaryotes and have been identified and classified in many species. bZIP proteins are known to regulate numerous biological processes, however, the role of bZIP in bud dodormancy has not been studied extensively. In total, 50 PpbZIP transcription factor-encoding genes were identified and categorized them into 10 groups (A-I and S). Similar intron/exon structures, additional conserved motifs, and DNA-binding site specificity supported our classification scheme. Additionally, chromosomal distribution and collinearity analyses suggested that expansion of the PpbZIP transcription factor family was due to segment/chromosomal duplications. We also predicted the dimerization properties based on characteristic features of the leucine zipper and classified PpbZIP proteins into 23 subfamilies. Furthermore, qRT-PCR results indicated that PpbZIPs genes may be involved in regulating dormancy. The same gene of different species might participate in different regulating networks through interactions with specific partners. Our expression profiling results complemented the microarray data, suggesting that co-expression patterns of bZIP transcription factors during dormancy differed among deciduous fruit trees. Our findings further clarify the molecular characteristics of the PpbZIP transcription factor family, including potential gene functions during dormancy. This information may facilitate further research on the evolutionary history and biological functions of bZIP proteins in peach and other rosaceae plants. PMID:27107182

  14. Selective utilization of basic helix-loop-helix-leucine zipper proteins at the immunoglobulin heavy-chain enhancer.

    PubMed

    Carter, R S; Ordentlich, P; Kadesch, T

    1997-01-01

    The microE3 E box within the immunoglobulin heavy-chain (IgH) enhancer binds several proteins of the basic helix-loop-helix-leucine zipper (bHLHzip) class, including TFE3, USF1, and Max. Both TFE3 and USF have been described as transcriptional activators, and so we investigated their possible roles in activating the IgH enhancer in vivo. Although TFE3 activated various enhancer-based reporters, both USF1 and Max effectively inhibited transcription. Inhibition by USF correlated with the lack of a strong activation domain and was the result of the protein neutralizing the microE3 site. The effects of dominant-negative derivatives of TFE3 and USF1 confirmed that TFE3, or a TFE3-like protein, is the primary cellular bHLHzip protein that activates the IgH enhancer. In addition to providing a physiological role for TFE3, our results call into question the traditional view of USF1 as an obligate transcriptional activator. PMID:8972181

  15. The basic leucine zipper transcription factor E4BP4 is essential for natural killer cell development.

    PubMed

    Gascoyne, Duncan M; Long, Elaine; Veiga-Fernandes, Henrique; de Boer, Jasper; Williams, Owen; Seddon, Benedict; Coles, Mark; Kioussis, Dimitris; Brady, Hugh J M

    2009-10-01

    Natural killer (NK) cells are a subset of lymphocytes crucial for innate immunity and modification of adaptive immune responses. In contrast to commitment to the T cell or B cell lineage, little is known about NK cell lineage commitment. Here we show that the basic leucine zipper (bZIP) transcription factor E4BP4 (also called NFIL3) is essential for generation of the NK cell lineage. E4BP4-deficient mice (Nfil3(-/-); called 'E4bp4(-/-)' here) had B cells, T cells and NKT cells but specifically lack NK cells and showed severely impaired NK cell-mediated cytotoxicity. Overexpression of E4bp4 was sufficient to increase NK cell production from hematopoietic progenitor cells. E4BP4 acted in a cell-intrinsic manner 'downstream' of the interleukin 15 receptor (IL-15R) and through the transcription factor Id2. E4bp4(-/-) mice may provide a model for definitive analysis of the contribution of NK cells to immune responses and pathologies. PMID:19749763

  16. Constitutive expression of the Poplar FD-like basic leucine zipper transcription factor alters growth and bud development.

    PubMed

    Parmentier-Line, Cécile M; Coleman, Gary D

    2016-01-01

    In poplar, the CO/FT regulatory module mediates seasonal growth cessation. Although FT interacts with the basic leucine zipper transcription factor FD, surprisingly little is known about the possible role of FD in bud development and growth cessation in trees. In this study, we examined the expression and localization of the poplar FD homolog, PtFD1, during short-day (SD)-induced bud development, and the consequences of overexpressing PtFD1 on bud development and shoot growth. PtFD1 was primarily expressed in apical and axillary buds and exhibited a transient increase in expression during the initial stages of SD-induced bud development. This transient increase declined with continued SD treatment. When PtFD1 was overexpressed in poplar, SD-induced growth cessation and bud formation were abolished. PTFD1 overexpression also resulted in precocious flowering of juvenile plants in long-day (LD) photoperiods. Because the phenotypes associated with overexpression of PtFD1 are similar to those observe when poplar FT1 is overexpressed (Science, 312, 2006, 1040), the expression and diurnal patterns of expression of both poplar FT1 and FT2 were characterized in PtFD1 overexpression poplars and found to be altered. DNA microarray analysis revealed few differences in gene expression between PtFD1 overexpressing poplars in LD conditions while extensive levels of differential gene expression occur in SD-treated plants. These results enforce the connection between the regulation of flowering and the regulation of growth cessation and bud development in poplar. PMID:25915693

  17. Influence of the Valine Zipper Region on the Structure and Aggregation of the Basic Leucine Zipper (bZIP) Domain of Activating Transcription Factor 5 (ATF5)

    PubMed Central

    Ciaccio, Natalie A.; Reynolds, T. Steele; Middaugh, C. Russell; Laurence, Jennifer S.

    2012-01-01

    Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of Activating Transcription Factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and non-reducing conditions. Our data indicate that removal of this region results in a loss of alpha-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation. PMID:23067245

  18. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    SciTech Connect

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

  19. A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

    PubMed Central

    Vicente-Carbajosa, Jesus; Moose, Stephen P.; Parsons, Ronald L.; Schmidt, Robert J.

    1997-01-01

    The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression. PMID:9207153

  20. The basic leucine zipper transcription factor ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 is an important transcriptional regulator of abscisic acid-dependent grape berry ripening processes.

    PubMed

    Nicolas, Philippe; Lecourieux, David; Kappel, Christian; Cluzet, Stéphanie; Cramer, Grant; Delrot, Serge; Lecourieux, Fatma

    2014-01-01

    In grape (Vitis vinifera), abscisic acid (ABA) accumulates during fruit ripening and is thought to play a pivotal role in this process, but the molecular basis of this control is poorly understood. This work characterizes ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 (VvABF2), a grape basic leucine zipper transcription factor belonging to a phylogenetic subgroup previously shown to be involved in ABA and abiotic stress signaling in other plant species. VvABF2 transcripts mainly accumulated in the berry, from the onset of ripening to the harvesting stage, and were up-regulated by ABA. Microarray analysis of transgenic grape cells overexpressing VvABF2 showed that this transcription factor up-regulates and/or modifies existing networks related to ABA responses. In addition, grape cells overexpressing VvABF2 exhibited enhanced responses to ABA treatment compared with control cells. Among the VvABF2-mediated responses highlighted in this study, the synthesis of phenolic compounds and cell wall softening were the most strongly affected. VvABF2 overexpression strongly increased the accumulation of stilbenes that play a role in plant defense and human health (resveratrol and piceid). In addition, the firmness of fruits from tomato (Solanum lycopersicum) plants overexpressing VvABF2 was strongly reduced. These data indicate that VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening. PMID:24276949

  1. Binding of the wheat basic leucine zipper protein EmBP-1 to nucleosomal binding sites is modulated by nucleosome positioning.

    PubMed Central

    Niu, X; Adams, C C; Workman, J L; Guiltinan, M J

    1996-01-01

    To investigate interactions of the basic leucine zipper transcription factor EmBP-1 with its recognition sites in nucleosomal DNA, we reconstituted an abscisic acid response element and a high-affinity binding site for EmBP-1 into human and wheat nucleosome cores in vitro. DNA binding studies demonstrated that nucleosomal elements can be bound by EmBP-1 at reduced affinities relative to naked DNA. EmBP-1 affinity was lowest when the recognition sites were positioned near the center of the nucleosome. Binding was achieved with a truncated DNA binding domain; however, binding of full-length EmBP-1 caused additional strong DNase I hypersensitivity flanking the binding sites. Similar results were observed with nucleosomes reconstituted with either human or wheat histones, demonstrating a conserved mechanism of transcription factor-nucleosome interactions. We conclude that positioning of recognition sequences on a nucleosome may play an important role in regulating interactions of EmBP-1 with its target sites in plant cells. PMID:8837510

  2. Leucine aminopeptidase blood test

    MedlinePlus

    Serum leucine aminopeptidase ... Leucine aminopeptidase is a type of protein called an enzyme . This enzyme is normally found in cells ... check if your liver is damaged. Too much leucine aminopeptidase is released into your blood when you ...

  3. The basic domain/leucine zipper protein hXBP-1 preferentially binds to and transactivates CRE-like sequences containing an ACGT core.

    PubMed Central

    Clauss, I M; Chu, M; Zhao, J L; Glimcher, L H

    1996-01-01

    The transcription factor hXBP-1 belongs to the family of basic region/leucine zipper (bZIP) proteins and interacts with the cAMP responsive element (CRE) of the major histocompatibility complex (MHC) class II A alpha, DR alpha and DP beta genes. However, the developmental expression of hXBP-1 as revealed by in situ hybridization in mouse embryos, has suggested that it interacts with the promoter of additional genes. To identify other potential target genes of this factor, we performed binding site selection experiments with recombinant hXBP-1 protein. The results indicated that hXBP-1 binds preferably to the CRE-like element GAT-GACGTG(T/G)NNN(A/T)T, wherein the core sequence ACGT is highly conserved, and that it also binds to some TPA response elements (TRE). hXBP-1 can transactivate multimers of the target sequences to which it binds in COS cells, and the level of transactivation directly correlates with the extent of binding as observed in gel retardation experiments. One target sequence that is strongly bound by hXBP-1 is the 21 bp repeat in the HTLV-1 LTR, and we demonstrate here that hXBP-1 can transactivate the HTLV-1 LTR. Further, the transactivation domain of hXBP-1 encompasses a large C-terminal region of the protein, containing domains rich in glutamine, serine and threonine, and proline and glutamine residues, as shown in transient transfection experiments using hXBP-1-GAL4 fusion proteins and a reporter gene under the control of GAL4-binding sites. PMID:8657566

  4. A Composite Element that Binds Basic Helix Loop Helix and Basic Leucine Zipper Transcription Factors Is Important for Gonadotropin-Releasing Hormone Regulation of the Follicle-Stimulating Hormone β Gene

    PubMed Central

    Ciccone, Nick A.; Lacza, Charlemagne T.; Hou, Melody Y.; Gregory, Susan J.; Kam, Kyung-Yoon; Xu, Shuyun; Kaiser, Ursula B.

    2008-01-01

    Although FSH plays an essential role in controlling gametogenesis, the biology of FSHβ transcription remains poorly understood, but is known to involve the complex interplay of multiple endocrine factors including GnRH. We have identified a GnRH-responsive element within the rat FSHβ promoter containing an E-box and partial cAMP response element site that are bound by the basic helix loop helix transcription factor family members, upstream stimulating factor (USF)-1/USF-2, and the basic leucine zipper member, cAMP response element-binding protein (CREB), respectively. Expression studies with CREB, USF-1/USF-2, and activating protein-1 demonstrated that the USF transcription factors increased basal transcription, an effect not observed if the cognate binding site was mutated. Conversely, expression of a dominant negative CREB mutant or CREB knockdown attenuated induction by GnRH, whereas dominant negative Fos or USF had no effect on the GnRH response. GnRH stimulation specifically induced an increase in phosphorylated CREB occupation of the FSHβ promoter, leading to the recruitment of CREB-binding protein to enhance gene transcription. In conclusion, a composite element bound by both USF and CREB serves to integrate signals for basal and GnRH-stimulated transcription of the rat FSHβ gene. PMID:18550775

  5. Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

    PubMed Central

    Böer, Ulrike; Eglins, Julia; Krause, Doris; Schnell, Susanne; Schöfl, Christof; Knepel, Willhart

    2007-01-01

    The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium. PMID:17696880

  6. Leucine aminopeptidase - urine

    MedlinePlus

    Leucine aminopeptidase is a type of protein called an enzyme. It is normally found in liver cells ... Increased levels of leucine aminopeptidase can be seen in ... Hepatitis Liver cancer Liver ischemia (reduced blood flow to the ...

  7. Leucine aminopeptidase - urine

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003617.htm Leucine aminopeptidase - urine To use the sharing features on this page, please enable JavaScript. Leucine aminopeptidase is a type of protein called an ...

  8. The Basic Leucine Zipper Transcription Factor ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 Is an Important Transcriptional Regulator of Abscisic Acid-Dependent Grape Berry Ripening Processes1[W][OPEN

    PubMed Central

    Nicolas, Philippe; Lecourieux, David; Kappel, Christian; Cluzet, Stéphanie; Cramer, Grant; Delrot, Serge; Lecourieux, Fatma

    2014-01-01

    In grape (Vitis vinifera), abscisic acid (ABA) accumulates during fruit ripening and is thought to play a pivotal role in this process, but the molecular basis of this control is poorly understood. This work characterizes ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 (VvABF2), a grape basic leucine zipper transcription factor belonging to a phylogenetic subgroup previously shown to be involved in ABA and abiotic stress signaling in other plant species. VvABF2 transcripts mainly accumulated in the berry, from the onset of ripening to the harvesting stage, and were up-regulated by ABA. Microarray analysis of transgenic grape cells overexpressing VvABF2 showed that this transcription factor up-regulates and/or modifies existing networks related to ABA responses. In addition, grape cells overexpressing VvABF2 exhibited enhanced responses to ABA treatment compared with control cells. Among the VvABF2-mediated responses highlighted in this study, the synthesis of phenolic compounds and cell wall softening were the most strongly affected. VvABF2 overexpression strongly increased the accumulation of stilbenes that play a role in plant defense and human health (resveratrol and piceid). In addition, the firmness of fruits from tomato (Solanum lycopersicum) plants overexpressing VvABF2 was strongly reduced. These data indicate that VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening. PMID:24276949

  9. Deciphering the Combinatorial DNA-binding Code of the CCAAT-binding Complex and the Iron-regulatory Basic Region Leucine Zipper (bZIP) Transcription Factor HapX*

    PubMed Central

    Hortschansky, Peter; Ando, Eriko; Tuppatsch, Katja; Arikawa, Hisashi; Kobayashi, Tetsuo; Kato, Masashi; Haas, Hubertus; Brakhage, Axel A.

    2015-01-01

    The heterotrimeric CCAAT-binding complex (CBC) is evolutionarily conserved in eukaryotic organisms, including fungi, plants, and mammals. The CBC consists of three subunits, which are named in the filamentous fungus Aspergillus nidulans HapB, HapC, and HapE. HapX, a fourth CBC subunit, was identified exclusively in fungi, except for Saccharomyces cerevisiae and the closely related Saccharomycotina species. The CBC-HapX complex acts as the master regulator of iron homeostasis. HapX belongs to the class of basic region leucine zipper transcription factors. We demonstrated that the CBC and HapX bind cooperatively to bipartite DNA motifs with a general HapX/CBC/DNA 2:1:1 stoichiometry in a class of genes that are repressed by HapX-CBC in A. nidulans during iron limitation. This combinatorial binding mode requires protein-protein interaction between the N-terminal domain of HapE and the N-terminal CBC binding domain of HapX as well as sequence-specific DNA binding of both the CBC and HapX. Initial binding of the CBC to CCAAT boxes is mandatory for DNA recognition of HapX. HapX specifically targets the minimal motif 5′-GAT-3′, which is located at a distance of 11–12 bp downstream of the respective CCAAT box. Single nucleotide substitutions at the 5′- and 3′-end of the GAT motif as well as different spacing between the CBC and HapX DNA-binding sites revealed a remarkable promiscuous DNA-recognition mode of HapX. This flexible DNA-binding code may have evolved as a mechanism for fine-tuning the transcriptional activity of CBC-HapX at distinct target promoters. PMID:25589790

  10. Leucine metabolism in human newborns

    SciTech Connect

    Denne, S.C.; Kalhan, S.C. )

    1987-12-01

    The present study was designed to (1) determine whether a relationship exists between newborn birth weight and leucine metabolism and (2) compare leucine and energy metabolism in a period of rapid growth and development (i.e., newborn) with a constant nongrowth period (i.e., adult). Leucine kinetics and energy expenditure were measured in the postabsorptive state in 12 normal full-term newborns in early neonatal life and in 11 normal adults using a primed constant L-(1-{sup 13}C)leucine infusion combined with respiratory calorimetry. A significant positive correlation between newborn birth weight and leucine flux was observed. These data suggest the following. (1) A relationship exists between newborn birth weight and protein metabolism, as reflected by the correlation between leucine flux when expressed as micromoles per kilogram per hour and birth weight. (2) The high rate of leucine flux measured in newborns probably reflects the rapid remodeling of protein that occurs in this period of development, even during fasting. (3) The similar values in newborns and adults of leucine kinetics and energy expenditure when normalized to metabolic body weight and the nearly equivalent allometric exponents relating body weight to leucine flux and energy expenditure support a close relationship between leucine and energy metabolism, at least at the extremes of human growth.

  11. Leucine metabolism in stable cirrhosis.

    PubMed

    Mullen, K D; Denne, S C; McCullough, A J; Savin, S M; Bruno, D; Tavill, A S; Kalhan, S C

    1986-01-01

    Alterations in protein and amino acid metabolism have been postulated to explain the frequent observations of muscle wasting and decreased plasma branched-chain amino acid concentrations in cirrhosis. In order to investigate the changes in protein metabolism, we have measured the rates of leucine turnover and oxidation in six stable, biopsy-proven cirrhotics and six age and sex-matched healthy control subjects after an overnight fast, using [1-13C]leucine tracer. Following a primed constant-rate infusion of [1-13C]leucine, the 13C enrichments of plasma leucine and expired CO2 were used to estimate leucine turnover and oxidation, respectively. Fat-free body mass was estimated from the measurements of total body water as quantified by H2[18O] tracer dilution. The rates of CO2 production and oxygen consumption were measured hourly during the study period, using open-circuit respiratory calorimetry. Urinary urea, ammonia and total nitrogen excretion rates were quantified from timed urine samples. Even though the plasma leucine levels were lower in cirrhotics as compared with controls (100.5 +/- 17.1 vs. 138.3 +/- 20.4 mumoles per liter, mean +/- S.D., p less than 0.001), the rates of leucine turnover were not significantly different in the two groups (89.4 +/- 19.0 vs. 87.8 +/- 19.0 mumoles per kg X hr). In contrast, the rates of leucine oxidation were significantly reduced in cirrhosis (8.1 +/- 2.5 vs. 12.7 +/- 3.1 mumoles per kg X hr, p less than 0.01). When all subjects were considered, the leucine oxidation rate was correlated with plasma leucine concentration (r = 0.62, p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3089896

  12. Leucine regulation of the ilvGEDA operon of Serratia marcescens by attenuation is modulated by a single leucine codon.

    PubMed Central

    Hsu, J H; Harms, E; Umbarger, H E

    1985-01-01

    The effect of leucine limitation and of restricted leucine tRNA charging on the expression of the ilvGEDA operon of Serratia marcescens was examined. In this organism, the ilv leader region specifies a putative peptide containing only a single leucine codon that could be involved in leucine-mediated control by attenuation (E. Harms, J.-H. Hsu, C. S. Subrahmanyam, and H. E. Umbarger, J. Bacteriol. 164:207-216, 1985). A plasmid (pPU134) containing the DNA of the S. marcescens ilv control region and three of the associated structural genes was studied as a single chromosomal copy in an Escherichia coli strain auxotrophic for all three branched-chain amino acids. The S. marcescens ilv genes responded to a multivalent control similar to that found in other enteric organisms. Furthermore, the S. marcescens ilv genes were derepressed when the charging of leucine tRNA was restricted in a leuS derivative of E. coli that had been transformed with pPU134. It was concluded that ribosome stalling leading to deattenuation is not dependent on either tandem or a consecutive series of codons for the regulatory amino acid. However, the fact that the single leucine codon is a less frequently used codon (CUA) may be important. The procedure for obtaining the cloned ilv genes in single chromosomal copy exploited the dependence of ColE1 replicons on the polA gene. The cloning experiments also revealed a branched-chain amino acid-glutamate transaminase in S. marcescens that is different from transaminase B. PMID:3900038

  13. Leucine in Obesity: Therapeutic Prospects.

    PubMed

    Yao, Kang; Duan, Yehui; Li, Fengna; Tan, Bie; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2016-08-01

    Obesity develops from an imbalance of energy homeostasis and is associated with chronic low-grade inflammation in white adipose tissues (WAT). Inflammation is involved in the pathophysiology of many obesity-induced disorders including insulin resistance and diabetes. Increasing evidence has shown that dietary leucine supplementation positively affects the parameters associated with obesity and obesity-related metabolic disorders. The beneficial effects include increased loss of body weight, reduced WAT inflammation, improved lipid and glucose metabolism, enhanced mitochondrial function, and preserved lean body mass. Although these beneficial effects have not been clearly established, dietary leucine supplementation, either alone or as part of a therapeutic regimen, may be a good nutritional tool in the prevention and management of obesity and obesity-induced metabolic disorders. PMID:27256112

  14. Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum.

    PubMed

    Tanaka, M; Mukohata, Y; Yuasa, S

    2000-04-01

    The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine. PMID:10779875

  15. Regulation of Leucine Biosynthesis in Bacillus subtilis

    PubMed Central

    Ward, Jonathan B.; Zahler, Stanley A.

    1973-01-01

    The biosynthesis of α-isopropylmalate (αIPM) synthetase, IPM isomerase, and βIPM dehydrogenase in Bacillus subtilis can be derepressed in leucine auxotrophs by limiting them for leucine. The derepression of the three enzymes is apparently coordinate. A class of mutants resistant to 4-azaleucine excretes leucine and has derepressed levels of all three enzymes. The azaleucine-resistance mutations may lie in a gene (azlA) encoding a repressor. Efforts to find mutations characteristic of a constitutive operator have been unsuccessful. No polar mutations have been found among nine leucine auxotrophs that have characteristics of frameshift mutations. The enzyme catalyzing the first step in leucine biosynthesis, αIPM synthetase, is sensitive to feedback inhibition by leucine. We conclude that leucine biosynthesis is controlled by the inhibition of the activity of the first biosynthetic enzyme by leucine, and by the repression of the synthesis of the first three biosynthetic enzymes by leucine. The repression of the three enzymes may be under the control of a single repressor and a single operator, or of a single repressor and a separate operator for each structural gene. PMID:4200854

  16. A Role for the Kp Leucine Zipper in Regulating P Element Transposition in Drosophila Melanogaster

    PubMed Central

    Andrews, J. D.; Gloor, G. B.

    1995-01-01

    The KP element can repress P element mobility in Drosophila melanogaster. Three mutant KP elements were made that had either two amino acid substitutions or a single amino acid deletion in the putative leucine zipper domain found in the KP polypeptide. Each KP element was expressed from the actin 5C proximal promoter. The wild-type control construct strongly repressed P element mobility, measured by the GD sterility and sn(w) mutability assays, in a position-independent manner. The single amino acid deletion mutant failed to repress P mobility regardless of its insertion site, while repression of P element mobility by the double amino acid substitution mutants was position dependent. The results show that the leucine zipper of the KP polypeptide is important for P element regulation. This supports the multimer-poisoning model of P element repression, because leucine zipper motifs are involved in protein-protein interactions. PMID:8647395

  17. Leucine metabolism in patients with Hepatic Encephalopathy

    SciTech Connect

    McGhee, A.S.; Kassouny, M.E.; Matthews, D.E.; Millikan, W.

    1986-03-01

    A primed continuous infusion of (/sup 15/N, 1-/sup 13/C)leucine was used to determine whether increased oxidation and/or protein synthesis of leucine occurs in patients with cirrhosis. Five controls and patients were equilibrated on a metabolic balance diet (0.6 g protein per kg ideal body weight (IBW)). An additional four patients were equilibrated in the same manner with the same type of diet with a protein level of 0.75 g per kg IBW. Plasma leucine and breath CO/sub 2/ enrichments were measured by mass spectrometry. Protein synthesis and leucine metabolism were identical in controls and patients when both were fed a diet with 0.6 g protein/kg IBW. Results indicate that systemic derangements of leucine metabolism are not the cause of Hepatic Encephalopathy.

  18. A Heterospecific Leucine Zipper Tetramer

    SciTech Connect

    Deng, Y.; Liu, J; Zheng, Q; Li, Q; Kallenbach, N; Lu, M

    2008-01-01

    Protein-protein interactions dictate the assembly of the macromolecular complexes essential for functional networks and cellular behavior. Elucidating principles of molecular recognition governing important interfaces such as coiled coils is a challenging goal for structural and systems biology. We report here that two valine-containing mutants of the GCN4 leucine zipper that fold individually as four-stranded coiled coils associate preferentially in mixtures to form an antiparallel, heterotetrameric structure. X-ray crystallographic analysis reveals that the coinciding hydrophobic interfaces of the hetero- and homotetramers differ in detail, explaining their partnering and structural specificity. Equilibrium disulfide exchange and thermal denaturation experiments show that the 50-fold preference for heterospecificity results from a combination of preferential packing and hydrophobicity. The extent of preference is sensitive to the side chains comprising the interface. Thus, heterotypic versus homotypic interaction specificity in coiled coils reflects a delicate balance in complementarity of shape and chemistry of the participating side chains.

  19. Compartmental model of leucine kinetics in humans.

    PubMed

    Cobelli, C; Saccomani, M P; Tessari, P; Biolo, G; Luzi, L; Matthews, D E

    1991-10-01

    The complexity of amino acid and protein metabolism has limited the development of comprehensive, accurate whole body kinetic models. For leucine, simplified approaches are in use to measure in vivo leucine fluxes, but their domain of validity is uncertain. We propose here a comprehensive compartmental model of the kinetics of leucine and alpha-ketoisocaproate (KIC) in humans. Data from a multiple-tracer administration were generated with a two-stage (I and II) experiment. Six normal subjects were studied. In experiment I, labeled leucine and KIC were simultaneously injected into plasma. Four plasma leucine and KIC tracer concentration curves and label in the expired CO2 were measured. In experiment II, labeled bicarbonate was injected into plasma, and labeled CO2 in the expired air was measured. Radioactive (L-[1-14C]leucine, [4,5-3H]KIC, [14C]bicarbonate) and stable isotope (L-[1-13C]leucine, [5,5,5-2H3]KIC, [13C]bicarbonate) tracers were employed. The input format was a bolus (impulse) dose in the radioactive case and a constant infusion in the stable isotope case. A number of physiologically based, linear time-invariant compartmental models were proposed and tested against the data. The model finally chosen for leucine-KIC kinetics has 10 compartments: 4 for leucine, 3 for KIC, and 3 for bicarbonate. The model is a priori uniquely identifiable, and its parameters were estimated with precision from the five curves of experiment I. The separate assessment of bicarbonate kinetics (experiment II) was shown to be unnecessary. The model defines masses and fluxes of leucine in the organism, in particular its intracellular appearance from protein breakdown, its oxidation, and its incorporation into proteins. An important feature of the model is its ability to estimate leucine oxidation by resolving the bicarbonate model in each individual subject. Finally, the model allows the assessment of the domain of validity of the simpler commonly used models. PMID:1928344

  20. 3D Printing of Protein Models in an Undergraduate Laboratory: Leucine Zippers

    ERIC Educational Resources Information Center

    Meyer, Scott C.

    2015-01-01

    An upper-division undergraduate laboratory experiment is described that explores the structure/function relationship of protein domains, namely leucine zippers, through a molecular graphics computer program and physical models fabricated by 3D printing. By generating solvent accessible surfaces and color-coding hydrophobic, basic, and acidic amino…

  1. Two Di-Leucine Motifs Regulate Trafficking of Mucolipin-1 to Lysosomes

    PubMed Central

    Vergarajauregui, Silvia; Puertollano, Rosa

    2006-01-01

    Mutations in the mucolipin-1 gene have been linked to mucolipidosis type IV, a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. Mucolipin-1 is a membrane protein containing six putative transmembrane domains with both its N- and C-termini localized facing the cytosol. To gain information on the sorting motifs that mediate the trafficking of this protein to lysosomes, we have generated chimeras in which the N- and C- terminal tail portions of mucolipin-1 were fused to a reporter gene. In this article, we report the identification of two separate di-leucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes. One di-leucine motif is positioned at the N-terminal cytosolic tail and mediates direct transport to lysosomes, whereas the other di-leucine motif is found at the C-terminal tail and functions as an adaptor protein 2-dependent internalization motif. We have also found that the C-terminal tail of mucolipin-1 is palmitoylated and that this modification might regulate the efficiency of endocytosis. Finally, the mutagenesis of both di-leucine motifs abrogated lysosomal accumulation and resulted in cell-surface redistribution of mucolipin-1. Taken together, these results reveal novel information regarding the motifs that regulate mucolipin-1 trafficking and suggest a role for palmitoylation in protein sorting. PMID:16497227

  2. Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis.

    PubMed

    Zhu, Lin; Wu, Zhe; Jin, Jian-Ming; Tang, Shuang-Yan

    2016-07-01

    L-tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare L-tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the L-tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of L-tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies (k cat/K m) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of L-tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme. PMID:26898942

  3. 21 CFR 582.5406 - Leucine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Leucine. 582.5406 Section 582.5406 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  4. 21 CFR 582.5406 - Leucine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Leucine. 582.5406 Section 582.5406 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  5. LEUCINE STIMULATION OF SKELETAL MUSCLE PROTEIN SYNTHESIS DURING PROLONGED LEUCINE INFUSION IS DEPENDENT ON AMINO ACID AVAILABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine stimulates protein synthesis in cultured cells, mature rats and neonatal pigs. We have reported that leucine infusion increases protein synthesis in skeletal muscle of neonatal pigs during a 60-min infusion. When leucine infusion was prolonged for 120 min, however, protein synthesis was no...

  6. Systemic exchangeability of enteral leucine: relationship to plasma flux

    SciTech Connect

    Istfan, N.W.; Ling, P.R.; Bistrian, B.R.; Blackburn, G.L.

    1988-04-01

    The exchangeability of enterally infused leucine within the systemic compartment was estimated in fasted and fed rats using L-(1-/sup 14/C)leucine as a tracer. The experimental design consisted of enteral and parenteral feedings with intravenous or intragastric tracer infusions. During continuous intragastric feeding, only 73 +/- 6% (SE) of the intragastric leucine tracer infusion was accounted for in the systemic circulation. When comparing intravenous vs. intragastric tracer, the estimate of the contribution of protein breakdown to plasma leucine flux was 6 +/- 1 (SE) mumol.h-1.100 g-1 and 18 +/- 3 (SE) mumol.h-1.100 g-1 (P less than 0.01), respectively, for the two routes of administration. Correction of enteral input (either isotope or total leucine), by a factor of 27% for first-pass extraction, eliminated all significant differences in plasma leucine kinetics. Of the 27% of enterally infused tracer not appearing systemically, only 3% could be accounted for in newly synthesized protein in the liver. The remainder is hypothesized to represent first-pass utilization of leucine in intestinal protein synthesis and other metabolic pathways in the splanchnic bed. In contrast, systemic appearance of enteral leucine was essentially complete in the fasted rats, indicating less splanchnic metabolism of leucine in this state. These data indicate that significant error can result in estimating the contribution of endogenous protein breakdown to plasma leucine flux during feeding if the systemic exchangeability of dietary leucine is not considered.

  7. Kinetics of sequential metabolism from D-leucine to L-leucine via alpha-ketoisocaproic acid in rat.

    PubMed

    Hasegawa, Hiroshi; Matsukawa, Takehisa; Shinohara, Yoshihiko; Hashimoto, Takao

    2002-12-01

    D-Leucine is considered to be converted into the L-enantiomer by two steps: oxidative deamination to form alpha-ketoisocaproic acid (KIC) and subsequent stereospecific reamination of KIC. We investigated the pharmacokinetics of leucine enantiomers and KIC in rats to evaluate how deamination of D-leucine, reamination of KIC, and decarboxylation of KIC were affected to the overall extent that converted D-leucine into the L-enantiomer. After intravenous administrations of D-[(2)H(7)]leucine, L-[(2)H(7)]leucine, or [(2)H(7)]KIC, their plasma concentrations together with endogenous L-leucine and KIC were determined by gas chromatography-mass spectrometry. The rapid appearances of [(2)H(7)]KIC and L-[(2)H(7)]leucine were observed after administration of D-[(2)H(7)]leucine, whereas no detectable amount of D-[(2)H(7)]leucine was found after administrations of [(2)H(7)]KIC or L-[(2)H(7)]leucine. The fraction of conversion from D-[(2)H(7)]leucine into [(2)H(7)]KIC (F(D-->KIC)) was estimated by using the area under the curve (AUC) of [(2)H(7)]KIC on the D-[(2)H(7)]leucine administration [AUC(KIC(D))] and that of [(2)H(7)]KIC on the [(2)H(7)]KIC administration (AUC(KIC)) to yield 70.1%. The fraction of conversion from [(2)H(7)]KIC to L-[(2)H(7)]leucine (F(KIC-->L)) was 40.2%. The fraction of conversion from D-leucine to the L-enantiomer (F(D-->L)) was considered to be the product of F(D-->KIC) and F(KIC-->L), indicating that 28.2% of D-[(2)H(7)]leucine was metabolized to L-[(2)H(7)]leucine via [(2)H(7)]KIC. These results suggested that the relatively low conversion of D-leucine into the L-enantiomer might depend on irreversible decarboxylation of KIC. Regardless of [(2)H(7)]KIC, F(D-->L) was also calculated directly using AUC(L(D)) and AUC(L) to yield 27.5%. There were no differences between the two F(D-->L) values, suggesting that almost all of the formation of L-[(2)H(7)]leucine from D-[(2)H(7)]leucine occurred via [(2)H(7)]KIC as an intermediate. PMID:12433816

  8. Age Attenuates Leucine Oxidation after Eccentric Exercise

    PubMed Central

    Kullman, E. L.; Campbell, W. W.; Krishnan, R. K.; Yarasheski, K. E.; Evans, W. J.; Kirwan, J. P.

    2013-01-01

    Aging may alter protein metabolism during periods of metabolic and physiologic challenge. The purpose of this study was to assess the effects of age on whole-body amino acid turnover in response to eccentric exercise and hyperglycemia-induced hyperinsulinemia. 16 healthy men were divided into young (N = 8) and older (N = 8) groups. Protein metabolism was assessed using a [1-13C]-leucine isotopic tracer approach. Measures were obtained under fasted basal conditions and during 3-h hyperglycemic clamps that were performed without (control) and 48 h after eccentric exercise. Exercise reduced leucine oxidation in the younger men (P < 0.05), but not in older men. Insulin sensitivity was inversely correlated with leucine oxidation (P < 0.05), and was lower in older men (P < 0.05). Healthy aging is associated with an impaired capacity to adjust protein oxidation in response to eccentric exercise. The decreased efficiency of protein utilization in older men may contribute to impaired maintenance, growth, and repair of body tissues with advancing age. PMID:23325713

  9. Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M.

    PubMed

    Moll, J R; Ruvinov, S B; Pastan, I; Vinson, C

    2001-03-01

    We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g <--> e' (i, i' +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (T(m) = 28 degrees C) or nanomolar (T(m) = 40 degrees C) range, respectively, but heterodimerize with a T(m) >90 degrees C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 microM and 6 degrees C but that heterodimerize with a T(m) = 74 degrees C or K(d(37)) = 1.1 x 10(-11) M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with T(m) = 73 degrees C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells. PMID:11344333

  10. Yeast membrane proteomics using leucine metabolic labelling: Bioinformatic data processing and exemplary application to the ER-intramembrane protease Ypf1.

    PubMed

    Nilse, Lars; Avci, Dönem; Heisterkamp, Patrick; Serang, Oliver; Lemberg, Marius K; Schilling, Oliver

    2016-10-01

    We describe in detail the usage of leucine metabolic labelling in yeast in order to monitor quantitative proteome alterations, e.g. upon removal of a protease. Since laboratory yeast strains are typically leucine auxotroph, metabolic labelling with trideuterated leucine (d3-leucine) is a straightforward, cost-effective, and ubiquitously applicable strategy for quantitative proteomic studies, similar to the widely used arginine/lysine metabolic labelling method for mammalian cells. We showcase the usage of advanced peptide quantification using the FeatureFinderMultiplex algorithm (part of the OpenMS software package) for robust and reliable quantification. Furthermore, we present an OpenMS bioinformatics data analysis workflow that combines accurate quantification with high proteome coverage. In order to enable visualization, peptide-mapping, and sharing of quantitative proteomic data, especially for membrane-spanning and cell-surface proteins, we further developed the web-application Proteator (http://proteator.appspot.com). Due to its simplicity and robustness, we expect metabolic leucine labelling in yeast to be of great interest to the research community. As an exemplary application, we show the identification of the copper transporter Ctr1 as a putative substrate of the ER-intramembrane protease Ypf1 by yeast membrane proteomics using d3-leucine isotopic labelling. PMID:27426920

  11. Pathway for isoleucine formation form pyruvate by leucine biosynthetic enzymes in leucine-accumulating isoleucine revertants of Serratia marcescens.

    PubMed

    Kisumi, M; Komatsubara, S; Chibata, I

    1977-07-01

    Leaky revertants isolated from isoleucine auxotrophs of Serratia marcescens mutant resistant to alpha-aminobutyric acid were previously reported to accumulate leucine in the medium, due to the absence of both feedback inhibition and repression of leucine biosynthesis. Growth of the revertant was accelerated by pyruvate, D(-)-citramalate, citraconate, and alpha-ketobutyrate, but not by threonine. Extracts of the revertant exhibited high activities of pyruvate-dependent coenzyme A liberation from acetyl-coenzyme A, hydration of citraconate, and conversion of citraconate to alpha-ketobutyrate, but showed no threonine-deaminating activity. In the leucine-accumulating revertants the above three activities were not affected by leucine, but in the wild strain and other revertants accumulating no leucine all or one of these activities was controlled by leucine. A leucine auxotroph isolated from the leucine-accumulating revertant showed isoleucine auxotrophy as well. From these data, it is concluded that, in leucine-accumulating revertants, of S. marcescent, isoleucine, is synthesized from alpha-ketobutyrate via citramalate formed from pyruvate annd acetyl-coenzyme A by leucine biosynthetic enzymes, as a result of desensitization of alpha-isopropylmalate synthetase to feedback inhibition. PMID:142769

  12. Potent anti-seizure effects of D-leucine.

    PubMed

    Hartman, Adam L; Santos, Polan; O'Riordan, Kenneth J; Stafstrom, Carl E; Hardwick, J Marie

    2015-10-01

    There are no effective treatments for millions of patients with intractable epilepsy. High-fat ketogenic diets may provide significant clinical benefit but are challenging to implement. Low carbohydrate levels appear to be essential for the ketogenic diet to work, but the active ingredients in dietary interventions remain elusive, and a role for ketogenesis has been challenged. A potential antiseizure role of dietary protein or of individual amino acids in the ketogenic diet is understudied. We investigated the two exclusively ketogenic amino acids, L-leucine and L-lysine, and found that only L-leucine potently protects mice when administered prior to the onset of seizures induced by kainic acid injection, but not by inducing ketosis. Unexpectedly, the D-enantiomer of leucine, which is found in trace amounts in the brain, worked as well or better than L-leucine against both kainic acid and 6Hz electroshock-induced seizures. However, unlike L-leucine, D-leucine potently terminated seizures even after the onset of seizure activity. Furthermore, D-leucine, but not L-leucine, reduced long-term potentiation but had no effect on basal synaptic transmission in vitro. In a screen of candidate neuronal receptors, D-leucine failed to compete for binding by cognate ligands, potentially suggesting a novel target. Even at low doses, D-leucine suppressed ongoing seizures at least as effectively as diazepam but without sedative effects. These studies raise the possibility that D-leucine may represent a new class of anti-seizure agents, and that D-leucine may have a previously unknown function in eukaryotes. PMID:26054437

  13. Protein and leucine metabolism in maple syrup urine disease

    SciTech Connect

    Thompson, G.N.; Bresson, J.L.; Pacy, P.J.; Bonnefont, J.P.; Walter, J.H.; Leonard, J.V.; Saudubray, J.M.; Halliday, D. )

    1990-04-01

    Constant infusions of (13C)leucine and (2H5)phenylalanine were used to trace leucine and protein kinetics, respectively, in seven children with maple syrup urine disease (MSUD) and eleven controls matched for age and dietary protein intake. Despite significant elevations of plasma leucine (mean 351 mumol/l, range 224-477) in MSUD subjects, mean whole body protein synthesis (3.78 +/- 0.42 (SD) g.kg-1. 24 h-1) and catabolism (4.07 +/- 0.46) were similar to control values (3.69 +/- 0.50 and 4.09 +/- 0.50, respectively). The relationship between phenylalanine and leucine fluxes was also similar in MSUD subjects (mean phenylalanine-leucine flux ratio 0.35 +/- 0.07) and previously reported adult controls (0.33 +/- 0.02). Leucine oxidation was undetectable in four of the MSUD subjects and very low in the other three (less than 4 mumol.kg-1.h-1; controls 13-20). These results show that persistent elevation in leucine concentration has no effect on protein synthesis. The marked disturbance in leucine metabolism in MSUD did not alter the relationship between rates of catabolism of protein to phenylalanine and leucine, which provides further support for the validity of the use of a single amino acid to trace whole body protein metabolism. The minimal leucine oxidation in MSUD differs from findings in other inborn metabolic errors and indicates that in patients with classical MSUD there is no significant route of leucine disposal other than through protein synthesis.

  14. Leucine acts as a nutrient signal to stimulate protein synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial rise in amino acids and insulin independently stimulates protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to amino acids. We have shown that the postprandial rise in leucine, but not isoleucine or valine, acutely stimulates muscle pro...

  15. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1460 Leucine aminopeptidase...

  16. Leucine for retention of lean mass on a hypocaloric diet.

    PubMed

    Jitomir, Jean; Willoughby, Darryn S

    2008-12-01

    As obesity rates continue to climb, there is a pressing need for novel weight loss techniques. However, the energy-restricted diets recommended for weight loss typically result in significant amounts of lean tissue loss, in addition to the desired body fat loss. Leucine, a supported anticatabolic agent, has shown promise in research at many levels. First, leucine is known to stimulate the mammalian target of rapamycin pathway, which initiates translation and protein synthesis in muscle cells. Furthermore, leucine may help to regulate blood glucose levels by promoting gluconeogenesis. Finally, several recent studies provide evidence that leucine aids in the retention of lean mass in a hypocaloric state. The aim of this paper is to review relevant leucine research in the three areas described and assess its potential as supplement for obese individuals. PMID:19053849

  17. MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae.

    PubMed

    Jarocki, Veronica M; Santos, Jerran; Tacchi, Jessica L; Raymond, Benjamin B A; Deutscher, Ania T; Jenkins, Cheryl; Padula, Matthew P; Djordjevic, Steven P

    2015-01-01

    Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae. PMID:25589579

  18. MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

    PubMed Central

    Jarocki, Veronica M.; Santos, Jerran; Tacchi, Jessica L.; Raymond, Benjamin B. A.; Deutscher, Ania T.; Jenkins, Cheryl; Padula, Matthew P.; Djordjevic, Steven P.

    2015-01-01

    Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae. PMID:25589579

  19. A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons.

    PubMed Central

    Matsuoka, K; Taoka, M; Satozawa, N; Nakayama, H; Ichimura, T; Takahashi, N; Yamakuni, T; Song, S Y; Isobe, T

    1994-01-01

    A nuclear protein, termed leucine-rich acidic nuclear protein (LANP), has been isolated from among rat cerebellar proteins whose expression was transiently increased during an early stage of postnatal development. The amino acid sequence, deduced from its cDNA, showed that LANP contains 247 amino acids consisting of two distinct structural domains: the N-terminal domain characterized by "leucine-rich repeat," which is found in many eukaryotic proteins and which potentially functions in mediating protein-protein interactions, and the C-terminal domain characterized by a cluster of acidic amino acids with a putative nuclear localization signal. Immunohistochemical study using an antibody against LANP revealed that the protein is localized mainly in nuclei of Purkinje cells. In the rat cerebellum on postnatal day 7, LANP mRNA was expressed moderately in the external granule and Purkinje cells and weakly in the internal granule cells. The expression in these cells, especially in Purkinje cells, increased in the second postnatal week and thereafter decreased to an adult level. The structural characteristics, localization, and the stage- and cell type-specific expression suggest a potential role of LANP in a signal transduction pathway that directs differentiation of cerebellar neurons. Images PMID:7937870

  20. Thermodynamic characteristics of the heparin-leucine-CaCl2 system in a diluted physiological solution

    NASA Astrophysics Data System (ADS)

    Nikolaeva, L. S.; Belov, G. V.; Rulev, Yu. A.; Semenov, A. N.

    2013-03-01

    Chemical equilibria in aqueous solutions of high-molecular weight heparin (Na4hep) and leucine (HLeu) are calculated through the mathematical modeling of chemical equilibria based on representative experimental pH titration data. In addition, chemical equilibria in the CaCl2-Na4hep-HLeu-H2O-NaCl system in the presence of 0.154M NaCl background electrolyte at a temperature of 37°C in the range of 2.30 ≤ pH ≤ 10.50 and initial concentrations of basic components n × 10-3 M ( n ≤ 4).

  1. Identification of a putative nuclear export signal motif in human NANOG homeobox domain

    SciTech Connect

    Park, Sung-Won; Do, Hyun-Jin; Huh, Sun-Hyung; Sung, Boreum; Uhm, Sang-Jun; Song, Hyuk; Kim, Nam-Hyung; Kim, Jae-Hwan

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We found the putative nuclear export signal motif within human NANOG homeodomain. Black-Right-Pointing-Pointer Leucine-rich residues are important for human NANOG homeodomain nuclear export. Black-Right-Pointing-Pointer CRM1-specific inhibitor LMB blocked the potent human NANOG NES-mediated nuclear export. -- Abstract: NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ({sup 125}MQELSNILNL{sup 134}) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-{Delta}NLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

  2. Leucine supplementation improves regeneration of skeletal muscles from old rats.

    PubMed

    Pereira, Marcelo G; Silva, Meiricris T; da Cunha, Fernanda M; Moriscot, Anselmo S; Aoki, Marcelo S; Miyabara, Elen H

    2015-12-01

    The decreased regenerative capacity of old skeletal muscles involves disrupted turnover of proteins. This study investigated whether leucine supplementation in old rats could improve muscle regenerative capacity. Young and old male Wistar rats were supplemented with leucine; then, the muscles were cryolesioned and examined after 3 and 10 days. Leucine supplementation attenuated the decrease in the expression of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4E (eIF4E) in young and old muscles on day 3 post-injury and promoted an increase in the cross-sectional area of regenerating myofibers from both young and old soleus muscles on day 10 post-injury. This supplementation decreased the levels of ubiquitinated proteins and increased the proteasome activity in young regenerating muscles, but the opposite effect was observed in old regenerating muscles. Moreover, leucine decreased the inflammation area and induced an increase in the number of proliferating satellite cells in both young and old muscles. Our results suggest that leucine supplementation improves the regeneration of skeletal muscles from old rats, through the preservation of certain biological responses upon leucine supplementation. Such responses comprise the decrease in the inflammation area, increase in the number of proliferating satellite cells and size of regenerating myofibers, combined with the modulation of components of the phosphoinositide 3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and ubiquitin-proteasome system. PMID:26481769

  3. Fetoplacental deamination and decarboxylation of leucine

    SciTech Connect

    Loy, G.L.; Quick, A.N. Jr.; Hay, W.W. Jr.; Meschia, G.; Battaglia, F.C.; Fennessey, P.V. )

    1990-10-01

    Fetal and placental metabolism of leucine (Leu) and ketoisocaproic acid (KIC) were studied in seven fetal lambs at 132 +/- 1.3-days gestation. Fetal infusions of (1-13C)Leu, (1-14C)Leu, and antipyrine were carried out for 4 h. Uterine and umbilical blood flows were measured using the antipyrine steady-state diffusion technique. Leu and KIC concentrations, (14C)Leu-specific activities, 14CO2, (13C)Leu, and (13C)KIC enrichment (mole percent enrichment) were measured in the maternal artery, uterine vein, and umbilical artery and vein to calculate net fluxes of tracee and tracer molecules between fetus and placenta and between the uteroplacenta and the maternal circulation. There were net Leu and KIC fluxes into the fetus from the placenta with the KIC flux equal to approximately 19% of the combined Leu plus KIC flux. In addition, there was a net KIC flux into the uterine circulation. The fraction of infused tracer Leu escaping the placenta into the mother was small (approximately 6%). By contrast, there was a rapid exchange of tracer Leu carbon between placenta and fetus resulting in a significant flux of labeled KIC from placenta to fetus. Approximately 20% of the infused tracer carbon was converted to CO2 within the fetus. This rate of conversion was greater than 80% of the total fetoplacental conversion rate and significantly higher than the flux of KIC tracer carbon from placenta to fetus. Fetal KIC decarboxylation rate, calculated from the fetal KIC enrichment data, was 2.83 +/- 0.40 mumol.min-1.kg fetus-1 and approximately 60% of the combined net Leu and KIC flux into the fetus from the placenta.

  4. Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2*

    PubMed Central

    Li, Xiaojie; Moore, Darren J.; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.

    2010-01-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity. PMID:20595391

  5. MicroRNA-27a is induced by leucine and contributes to leucine-induced proliferation promotion in C2C12 cells.

    PubMed

    Chen, Xiaoling; Huang, Zhiqing; Chen, Daiwen; Yang, Ting; Liu, Guangmang

    2013-01-01

    Leucine, a branched chain amino acid, is well known to stimulate protein synthesis in skeletal muscle. However, the role of leucine in myoblast proliferation remains unclear. In this study, we found that leucine could promote proliferation of C2C12 cells. Moreover, expressions of miR-27a and myostatin (a bona fide target of miR-27a) were upregulated and downregulated, respectively, following leucine treatment. We also found that miR-27a loss-of-function by transfection of a miR-27a inhibitor suppressed the promotion of myoblast proliferation caused by leucine. Our results suggest that miR-27a is induced by leucine and contributes to leucine-induced proliferation promotion of myoblast. PMID:23880856

  6. Leucine metabolism in cirrhotic patients with hepatic encephalopathy

    SciTech Connect

    McGhee, A.S.

    1985-01-01

    The purpose of this study was to determine whether increased oxidation of or protein synthesis requiring leucine occurs in cirrhotic patients. Five control subjects and four subjects with cirrhosis were equilibrated on a baseline diet (0.6 g protein per kg ideal body weight (IBW)) with sufficient nonprotein calories to preclude negative nitrogen balance. An additional four patients were equilibrated on the same type of diet with a higher protein level (0.75 g per kg IBW). Control subjects and the patients were then studied during continuous infusion of L-(/sup 15/N, 1-/sup 13/C) leucine in the fasted state and, in the fed state, with a Propac diet which had the same distribution of energy nutrients as the baseline diets. Plasma levels of L-(/sup 15/N, 1-/sup 13/C), L-(1-/sup 13/C) and L-(/sup 15/N) leucine were measured during isotopic steady state by gas chromatography-mass spectrometry and fractional excretion of /sup 13/CO/sup 2/ in breath samples were analyzed by isotopic ratio mass spectrometry. During the fasted and fed states leucine metabolism was measured to quantitate rates of nitrogen flux (Q/sub N/), carbon flux (Q/sub c/) and oxidation to carbon dioxide and water (C). From these measured values, proteins breakdown (B), protein synthesis (S), deamination (X/sup 0/) and reamination (X/sub N/) were calculated. The results showed that protein synthesis and leucine metabolism were identical in controls and patients when both were fed a diet with 0.6 g protein/kg IBW and maintenance level of nonprotein calories. The data also showed that leucine metabolism can be quantitatively and reproducibly measured in subjects with cirrhosis.

  7. Dietary leucine requirement of juvenile Japanese seabass ( Lateolabrax japonicus)

    NASA Astrophysics Data System (ADS)

    Li, Yan; Cheng, Zhenyan; Mai, Kangsen; Ai, Qinghui

    2015-02-01

    A 56-day feeding trial was conducted to examine the dietary leucine requirement of juvenile Japanese seabass in seawater floating net cages (1.5 m × 1.5 m × 2.0 m). Six isonitrogenous (crude protein 40%) and isoenergetic (gross energy 20 kJ g-1) diets were formulated to contain different concentrations of leucine (0.9%, 1.49%, 2.07%, 2.70%, 3.30% and 3.88% of dry matter). Crystalline L-amino acids were supplemented to simulate the whole body amino acid pattern of Japanese seabass except for leucine. Three groups (30 fish individuals each, 8.0 g ± 0.20 g in initial weight) were fed to apparent satiation at 5:00 and 17:30 every day. During the experimental period, the water temperature ranged from 26 to 32δC and salinity from 26 to 30, and the dissolved oxygen was maintained at 7 mg L-1. The results showed that weight gain ( WG), nitrogen retention ( NR), feed efficiency ( FE) and protein efficiency ratio ( PER) were significantly increased when dietary leucine was increased from 0.90% to 2.70% of dry matter, and then declined. WG was the highest when fish were fed D4 containing 2.70% of leucine. No significant differences were observed in body composition among dietary treatments ( P > 0.05). Considering the change of WG, the optimum dietary leucine requirement of juvenile Japanese seabass was either 2.39% of dry matter or 5.68% of dietary protein.

  8. Phosphate depletion impairs leucine-induced insulin secretion.

    PubMed

    Oh, H Y; Fadda, G Z; Smogorzewski, M; Liou, H H; Massry, S G

    1994-11-01

    Phosphate depletion (PD) in vivo causes a sundry of abnormalities in pancreatic islets including a rise in cytosolic calcium, low ATP content, reduced Ca2+ ATPase and Na(+)-K+ ATPase activity, and impaired insulin secretion in response to glucose or potassium. L-Leucine is a strong secretagogue that triggers insulin secretion by deamination to alpha-ketoisocaproic acid (KIC) and the subsequent metabolism of the latter to ATP and by the activation of glutamate dehydrogenase (GLDH), which acts on glutamate to generate alpha-ketoglutarate, the metabolism of which results in ATP production. The generation of ATP triggers events that lead to insulin secretion. It is not known whether PD impairs leucine-induced insulin secretion, and the cellular derangements that are involved in such an abnormality are not defined. These issues were studied in PD rats and in pair-weighed normal animals as controls. D-Leucine uptake by islets from PD rats is normal, but both leucine- and KIC-induced insulin secretions are impaired and the activity of branched-chain keto acid dehydrogenase, which facilitates the metabolism of KIC, is reduced. Both leucine and 2-aminobicyclo (2-2-1) haptene failed to stimulate GLDH and to augment the generation of alpha-ketoglutarate in the islets of PD rats. Also, the concentration of basal alpha-ketoglutarate was significantly higher in the islets of PD rats, suggesting that its metabolism is impaired. In addition, the activity of glutaminase is significantly reduced, an abnormality that would result in decreased production of glutamate, the substrate for GLDH. The data show that PD impairs leucine-induced insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873737

  9. Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF.

    PubMed

    Pogenberg, Vivian; Ogmundsdóttir, Margrét H; Bergsteinsdóttir, Kristín; Schepsky, Alexander; Phung, Bengt; Deineko, Viktor; Milewski, Morlin; Steingrímsson, Eiríkur; Wilmanns, Matthias

    2012-12-01

    Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix-loop-helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer. PMID:23207919

  10. Enteral leucine and protein synthesis in skeletal and cardiac muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are three members of the Branch Chain Amino Acids: leucine, isoleucine, and valine. As essential amino acids, these amino acids have important functions which include a primary role in protein structure and metabolism. It is intriguing that the requirement for BCAA in humans comprise about 40–...

  11. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  12. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  13. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  14. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  15. Plasma reciprocal pool specific activity predicts that of intracellular free leucine for protein synthesis

    SciTech Connect

    Horber, F.F.; Horber-Feyder, C.M.; Krayer, S.; Schwenk, W.F.; Haymond, M.W. )

    1989-09-01

    We previously proposed that, during the infusion of either labeled leucine or its alpha-ketoacid, alpha-ketoisocaproate (KIC), the plasma specific activity (SA) of the transaminated product of the infused tracer (reciprocal pool SA) may better reflect the intracellular leucine SA than the plasma SA of either infused tracer (primary pool SA). To test this hypothesis, 14 dogs were simultaneously infused intravenously with (3H)leucine and (14C)KIC, and blood and tissue compartments were sampled. The ratios of (3H)-leucine to (14C)leucine (3H)/(14C)leucine in mixed tissue proteins and in the intracellular space of striated muscle were the same as the ratio of the isotope infusion rates and similar, although slightly lower (P less than 0.01), than (3H)KIC/(14C)leucine SA (ratio of reciprocal pool SA) in plasma. Plasma (3H)KIC/(14C)leucine SA were essentially identical to the (3H)/(14C) of leucine in (1) mixed liver proteins, (2) intrahepatic free leucine, and (3) fibrin. The (3H)/(14C)leucine in mixed renal proteins and in the intracellular space of kidney and erythrocytes were similar to those of the venous plasma (3H)/(14C)leucine SA. The plasma (3H)KIC and (14C)leucine SA (the reciprocal pool SA) were similar to the SA of (3H)- and (14C)leucine in the intracellular space of all organs investigated with the exception of kidney. Therefore, in postabsorptive dogs, the plasma SA of the transaminated product of the infused labeled KIC or leucine is an excellent predictor of the intracellular leucine SA in all tissues investigated with the exception of kidney.

  16. Leucine kinetics from (2H3)- and ( sup 13 C)leucine infused simultaneously by gut and vein

    SciTech Connect

    Hoerr, R.A.; Matthews, D.E.; Bier, D.M.; Young, V.R. )

    1991-01-01

    In amino acid tracer kinetic studies of the fed state, ingested amino acid may be taken up during its initial transit through splanchnic tissues and thus not enter the plasma compartment where tracer is infused. To investigate this possibility, adult human subjects received simultaneous intravenous (iv) and intragastric (ig) leucine tracer infusions, first during a postabsorptive (PA) 4-h primed continuous ig infusion of L-(1-13C)-leucine and L-(5,5,5-2H3)leucine iv, followed on a separate day by a fed infusion, in which an ig infusion of a liquid formula was started 2 h before the tracer infusion and continued throughout the tracer study. Subjects were accustomed to a constant experimental diet supplying 1.5 g protein.kg-1.day-1 and 41-45 kcal.kg-1.day-1 for 7 and 12 days before the PA and fed studies, respectively. For the PA study, plasma enrichment for the ig tracer was 3.34 +/- 0.27 (SE) mol + excess and for the iv tracer it was 4.18 +/- 0.10 (P less than 0.02). Enrichments of alpha-keto-isocaproic acid (KIC) were 3.24 +/- 0.16 (ig) and 3.02 +/- 0.14 (iv), respectively (not significant (NS)). For the fed study, plasma leucine enrichment for the ig tracer was 2.15 +/- 0.14 and for the iv tracer was 2.84 +/- 0.09 (P less than 0.02). KIC enrichments were 2.02 +/- 0.08 (ig) and 2.24 +/- 0.08 (iv), respectively (NS). In the PA study, the ratio of the plasma leucine enrichments for the ig and iv tracers was 0.80 +/- 0.06 and in the fed experiment, 0.76 +/- 0.05, respectively.

  17. Relationship between surface concentration of L-leucine and bulk powder properties in spray dried formulations.

    PubMed

    Mangal, Sharad; Meiser, Felix; Tan, Geoffrey; Gengenbach, Thomas; Denman, John; Rowles, Matthew R; Larson, Ian; Morton, David A V

    2015-08-01

    The amino acid L-leucine has been demonstrated to act as a lubricant and improve the dispersibility of otherwise cohesive fine particles. It was hypothesized that optimum surface L-leucine concentration is necessary to achieve optimal surface and bulk powder properties. Polyvinylpyrrolidone was spray dried with different concentration of L-leucine and the change in surface composition of the formulations was determined using X-ray photoelectron spectroscopy (XPS) and time of flight-secondary ion mass spectrometry (ToF-SIMS). The formulations were also subjected to powder X-ray diffraction analysis in order to understand the relationship between surface concentration and solid-state properties of L-leucine. In addition, the morphology, surface energy and bulk cohesion of spray dried formulations were also assessed to understand the relation between surface L-leucine concentration and surface and bulk properties. The surface concentration of L-leucine increased with higher feed concentrations and plateaued at about 10% L-leucine. Higher surface L-leucine concentration also resulted in the formation of larger L-leucine crystals and not much change in crystal size was noted above 10% L-leucine. A change in surface morphology of particles from spherical to increasingly corrugated was also observed with increasing surface l-leucine concentration. Specific collapsed/folded over particles were only seen in formulations with 10% or higher l-leucine feed concentration suggesting a change in particle surface formation process. In addition, bulk cohesion also reduced and approached a minimum with 10% L-leucine concentration. Thus, the surface concentration of L-leucine governs particle formation and optimum surface L-leucine concentration results in optimum surface and bulk powder properties. PMID:26007290

  18. Vibrational absorption and vibrational circular dichroism spectra of leucine in water under different pH conditions: hydrogen-bonding interactions with water.

    PubMed

    Poopari, Mohammad Reza; Zhu, Peiyan; Dezhahang, Zahra; Xu, Yunjie

    2012-11-21

    Vibrational absorption (VA) and vibrational circular dichroism (VCD) spectroscopy have been used to study leucine, a flexible branched-chain amino acid, in aqueous solution. The VA spectra in the range of 1800-1250 cm(-1) of leucine in D(2)O under three representative pHs from strongly acidic (pH = 1), near neutral (pH = 6), to strongly basic (pH = 13), have been measured. The related VCD spectrum has been obtained under near neutral condition. Searches have been carried out to identify the most stable conformers of the Zwitterionic, protonated, and deprotonated forms of leucine in water. The geometry optimization, harmonic frequency calculations, and VA and VCD intensities have been computed at the B3LYP/6-311++G(d,p) level with the implicit polarizable continuum solvation model. While the observed VA spectra under three pHs can be well interpreted with the inclusion of the implicit solvation model, both implicit and explicit solvation models have been found to be crucial for the adequate interpretation of the complex VCD features observed. Molecular dynamics simulations and radial distribution functions have been used to aid the modeling of the leucine-(water)(N) clusters. It has been recognized that the insertion of a water molecule between the COO(-) and NH(3) (+) functional groups in the explicit solvated clusters is critical to reproduce the VCD signatures observed. Furthermore, the inclusion of the implicit bulk water environment has been found to be essential to lock water molecules, which are directly hydrogen bonded to leucine, into the positions expected in solution. The application of the explicit and implicit solvation models simultaneously allows new insights into the hydrogen bonding network surrounding leucine in aqueous solution and the role of the surrounding bulk water in stabilizing such hydrogen-bonding network. PMID:23181307

  19. Vibrational absorption and vibrational circular dichroism spectra of leucine in water under different pH conditions: Hydrogen-bonding interactions with water

    NASA Astrophysics Data System (ADS)

    Poopari, Mohammad Reza; Zhu, Peiyan; Dezhahang, Zahra; Xu, Yunjie

    2012-11-01

    Vibrational absorption (VA) and vibrational circular dichroism (VCD) spectroscopy have been used to study leucine, a flexible branched-chain amino acid, in aqueous solution. The VA spectra in the range of 1800-1250 cm-1 of leucine in D2O under three representative pHs from strongly acidic (pH = 1), near neutral (pH = 6), to strongly basic (pH = 13), have been measured. The related VCD spectrum has been obtained under near neutral condition. Searches have been carried out to identify the most stable conformers of the Zwitterionic, protonated, and deprotonated forms of leucine in water. The geometry optimization, harmonic frequency calculations, and VA and VCD intensities have been computed at the B3LYP/6-311++G(d,p) level with the implicit polarizable continuum solvation model. While the observed VA spectra under three pHs can be well interpreted with the inclusion of the implicit solvation model, both implicit and explicit solvation models have been found to be crucial for the adequate interpretation of the complex VCD features observed. Molecular dynamics simulations and radial distribution functions have been used to aid the modeling of the leucine-(water)N clusters. It has been recognized that the insertion of a water molecule between the COO- and NH3+ functional groups in the explicit solvated clusters is critical to reproduce the VCD signatures observed. Furthermore, the inclusion of the implicit bulk water environment has been found to be essential to lock water molecules, which are directly hydrogen bonded to leucine, into the positions expected in solution. The application of the explicit and implicit solvation models simultaneously allows new insights into the hydrogen bonding network surrounding leucine in aqueous solution and the role of the surrounding bulk water in stabilizing such hydrogen-bonding network.

  20. Paramagnetic molecular centers in the gamma-irradiated novel compound of aluminum and leucine, Al 6O 4(OH) 10(leucine) 2·5H 2O

    NASA Astrophysics Data System (ADS)

    Nothig-Laslo, Vesna; Himdan, Takialdin A.; Bilinski, Halka

    A new microcrystalline compound Al 6O 4(OH) 10(leucine) 2·5H 2O of possible biological and biochemical interest has been prepared and characterized by chemical analysis, i.r. spectrum, X-ray diffraction and thermogravimetric analysis. It was exposed to γ-irradiation at 77 K and at room temperature. Paramagnetic species formed were studied by ESR spectroscopy. The leucine radical ? has been identified which seems to be stabilized in the aluminium leucine compound by crystalline water. Coordinated leucine molecule in aluminium hydroxide acts as a trap for γ-irradiation energy.

  1. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis. PMID:22100731

  2. Leucine Metabolism in T Cell Activation: mTOR Signaling and Beyond.

    PubMed

    Ananieva, Elitsa A; Powell, Jonathan D; Hutson, Susan M

    2016-07-01

    In connection with the increasing interest in metabolic regulation of the immune response, this review discusses current advances in understanding the role of leucine and leucine metabolism in T lymphocyte (T cell) activation. T cell activation during the development of an immune response depends on metabolic reprogramming to ensure that sufficient nutrients and energy are taken up by the highly proliferating T cells. Leucine has been described as an important essential amino acid and a nutrient signal that activates complex 1 of the mammalian target of rapamycin (mTORC1), which is a critical regulator of T cell proliferation, differentiation, and function. The role of leucine in these processes is further discussed in relation to amino acid transporters, leucine-degrading enzymes, and other metabolites of leucine metabolism. A new model of T cell regulation by leucine is proposed and outlines a chain of events that leads to the activation of mTORC1 in T cells. PMID:27422517

  3. Characterization of Leucine Auxotrophs of the White Rot Basidiomycete Phanerochaete chrysosporium

    PubMed Central

    Molskness, Theodore A.; Alic, Margaret; Gold, Michael H.

    1986-01-01

    Six leucine auxotrophic strains of the white rot basidiomycete Phanerochaete chrysosporium were characterized genetically and biochemically. Complementation studies involving the use of heterokaryons identified three leucine complementation groups. Since all of the leucine auxotrophs grew on minimal medium supplemented with α-ketoisocaproate as well as with leucine, the transaminase catalyzing the last step in the leucine pathway was apparently normal in all strains. Therefore, the wild-type, auxotrophic, and several heterokaryotic strains were assayed for the activities of the other enzymes specific to leucine biosynthesis. Leu2 and Leu4 strains (complementation group I) lacked only α-isopropylmalate synthase activity; Leu3 and Leu6 strains (group III) lacked isopropylmalate isomerase activity; and Leu1 and Leu5 strains (group II) lacked β-isopropylmalate dehydrogenase. Heterokaryons formed from leucine auxotrophs of different complementation groups had levels of activity for all three enzymes similar to those found in the wild-type strain. PMID:16347073

  4. Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway

    PubMed Central

    Saxton, Robert A.; Knockenhauer, Kevin E.; Wolfson, Rachel L.; Chantranupong, Lynne; Pacold, Michael E.; Wang, Tim; Schwartz, Thomas U.; Sabatini, David M.

    2015-01-01

    Eukaryotic cells coordinate growth with the availability of nutrients through mTOR complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag GTPases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. We present the 2.7-Å crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway. PMID:26586190

  5. The maternal embryonic leucine zipper kinase (MELK) is upregulated in high-grade prostate cancer.

    PubMed

    Kuner, Ruprecht; Fälth, Maria; Pressinotti, Nicole Chui; Brase, Jan C; Puig, Sabrina Balaguer; Metzger, Jennifer; Gade, Stephan; Schäfer, Georg; Bartsch, Georg; Steiner, Eberhard; Klocker, Helmut; Sültmann, Holger

    2013-02-01

    Loss of cell cycle control is a prerequisite for cancer onset and progression. In prostate cancer, increased activity of cell cycle genes has been associated with prognostic parameters such as biochemical relapse and survival. The identification of novel oncogenic and druggable targets in patient subgroups with poor prognosis may help to develop targeted therapy approaches. We analyzed prostate cancer and corresponding benign tissues (n = 98) using microarrays. The comparison of high- and low-grade tumors (Gleason score ≥ 4 + 3 vs. ≤ 3 + 4) revealed 144 differentially expressed genes (p < 0.05). Out of these, 15 genes were involved in the cell cycle process. The gene maternal embryonic leucine zipper kinase (MELK) was identified to be highly correlated with cell cycle genes like UBE2C, TOP2A, CCNB2, and AURKB. Increased MELK gene expression in high-risk prostate cancer was validated by qPCR in an independent patient cohort (p < 0.005, n = 79). Immunohistochemistry analysis using a tissue microarray (n = 94) revealed increased MELK protein expression in prostate cancer tissues of high Gleason scores. RNAi-based inhibition of MELK in PC3 and LNCaP cells suggested putative function in chromatin modification, embryonic development and cell migration. The concerted inhibition of MELK and other cell cycle targets by the antibiotic siomycin A strongly impaired cell viability of prostate cancer cells, and may point to a novel therapy approach for a subset of high-risk prostate cancer patients. PMID:22945237

  6. Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein.

    PubMed Central

    Qian, Z; Brunovskis, P; Lee, L; Vogt, P K; Kung, H J

    1996-01-01

    Marek's disease virus is a highly oncogenic herpesvirus that can cause T lymphomas and peripheral nerve demyelination in chickens. meq, a candidate oncogene of Marek's disease virus, encodes a basic leucine zipper (bZIP) transcription factor which contains a large proline-rich domain in its C terminus. On the basis of its bZIP structural homology, meq is perhaps the only member of the jun-fos gene family completely viral in origin. We previously showed that Meq's C-terminal domain has potent transactivation activity and that its bZIP domain can dimerize with itself and with c-Jun also. In an effort to identify viral and cellular targets of Meq, we have determined the optimal binding sites for Meq-Jun heterodimers and Meq-Meq homodimers. By a PCR-based approach using cyclic amplification of selected targets, Meq-Jun heterodimers were found to optimally bind tetradecanoylphorbol acetate response element (TRE) and cyclic AMP response element (CRE) consensus sequences. This result was consistent with the results of our previous functional analysis implicating Meq-Jun heterodimers in the transactivation of the Meq promoter through a TRE- or CRE-like sequence. Interestingly, Meq-Meq homodimers were found to bind two distinct motif elements. The first [GAGTGATG AC(G)TCATC] has a consensus which includes a TRE or CRE core flanked by additional nucleotides critical for tight binding. Methylation interference and mutational analyses confirmed the importance of the flanking residues. The sequences of a subset of TRE and CRE sites selected by Meq-Meq are closely related to the binding motif of Maf, another bZIP oncoprotein. The second putative Meq binding site (RACACACAY) bears a completely different consensus not shared by other bZIP proteins. Binding to this consensus sequence also requires secondary structure characteristics associated with DNA bending. CACA motifs are known to promote DNA curvature and function in a number of special biological processes. Our results lend

  7. Leucine incorporation into mixed skeletal muscle protein in humans

    SciTech Connect

    Nair, K.S.; Halliday, D.; Griggs, R.C. Clinical Research Centre, Harrow )

    1988-02-01

    Fractional mixed skeletal muscle protein synthesis (FMPS) was estimated in 10 postabsorptive healthy men by determining the increment in the abundance of ({sup 13}C)-leucine in quadriceps muscle protein during an intravenous infusion of L-(1-{sup 13}C)leucine. Whole-body muscle protein synthesis (MPS) was calculated based on the estimation of muscle mass from creatinine excretion and compared with whole-body protein synthesis (WBPS) calculated from the nonoxidative portion of leucine flux. A significant correlation was found between MPS. The contribution of MPS to WBPS was 27 {plus minus} 1%, which is comparable to the reports in other species. Morphometric analyses of adjacent muscle samples in eight subjects demonstrated that the biopsy specimens consisted of 86.5 {plus minus} 2% muscular as opposed to other tissues. Because fiber type composition varies between biopsies, the authors examined the relationship between proportions of each fiber type and FMPS. Variation in the composition of biopsies and in fiber-type proportion did not affect the estimation of muscle protein synthesis rate. They conclude that stable isotope techniques using serial needle biopsies permit the direct measurement of FMPS in humans and that this estimation is correlated with an indirect estimation of WBPS.

  8. The Biogeography of Putative Microbial Antibiotic Production.

    PubMed

    Morlon, Hélène; O'Connor, Timothy K; Bryant, Jessica A; Charkoudian, Louise K; Docherty, Kathryn M; Jones, Evan; Kembel, Steven W; Green, Jessica L; Bohannan, Brendan J M

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  9. The Biogeography of Putative Microbial Antibiotic Production

    PubMed Central

    Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  10. BASIC Programming.

    ERIC Educational Resources Information Center

    Jennings, Carol Ann

    Designed for use by both secondary- and postsecondary-level business teachers, this curriculum guide consists of 10 units of instructional materials dealing with Beginners All-Purpose Symbol Instruction Code (BASIC) programing. Topics of the individual lessons are numbering BASIC programs and using the PRINT, END, and REM statements; system…

  11. Basic Warehousing.

    ERIC Educational Resources Information Center

    Marine Corps Inst., Washington, DC.

    Developed as part of the Marine Corps Institute (MCI) correspondence training program, this course on basic warehousing is designed to provide Marines with Military Occupation Speciality 3051 in the rank of private through corporal with instruction in those basic principles, methods, and procedures that can be applied to any warehousing or storage…

  12. Transcription attenuation in Salmonella typhimurium: the significance of rare leucine codons in the leu leader.

    PubMed Central

    Carter, P W; Bartkus, J M; Calvo, J M

    1986-01-01

    The leucine operon of Salmonella typhimurium is controlled by a transcription attenuation mechanism. Four adjacent leucine codons within a 160-nucleotide leu leader RNA are thought to play a central role in this mechanism. Three of the four codons are CUA, a rarely used leucine codon within enteric bacteria. To determine whether the nature of the leucine codon affects the regulation of the leucine operon, we used oligonucleotide-directed mutagenesis to first convert one CUA of the leader to CUG and then convert all three CUA codons to CUG. CUG is the most frequently used leucine codon in enteric bacteria. A mutant having (CUA)2CUGCUC in place of (CUA)3CUC has an altered response to leucine limitation, requiring a slightly higher degree of limitation to effect derepression. Changing (CUA)3CUC to (CUG)3CUC has more dramatic effects upon operon expression. First, the basal level of expression is lowered to the point that the mutant grows more slowly than the parent in a minimal medium lacking leucine. Second, the response of the mutant to a leucine limitation is dramatically altered such that even a strong limitation elicits only a modest degree of derepression. If the mutant is grown under conditions of leucyl-tRNA limitation rather than leucine limitation, complete derepression can be achieved, but only at a much higher degree of limitation than for the wild-type operon. These results provide a clear-cut example of codon usage having a dramatic effect upon gene expression. PMID:3534884

  13. French Basic Course: Basic Situations.

    ERIC Educational Resources Information Center

    Defense Language Inst., Monterey, CA.

    This volume of the French Basic Course contains ten situations from daily life, each divided into five sub-situations. The material for each situation consists of cartoons and lists of selected words. The purpose of the volume is to provide a vehicle for reviewing the grammar and vocabulary of lessons 1-85 of the Basic Course and adding new words…

  14. Schizophrenia Basics

    MedlinePlus

    ... I know with schizophrenia? For More Information Share Schizophrenia Basics Download PDF Download ePub Order a free hardcopy What is schizophrenia? Schizophrenia is a serious mental disorder that affects ...

  15. Cancer Basics

    MedlinePlus

    ... Cancer? Breast Cancer Colon/Rectum Cancer Lung Cancer Prostate Cancer Skin Cancer Show All Cancer Types News and Features Cancer Glossary ACS Bookstore Cancer Information Cancer Basics Cancer Prevention & Detection Signs & Symptoms of Cancer Treatments & Side Effects ...

  16. Basic Finance

    NASA Technical Reports Server (NTRS)

    Vittek, J. F.

    1972-01-01

    A discussion of the basic measures of corporate financial strength, and the sources of the information is reported. Considered are: balance sheet, income statement, funds and cash flow, and financial ratios.

  17. Fluoridation Basics

    MedlinePlus

    ... Water Fluoridation Journal Articles for Community Water Fluoridation Water Fluoridation Basics Recommend on Facebook Tweet Share Compartir ... because of tooth decay. History of Fluoride in Water In the 1930s, scientists examined the relationship between ...

  18. The Basics

    ERIC Educational Resources Information Center

    Indrisano, Roselmina; And Others

    1976-01-01

    These articles are presented as an aide in teaching basic subjects. This issue examines reading diagnosis, food preservation, prime numbers, electromagnets, acting out in language arts, self-directed spelling activities, and resources for environmental education. (Editor/RK)

  19. Glucose and leucine kinetics in idiopathic ketotic hypoglycaemia

    PubMed Central

    Bodamer, O A; Hussein, K; Morris, A A; Langhans, C‐D; Rating, D; Mayatepek, E; Leonard, J V

    2006-01-01

    Aims To investigate glucose and leucine kinetics in association with metabolic and endocrine investigations in children with ketotic hypoglycaemia (KH) in order to elucidate the underlying pathophysiology. Methods Prospective interventional study using stable isotope tracer in nine children (mean age 4.23 years, range 0.9–9.8 years; seven males) with KH and 11 controls (mean age 4.57 years, range 0.16–12.3 years; four males). Results Plasma insulin levels were significantly lower in KH compared to subjects in the non‐KH group. Plasma ketone body levels were significantly higher in KH than in non‐KH. Basal metabolic rate was significantly higher in subjects with KH (45.48±7.41 v 31.81±6.72 kcal/kg/day) but the respiratory quotients were similar in both groups (KH v non‐KH, 0.84±0.05 v 0.8±0.04. Leucine oxidation rates were significantly lower in children with KH (12.25±6.25 v 31.96±8.59 μmol/kg/h). Hepatic glucose production rates were also significantly lower in KH (3.84±0.46 v 6.6±0.59 mg/kg/min). Conclusions KH is caused by a failure to sustain hepatic glucose production rather than by increased glucose oxidation rates. Energy demand is significantly increased, whereas leucine oxidation is reduced. PMID:16443613

  20. Role of renal D-amino-acid oxidase in pharmacokinetics of D-leucine.

    PubMed

    Hasegawa, Hiroshi; Matsukawa, Takehisa; Shinohara, Yoshihiko; Konno, Ryuichi; Hashimoto, Takao

    2004-07-01

    d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding alpha-keto acids, but its contribution in vivo is poorly understood because the alpha-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their alpha-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[(2)H(7)]leucine to mutant mice lacking DAO activity (ddY/DAO(-)) and normal mice (ddY/DAO(+)), elimination of d-[(2)H(7)]leucine and formation of alpha-[(2)H(7)]ketoisocaproic acid ([(2)H(7)]KIC) and l-[(2)H(7)]leucine in plasma were determined. The ddY/DAO(-) mice, in contrast to ddY/DAO(+) mice, failed to convert d-[(2)H(7)]leucine to [(2)H(7)]KIC and l-[(2)H(7)]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[(2)H(7)]leucine and formation of [(2)H(7)]KIC and l-[(2)H(7)]leucine. Renal mass reduction slowed down the elimination of d-[(2)H(7)]leucine. The fraction of conversion of d-[(2)H(7)]leucine to [(2)H(7)]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[(2)H(7)]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC. PMID:15026304

  1. Effects of leucine supplemented diet on intestinal absorption in tumor bearing pregnant rats

    PubMed Central

    Ventrucci, Gislaine; de Mello, Maria Alice Roston; Gomes-Marcondes, Maria Cristina Cintra

    2002-01-01

    Background It is known that amino acid oxidation is increased in tumor-bearing rat muscles and that leucine is an important ketogenic amino acid that provides energy to the skeletal muscle. Methods To evaluate the effects of a leucine supplemented diet on the intestinal absorption alterations produced by Walker 256, growing pregnant rats were distributed into six groups. Three pregnant groups received a normal protein diet (18% protein): pregnant (N), tumor-bearing (WN), pair-fed rats (Np). Three other pregnant groups were fed a diet supplemented with 3% leucine (15% protein plus 3% leucine): leucine (L), tumor-bearing (WL) and pair-fed with leucine (Lp). Non pregnant rats (C), which received a normal protein diet, were used as a control group. After 20 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. Results Tumor-bearing pregnant rats showed impairment in food intake, body weight gain and muscle protein content, which were less accentuated in WL than in WN rats. These metabolic changes led to reduction in both fetal and tumor development. Leucine absorption slightly increased in WN group. In spite of having a significant decrease in leucine and methionine absorption compared to L, the WL group has shown a higher absorption rate of methionine than WN group, probably due to the ingestion of the leucine supplemented diet inducing this amino acid uptake. Glucose absorption was reduced in both tumor-bearing groups. Conclusions Leucine supplementation during pregnancy in tumor-bearing rats promoted high leucine absorption, increasing the availability of the amino acid for neoplasic cells and, mainly, for fetus and host utilization. This may have contributed to the better preservation of body weight gain, food intake and muscle protein observed in the supplemented rats in relation to the non-supplemented ones. PMID:11955290

  2. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii. PMID

  3. Histochemistry of leucine aminonaphthylamidase (LAN) in rainbow trout (Salmo gairdneri)

    USGS Publications Warehouse

    Bouck, Gerald R.

    1979-01-01

    The histochemistry of leucine aminonaphthylamidase (LAN) was studied in frozen tissue sections of rainbow trout both in yearling and adult fish. Age of fish had relatively little effect upon the results. The most intense LAN color production was in epithelial cells of midgut, pyloric ceca, hindgut, and in some segments of kidney tubules. Lower levels of LAN were evident in liver cells of Kupffer, and still lower or slight levels of LAN activity were found in blood cells, muscle, nerve, connective tissue, gonad, and pancreas. The results indicate that LAN might be useful in assessing histotoxicity to LAN-rich areas of the body.

  4. Exogenous valine reduces conversion of leucine to 3-methyl-1-butanol in Saccharomyces cerevisiae

    SciTech Connect

    Bigelis, R.; Weir, P.D.; Jones, R.R.M.; Umbarger, H.E.

    1983-02-01

    Mutant strains of the yeast Saccharomyces cerevisiae that require branched-chain amino acids must be supplemented with large concentrations (up to 10 mM) of these amino acids to satisfy their nutritional requirements. The utilization of one branched-chain amino acid, leucine, was examined in several leul strains of yeast grown aerobically in a glucose-ammonium salts minimal medium containing a limiting concentration (0.2 mM) of leucine. In this medium, the leucine requirement of the auxotrophic strains could be reduced by valine, another branched-chain amino acid. Increasing the valine concentration increased the cell yields of cultures and also reduced the levels of 3-methyl-1-butanol detected in the medium by gas chromatography. The concentration of 3-methyl-1-butanol was reduced from 122.0 to 48.9 ..mu..M when 5.0 mM valine was supplemented to limiting-leucine cultures. The amino acids isoleucine, threonine, norleucine, norvaline, ..cap alpha..-amino-butyrate, alanine, and glycine also spared the leucine requirement of leucine auxotrophs, most likely because they resemble leucine and competed for its uptake. We propose that leucine analogs restrict the entry and degradation of leucine and thus reduce its conversion to 3-methyl-1-butanol, a major component of fuel oil.

  5. Failure of dietary leucine to influence the tryptophan-niacin pathway in the chicken.

    PubMed

    Penz, A M; Clifford, A J; Rogers, Q R; Kratzer, F H

    1984-01-01

    Three experiments were conducted with young chicks to determine whether dietary leucine affects the conversion of tryptophan to niacin. Either tryptophan or niacin improved growth and reduced perosis when chicks were fed a purified diet marginal in tryptophan and deficient in niacin. Addition of 4.8% L-leucine to the diet did not alter the growth and perosis prevention response obtained with tryptophan. Liver weight was slightly increased by the addition of 5.4% L-leucine to the diet. Plasma insulin was slightly reduced by leucine and by isoleucine and valine. Picolinic carboxylase in the kidney was reduced in chicks fed 0.2% tryptophan with no niacin and was also reduced when isoleucine and valine were added to the diets. Liver picolinic carboxylase activity was not influenced by diet. Plasma isoleucine and valine were reduced by the addition of leucine to the diet and were increased again when isoleucine and valine were added to the diet. Plasma leucine was increased by the addition of leucine but was not altered by valine and isoleucine. Plasma tryptophan was not influenced by dietary supplements of leucine or isoleucine and valine. The results show that in the chick there is no evidence for an effect of leucine on the tryptophan to niacin pathway. PMID:6693983

  6. The thermodynamic characteristics of complex formation between calcium ions and L-leucine in aqueous solution

    NASA Astrophysics Data System (ADS)

    Kurochkin, V. Yu.; Chernikov, V. V.; Orlova, T. D.

    2011-04-01

    Complex formation of L-leucine with calcium ions in aqueous solution was studied by potentiometric titration at 298.15 K and ionic strength values I = 0.5, 1.0, and 1.5 (KNO3). The formation of the CaL+ and CaHL2+ complex particles was established and their stability constants were determined. The enthalpies of protolytic equilibria of leucine and formation of calcium ion complexes with leucine were determined calorimetrically at 298.15 K and I = 0.5 (KNO3). The thermodynamic characteristics of complex formation between calcium ions and L-leucine were calculated.

  7. Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism.

    PubMed

    Nemecek-Marshall, M; Wojciechowski, C; Wagner, W P; Fall, R

    1999-12-01

    There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads. PMID:10601206

  8. Leucine Transport in Cells Isolated from Cold-Hardened and Nonhardened Winter Rye 1

    PubMed Central

    Barran, Leslie R.; Singh, Jas

    1982-01-01

    The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25°C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5°C efflux from the cells was inhibited. The apparent Km and Vmax for leucine uptake for both types of cells were of the order of 20 to 60 micromolar and 0.5 to 1.3 nanomoles per minute per 106 cells. The pH and temperature optima for both types of cells were 5 and 25°C, respectively. The leucine transport system for these cells was relatively specific for amino acids lacking either bulky or charged groups on the amino acid side chains. Arrhenius plots for leucine uptake by hardened and nonhardened cells showed discontinuities at 13°C, and the energies of activation were similar. The results suggests that biochemical changes which occur in rye cells upon cold hardening did not result in an observable perturbation of the properties of the leucine transport system. PMID:16662298

  9. Comparative analysis of pharmacological treatments with N-acetyl-dl-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

    PubMed

    Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

    2015-12-15

    Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. PMID:26607469

  10. DOS basics

    SciTech Connect

    O`Connor, P.

    1994-09-01

    DOS is an acronym for Disk Operating System. It is actually a set of programs that allows you to control your personal computer. DOS offers the capabilities to create and manage files; organize and maintain information placed on disks; use application programs such as WordPerfect, Lotus 123, Excel, Windows, etc. In addition, DOS provides the basic utilities needed to copy files from one area to another, delete files and list files. The latest version of DOS also offers more advanced features that include hard disk compression and memory management. Basic DOS commands are discussed.

  11. Sestrin regulation of TORC1: Is Sestrin a leucine sensor?

    PubMed

    Lee, Jun Hee; Cho, Uhn-Soo; Karin, Michael

    2016-01-01

    Sestrins are highly conserved, stress-inducible proteins that inhibit target of rapamycin complex 1 (TORC1) signaling. After their transcriptional induction, both vertebrate and invertebrate Sestrins turn on the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which activates the tuberous sclerosis complex (TSC), a key inhibitor of TORC1 activation. However, Sestrin overexpression, on occasion, can result in TORC1 inhibition even in AMPK-deficient cells. This effect has been attributed to Sestrin's ability to bind the TORC1-regulating GATOR2 protein complex, which was postulated to control trafficking of TORC1 to lysosomes. How the binding of Sestrins to GATOR2 is regulated and how it contributes to TORC1 inhibition are unknown. New findings suggest that the amino acid leucine specifically disrupts the association of Sestrin2 with GATOR2, thus explaining how leucine and related amino acids stimulate TORC1 activity. We discuss whether and how these findings fit what has already been learned about Sestrin-mediated TORC1 inhibition from genetic studies conducted in fruit flies and mammals. PMID:27273098

  12. Ethanol Basics

    SciTech Connect

    2015-01-30

    Ethanol is a widely-used, domestically-produced renewable fuel made from corn and other plant materials. More than 96% of gasoline sold in the United States contains ethanol. Learn more about this alternative fuel in the Ethanol Basics Fact Sheet, produced by the U.S. Department of Energy's Clean Cities program.

  13. Basic Science.

    ERIC Educational Resources Information Center

    Mercer County Community Coll., Trenton, NJ.

    Instructional materials are provided for a course that covers basic concepts of physics and chemistry. Designed for use in a workplace literacy project developed by Mercer County Community College (New Jersey) and its partners, the course describes applications of these concepts to real-life situations, with an emphasis on applications of…

  14. Basic Skills.

    ERIC Educational Resources Information Center

    Luparelli, Augustus N.; And Others

    1981-01-01

    These four articles focus on developing basic reading, science, and job search skills: "Reading Program for Vocational Classes" by Augustus Luparelli; "Why Teach Employability Skills?" by Larry Siefferman; "Improving Vocabulary and Reading Skills" by Edythe Conway; and "Science in Everyday Life" by Virginia Eleazer and George Carney. (SK)

  15. Body Basics

    MedlinePlus

    ... more about how the body works, what basic human anatomy is, and what happens when parts of the body don't function properly. Blood Bones, Muscles, and Joints Brain and Nervous System Digestive System Endocrine System Eyes Female Reproductive System ...

  16. Basic Backwardness.

    ERIC Educational Resources Information Center

    Weingartner, Charles

    This paper argues that the "back to basics" movement is regressive and that regression is the characteristic mode of fear-ridden personalities. It is argued that many people in American society today have lost their ability to laugh and do not have the sense of humor which is crucial to a healthy mental state. Such topics as necrophilia, mental…

  17. Armchair BASIC.

    ERIC Educational Resources Information Center

    Fox, Annie; Fox, David

    1983-01-01

    A first lesson in learning the computer programing language BASIC, this article explains how to give instructions to the computer; the commands PRINT, NEW, LIST, and RUN; and how to do simple line editing. There is a short quiz at the end. (EAO)

  18. Basic Education.

    ERIC Educational Resources Information Center

    Robinson, Virginia, Ed.

    1984-01-01

    This issue of "Basic Education" is devoted to the arts in education as a concern that should be addressed in a time of new priorities for the curriculum. Five articles and a book review are included. The opening article, "The State of the Arts in Education: Envisioning Active Participation By All" (Virginia Robinson), emphasizes that the study of…

  19. Basic Horticulture.

    ERIC Educational Resources Information Center

    Geer, Barbra Farabough

    This learning packet contains teaching suggestions and student learning materials for a course in basic horticulture aimed at preparing students for employment in a number of horticulture areas. The packet includes nine sections and twenty instructional units. Following the standard format established for Oklahoma vocational education materials in…

  20. Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

    PubMed

    Wallbach, Manuel; Duque Escobar, Jorge; Babaeikelishomi, Rohollah; Stahnke, Marie-Jeannette; Blume, Roland; Schröder, Sabine; Kruegel, Jenny; Maedler, Kathrin; Kluth, Oliver; Kehlenbach, Ralph H; Miosge, Nicolai; Oetjen, Elke

    2016-04-01

    The dual leucine zipper kinase DLK induces β-cell apoptosis by inhibiting the transcriptional activity conferred by the β-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to β-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1β, known prediabetic signals. In the present study, the regulation of DLK in β-cells by these cytokines was investigated. Both, TNFα and IL-1β induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the β-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of β-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus. PMID:26776303

  1. Triennial growth symposium: Leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial increases in AA and insulin independently stimulate protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to AA. We have shown that the postprandial increase in leucine, but not isoleucine or valine, acutely stimulates muscle protein synth...

  2. Stimulation of muscle protein synthesis by leucine is dependent on plasma amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported that a physiological increase in plasma leucine increased translation initiation factor activity during 60- and 120-min leucine infusion. Muscle protein synthesis was stimulated at 60 min but not at 120 min, perhaps due to the decrease (-50%) in plasma essential amino acids (AA). ...

  3. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain.

    PubMed

    Gui, Yongli; Ma, Yuechao; Xu, Qingyang; Zhang, Chenglin; Xie, Xixian; Chen, Ning

    2016-02-20

    Here, we report the complete genome sequence of Corynebacterium glutamicum CP, an industrial l-leucine producing strain in China. The whole genome consists of a circular chromosome and a plasmid. The comparative genomics analysis shows that there are many mutations in the key enzyme coding genes relevant to l-leucine biosynthesis compared to C. glutamicum ATCC 13032. PMID:26784991

  4. Long-term leucine induced stimulation of muscle protein synthesis is amino acid dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infusing leucine for 1 h increases skeletal muscle protein synthesis in the neonate, but this is not sustained for 2 h unless the corresponding fall in amino acids is prevented. This study aimed to determine whether a continuous leucine infusion can stimulate protein synthesis for a prolonged period...

  5. Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal...

  6. Basic Skills--Basic Business.

    ERIC Educational Resources Information Center

    Conference Board of Canada, Ottawa (Ontario).

    The experience of eight prominent Canadian business organizations was examined in terms of how basic skills deficits are identified in their work force, the impact of those deficiencies on organizational competitiveness, and why corporate programs are developed in response to the issue. Some of the key findings were as follows: (1) employee…

  7. Transcriptome-Wide Survey and Expression Profile Analysis of Putative Chrysanthemum HD-Zip I and II Genes.

    PubMed

    Song, Aiping; Li, Peiling; Xin, Jingjing; Chen, Sumei; Zhao, Kunkun; Wu, Dan; Fan, Qingqing; Gao, Tianwei; Chen, Fadi; Guan, Zhiyong

    2016-01-01

    The homeodomain-leucine zipper (HD-Zip) transcription factor family is a key transcription factor family and unique to the plant kingdom. It consists of a homeodomain and a leucine zipper that serve in combination as a dimerization motif. The family can be classified into four subfamilies, and these subfamilies participate in the development of hormones and mediation of hormone action and are involved in plant responses to environmental conditions. However, limited information on this gene family is available for the important chrysanthemum ornamental species (Chrysanthemum morifolium). Here, we characterized 17 chrysanthemum HD-Zip genes based on transcriptome sequences. Phylogenetic analyses revealed that 17 CmHB genes were distributed in the HD-Zip subfamilies I and II and identified two pairs of putative orthologous proteins in Arabidopsis and chrysanthemum and four pairs of paralogous proteins in chrysanthemum. The software MEME was used to identify 7 putative motifs with E values less than 1e-3 in the chrysanthemum HD-Zip factors, and they can be clearly classified into two groups based on the composition of the motifs. A bioinformatics analysis predicted that 8 CmHB genes could be targeted by 10 miRNA families, and the expression of these 17 genes in response to phytohormone treatments and abiotic stresses was characterized. The results presented here will promote research on the various functions of the HD-Zip gene family members in plant hormones and stress responses. PMID:27196930

  8. Transcriptome-Wide Survey and Expression Profile Analysis of Putative Chrysanthemum HD-Zip I and II Genes

    PubMed Central

    Song, Aiping; Li, Peiling; Xin, Jingjing; Chen, Sumei; Zhao, Kunkun; Wu, Dan; Fan, Qingqing; Gao, Tianwei; Chen, Fadi; Guan, Zhiyong

    2016-01-01

    The homeodomain-leucine zipper (HD-Zip) transcription factor family is a key transcription factor family and unique to the plant kingdom. It consists of a homeodomain and a leucine zipper that serve in combination as a dimerization motif. The family can be classified into four subfamilies, and these subfamilies participate in the development of hormones and mediation of hormone action and are involved in plant responses to environmental conditions. However, limited information on this gene family is available for the important chrysanthemum ornamental species (Chrysanthemum morifolium). Here, we characterized 17 chrysanthemum HD-Zip genes based on transcriptome sequences. Phylogenetic analyses revealed that 17 CmHB genes were distributed in the HD-Zip subfamilies I and II and identified two pairs of putative orthologous proteins in Arabidopsis and chrysanthemum and four pairs of paralogous proteins in chrysanthemum. The software MEME was used to identify 7 putative motifs with E values less than 1e-3 in the chrysanthemum HD-Zip factors, and they can be clearly classified into two groups based on the composition of the motifs. A bioinformatics analysis predicted that 8 CmHB genes could be targeted by 10 miRNA families, and the expression of these 17 genes in response to phytohormone treatments and abiotic stresses was characterized. The results presented here will promote research on the various functions of the HD-Zip gene family members in plant hormones and stress responses. PMID:27196930

  9. Leucine-rich glioma-inactivated protein 1 antibody encephalitis

    PubMed Central

    Mayasi, Yunis; Takhtani, Deepak

    2014-01-01

    Objective: To describe a case of leucine-rich glioma-inactivated protein 1 (LGI1) antibody–associated encephalitis. Methods: The clinical and ancillary data and brain MRIs were gathered retrospectively by chart review. Relevant literature on similar cases was also reviewed. Results: The diagnosis of LGI1 antibody–associated autoimmune encephalitis was based on the typical clinical presentation of seizures, psychiatric symptoms, and memory loss as well as negative diagnostic testing for cancer; the diagnosis was confirmed by positive LGI1 antibody. The patient responded favorably to treatment with IV immunoglobulin and continues to do well. Conclusion: LGI1 antibody–associated encephalitis has increasingly been recognized as a primary autoimmune disorder with good prognosis and response to treatment. PMID:25520958

  10. Chiral conducting surfaces via electrochemical oxidation of L-leucine-oligothiophenes.

    PubMed

    McTiernan, Christopher D; Omri, Karim; Chahma, M'hamed

    2010-09-17

    Polythiophenes bearing a specific chiral center such as L-leucine have been prepared via the electrochemical oxidation of a series of L-leucine functionalized oligothiophenes (monothiophenes and terthiophenes). These oligothiophenes have been prepared through the condensation of L-leucine methyl ester and the corresponding thiophene monomers in the presence of hydroxybenzotriazole (HOBt) and N,N'-dicyclohexylcarbodiimide (DCC) followed by hydrolysis of the esters. The electroactive polymers are electrochemically stable and exhibit excellent adhesive properties on electrode surfaces (platinum, gold, and glassy carbon) as well as interesting optical properties in both doped and undoped states. Hydrogen bonds between a free amino acid (L-leucine, D-leucine, L-alanine, D-alanine, and D/L-alanine) and the L-leucine based polythiophenes (chiral conducting surface) were probed using cyclic voltammetry. Preliminary results show that the capacitive current of a modified L-leucine-polythiophene electrode decreases as a result of the formation of a hydrogen bond barrier on the surface of the chiral conducting surface accompanied with a shift of the oxidation potential. Cyclic voltammetry responses resulting from the interaction of the chiral conducting surface with L and Dfree amino acid isomers are similar. The formation of hydrogen bonds between the chiral conducting surfaces and the free amino acids was characterized by (1)H NMR. A chemical shift was observed for the N-H group in monomer 6 as a result of the hydrogen bond formation between the L-leucine methyl ester (D-leucine methyl ester, D/L-leucine methyl ester) and monomer 6. PMID:20718451

  11. Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice

    PubMed Central

    Zhao, Yang; Dai, Xiao-yan; Zhou, Zhou; Zhao, Ge-xin; Wang, Xian; Xu, Ming-jiang

    2016-01-01

    Aim: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice. Methods: ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses. Results: Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1. Conclusion: Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation. PMID:26687933

  12. Structural basis for leucine-rich nuclear export signal recognition by CRM1

    SciTech Connect

    Dong, Xiuhua; Biswas, Anindita; Süel, Katherine E.; Jackson, Laurie K.; Martinez, Rita; Gu, Hongmei; Chook, Yuh Min

    2009-07-10

    CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9 {angstrom} structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined {alpha}-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

  13. Homeodomain-Leucine zipper II family of transcription factors to the limelight

    PubMed Central

    Carabelli, Monica; Turchi, Luana; Ruzza, Valentino; Morelli, Giorgio; Ruberti, Ida

    2013-01-01

    The Arabidopsis genome encodes 10 Homeodomain-Leucine Zipper (HD-Zip) II transcription factors that can be subdivided into 4 clades (α–δ). All the γ (ARABIDOPSIS THALIANA HOMEOBOX 2 [ATHB2], HOMEOBOX ARABIDOPSIS THALIANA 1 [HAT1], HAT2) and δ (HAT3, ATHB4) genes are regulated by light quality changes (Low Red [R]/Far-Red [FR]) that induce the shade avoidance response in most of the angiosperms. HD-Zip IIγ and HD-Zip IIδ transcription factors function as positive regulators of shade avoidance, and there is evidence that at least ATHB2 is directly positively regulated by the basic Helix-Loop-Helix (bHLH) proteins PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5. Recent evidence demonstrate that, in addition to their function in shade avoidance, HD-Zip IIγ and HD-Zip IIδ proteins play an essential role in plant development from embryogenesis onwards in a white light environment. PMID:23838958

  14. Role of Insulin in the Regulation of Leucine Kinetics in the Conscious Dog

    PubMed Central

    Abumrad, Naji N.; Jefferson, L. S.; Rannels, S. R.; Williams, P. E.; Cherrington, A. D.; Lacy, W. W.

    1982-01-01

    To study the effect of insulin on leucine kinetics, three groups of conscious dogs were studied after an overnight fast (16-18 h). One, saline-infused group (n = 5), served as control. The other two groups were infused with somatostatin and constant replacement amount of glucagon; one group (n = 6) received no insulin replacement, to produce acute insulin deficiency, and the other (n = 6) was constantly replaced with 600 μU/kg per min insulin, to produce twice basal hyperinsulinemia. Hepatic and extrahepatic splanchnic (gut) balance of leucine and α-ketoisocaproate (KIC) were calculated using the arteriovenous difference technique. l,4,5,[3H]Leucine was used to measure the rates (micromoles per kilogram per minute) of appearance (Ra) and disappearance (Rd), and clearance (Cl) of plasma leucine (milliliters per kilogram per minute). Saline infusion for 7 h resulted in isotopic steady state, where Ra and Rd were equal (3.2±0.2 μmol/kg per min). Acute insulin withdrawal of 4-h duration caused the plasma leucine to increase by 40% (P < 0.005). This change was caused by a decrease in the outflow of leucine (Cl) from the plasma, since Ra did not change. The net hepatic release of the amino acid (0.24±0.03 μmol/kg per min) did not change significantly; the arterio-deep femoral venous differences of leucine (−10±1 μmol/liter) and KIC (−12±2 μmol/liter) did not change significantly indicating net release of the amino and ketoacids across the hindlimb. Selective twice basal hyperinsulinemia resulted in a 36% drop in plasma leucine (from control levels of 128±8 to 82±7 μmol/liter, P < 0.005) within 4 h. This was accompanied by a 15% reduction in Ra and a 56% rise in clearance (P < 0.001, both). Net hepatic leucine production and net release of leucine and KIC across the hindlimb fell markedly. These studies indicate that physiologic changes in circulating insulin levels result in a differential dose-dependent effect on total body leucine metabolism in the

  15. Stimulation of skeletal muscle protein synthesis in neonatal pigs by long-term infusion of leucine is amino acid dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infusing leucine for 1 hr increases skeletal muscle protein synthesis in neonatal pigs, but this is not sustained for 2 h unless the leucine-induced fall in amino acids is prevented. We aimed to determine whether continuous leucine infusion can stimulate protein synthesis for a prolonged period whe...

  16. Stimulation of muscle protein synthesis by prolonged parenteral infusion of leucine is dependent on amino acid availability in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial rise in amino acids, particularly leucine, stimulates muscle protein synthesis in neonates. Previously, we showed that a 1-h infusion of leucine increased protein synthesis, but this response was not sustained for 2 h unless the leucine-induced decrease in amino acids was prevented....

  17. Structure and dissolution of L-leucine-coated salbutamol sulphate aerosol particles.

    PubMed

    Raula, Janne; Seppälä, Jukka; Malm, Jari; Karppinen, Maarit; Kauppinen, Esko I

    2012-06-01

    L-Leucine formed different crystalline coatings on salbutamol sulphate aerosol particles depending on the saturation conditions of L-leucine. The work emphasizes a careful characterization of powders where structural compartments such as crystal size and particle coating may affect the performance of drug when administered. The sublimation of L-leucine from the aerosol particles took place 90°C lower temperature than the bulk L-leucine which was attributed to result from the sublimation of L-leucine from nano-sized crystalline domains. The dissolution slowed down and initial dissolution rate decreased with increasing L-leucine content. Decreasing crystalline domains to nano-scale improve heat and mass transfer which was observed as the lowered decomposition temperature of the drug salbutamol sulphate and the sublimation temperature of surface material L-leucine as well as the altered dissolution characteristics of the drug. The structure of the coated drug particles was studied by means of thermal analysis techniques (DSC and TG), and the dissolution of salbutamol sulphate was studied as an on-line measurement in a diffusion cell. PMID:22562614

  18. Purification and identification of a novel leucine aminopeptidase from Bacillus thuringiensis israelensis.

    PubMed

    Cahan, Rivka; Hetzroni, Efrat; Nisnevitch, Marina; Nitzan, Yeshayahu

    2007-11-01

    A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65 degrees C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65 degrees C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase's effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin. PMID:17682820

  19. Leucine supplementation accelerates connective tissue repair of injured tibialis anterior muscle.

    PubMed

    Pereira, Marcelo G; Silva, Meiricris T; Carlassara, Eduardo O C; Gonçalves, Dawit A; Abrahamsohn, Paulo A; Kettelhut, Isis C; Moriscot, Anselmo S; Aoki, Marcelo S; Miyabara, Elen H

    2014-10-01

    This study investigated the effect of leucine supplementation on the skeletal muscle regenerative process, focusing on the remodeling of connective tissue of the fast twitch muscle tibialis anterior (TA). Young male Wistar rats were supplemented with leucine (1.35 g/kg per day); then, TA muscles from the left hind limb were cryolesioned and examined after 10 days. Although leucine supplementation induced increased protein synthesis, it was not sufficient to promote an increase in the cross-sectional area (CSA) of regenerating myofibers (p > 0.05) from TA muscles. However, leucine supplementation reduced the amount of collagen and the activation of phosphorylated transforming growth factor-β receptor type I (TβR-I) and Smad2/3 in regenerating muscles (p < 0.05). Leucine also reduced neonatal myosin heavy chain (MyHC-n) (p < 0.05), increased adult MyHC-II expression (p < 0.05) and prevented the decrease in maximum tetanic strength in regenerating TA muscles (p < 0.05). Our results suggest that leucine supplementation accelerates connective tissue repair and consequent function of regenerating TA through the attenuation of TβR-I and Smad2/3 activation. Therefore, future studies are warranted to investigate leucine supplementation as a nutritional strategy to prevent or attenuate muscle fibrosis in patients with several muscle diseases. PMID:25268835

  20. Leucine Supplementation Accelerates Connective Tissue Repair of Injured Tibialis Anterior Muscle

    PubMed Central

    Pereira, Marcelo G.; Silva, Meiricris T.; Carlassara, Eduardo O. C.; Gonçalves, Dawit A.; Abrahamsohn, Paulo A.; Kettelhut, Isis C.; Moriscot, Anselmo S.; Aoki, Marcelo S.; Miyabara, Elen H.

    2014-01-01

    This study investigated the effect of leucine supplementation on the skeletal muscle regenerative process, focusing on the remodeling of connective tissue of the fast twitch muscle tibialis anterior (TA). Young male Wistar rats were supplemented with leucine (1.35 g/kg per day); then, TA muscles from the left hind limb were cryolesioned and examined after 10 days. Although leucine supplementation induced increased protein synthesis, it was not sufficient to promote an increase in the cross-sectional area (CSA) of regenerating myofibers (p > 0.05) from TA muscles. However, leucine supplementation reduced the amount of collagen and the activation of phosphorylated transforming growth factor-β receptor type I (TβR-I) and Smad2/3 in regenerating muscles (p < 0.05). Leucine also reduced neonatal myosin heavy chain (MyHC-n) (p < 0.05), increased adult MyHC-II expression (p < 0.05) and prevented the decrease in maximum tetanic strength in regenerating TA muscles (p < 0.05). Our results suggest that leucine supplementation accelerates connective tissue repair and consequent function of regenerating TA through the attenuation of TβR-I and Smad2/3 activation. Therefore, future studies are warranted to investigate leucine supplementation as a nutritional strategy to prevent or attenuate muscle fibrosis in patients with several muscle diseases. PMID:25268835

  1. Sunspace basics

    SciTech Connect

    Not Available

    1994-11-01

    Anyone who lives in a home with a sunspace will tell you that the sunspace is the most enjoyable room in the house. Many times the homeowner`s only regret is that the sunspace is not larger. Although aesthetics often drive the decision to add a sunspace or include one in a new home design, sunspaces can also provide supplemental space heating and a healthy environment for plants and people. In fact, a well-designed sunspace can provide up to 60% of a home`s winter heating requirements. This publication addresses basic elements of sunspace design; design considerations for supplemental space heating, growing plants, and use as a living space; design guidelines including siting, heat distribution, and glazing angles; and major sunspace components including glazing options, thermal mass, insulation, and climate controls. A list of sources for more information is also provided.

  2. Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms.

    PubMed

    Ahmad, Aqeel; Azmi, Sarfuddin; Srivastava, Saurabh; Kumar, Amit; Tripathi, Jitendra Kumar; Mishra, Nripendra N; Shukla, Praveen K; Ghosh, Jimut Kanti

    2014-11-01

    Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at 'a' and/or 'd' position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its 'a' and 'd' positions with D-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its D-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show

  3. Some non-anomerically C-C-linked carbohydrate amino acids related to leucine-synthesis and structure determination.

    PubMed

    Steiner, Bohumil; Micová, Júlia; Koós, Miroslav; Langer, Vratislav; Gyepesová, Dalma

    2003-06-23

    (5'R)-5'-Isobutyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione was synthesised starting from methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside via methyl 6-deoxy-6-isopropyl-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose applying the Bucherer-Bergs reaction. Its 5'-R configuration was confirmed by X-ray crystallography. Corresponding alpha-amino acid-methyl (5R)-5-amino-5-C-carboxy-5,6-dideoxy-6-isopropyl-alpha-D-lyxo-hexofuranoside (alternative name: 2-[methyl (4R)-beta-L-erythrofuranosid-4-C-yl]-D-leucine) was obtained from the above hydantoin by acid hydrolysis of the isopropylidene group followed by basic hydrolysis of the hydantoin ring. Analogous derivatives with 5S configuration, formed in a minority, were also isolated and characterised. PMID:12801708

  4. Homeodomain-Leucine Zipper II family of transcription factors to the limelight: central regulators of plant development.

    PubMed

    Carabelli, Monica; Turchi, Luana; Ruzza, Valentino; Morelli, Giorgio; Ruberti, Ida

    2013-09-01

    The Arabidopsis genome encodes 10 Homeodomain-Leucine Zipper (HD-Zip) II transcription factors that can be subdivided into 4 clades (α-δ). All the γ (ARABIDOPSIS THALIANA HOMEOBOX 2 [ATHB2], HOMEOBOX ARABIDOPSIS THALIANA 1 [HAT1], HAT2) and δ (HAT3, ATHB4) genes are regulated by light quality changes (Low Red [R]/Far-Red [FR]) that induce the shade avoidance response in most of the angiosperms. HD-Zip IIγ and HD-Zip IIδ transcription factors function as positive regulators of shade avoidance, and there is evidence that at least ATHB2 is directly positively regulated by the basic Helix-Loop-Helix (bHLH) proteins PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5. Recent evidence demonstrate that, in addition to their function in shade avoidance, HD-Zip IIγ and HD-Zip IIδ proteins play an essential role in plant development from embryogenesis onwards in a white light environment. PMID:23838958

  5. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    PubMed

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching. PMID:25731909

  6. Determination of the safety of leucine supplementation in healthy elderly men.

    PubMed

    Rasmussen, Betina; Gilbert, Erin; Turki, Abrar; Madden, Kenneth; Elango, Rajavel

    2016-07-01

    Leucine, a branched-chain amino acid (BCAA), has been shown to stimulate muscle protein synthesis, and thus has been proposed to prevent age-related muscle atrophy (sarcopenia). Therefore, leucine supplementation may have potential benefits in elderly populations to preserve muscle mass. The tolerable upper intake level (UL) for leucine intake in young men has recently been determined to be 500 mg kg(-1) day(-1), and increases in blood ammonia concentrations were seen at intake levels above 500 mg kg(-1) day(-1); the UL for leucine in elderly is unknown. The objective of the current study was to determine the safety of leucine supplementation in healthy elderly men. Six healthy elderly men (72.2 ± 3.5 years) received graded stepwise increases in leucine intakes ranging from 50 to 750 mg kg(-1) day(-1), on eight separate study days. Plasma and urinary biochemical variables, including blood ammonia, and an oral primed-continuous protocol of L-1-(13)C-Leucine was performed. Blood ammonia concentrations above normal values (35 µmol/L) were observed at leucine intakes >550 mg kg(-1) day(-1). Leucine oxidation measured as a F(13)CO2 (rate of label tracer oxidation) increased with increasing leucine intakes and started to plateau after 450 mg kg(-1) day(-1). Two-phased linear regression analysis of the F(13)CO2 data revealed a breakpoint of 431 mg kg(-1) day(-1) (R (2) = 0.73), suggesting that the upper limit to oxidize leucine was reached at that point. Taking the data together the upper limit for leucine intake in healthy elderly could be set similar to young men at 500 mg kg(-1) day(-1) or ~35 g/day for an individual weighing 70 kg. PMID:27138628

  7. Transactivation and transformation by Myb are negatively regulated by a leucine-zipper structure.

    PubMed Central

    Kanei-Ishii, C; MacMillan, E M; Nomura, T; Sarai, A; Ramsay, R G; Aimoto, S; Ishii, S; Gonda, T J

    1992-01-01

    The negative regulatory domain of the c-myb protooncogene product (c-Myb) normally represses transcriptional activation by c-Myb. We show here that a leucine-zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. We also demonstrate that this leucine-zipper structure can interact with cellular proteins. Our results suggest that an inhibitor that suppresses transactivation binds to c-Myb through the leucine zipper and that c-Myb can be oncogenically activated by missense mutation. Images PMID:1557416

  8. Leucine and Protein Metabolism in Obese Zucker Rats

    PubMed Central

    She, Pengxiang; Olson, Kristine C.; Kadota, Yoshihiro; Inukai, Ayami; Shimomura, Yoshiharu; Hoppel, Charles L.; Adams, Sean H.; Kawamata, Yasuko; Matsumoto, Hideki; Sakai, Ryosei; Lang, Charles H.; Lynch, Christopher J.

    2013-01-01

    Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-14C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (−21–24%). Plasma BCAAs and BCKAs were elevated 45–69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (−47–66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23–29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193–418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and

  9. In vitro transcriptional studies of the bkd operon of Pseudomonas putida: L-branched-chain amino acids and D-leucine are the inducers.

    PubMed

    Madhusudhan, K T; Luo, J; Sokatch, J R

    1999-05-01

    BkdR is the transcriptional activator of the bkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5' region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative rho-independent terminator of the bkd operon. Substrate DNA, BkdR, and any of the L-branched-chain amino acids or D-leucine was required for transcription. Branched-chain keto acids, D-valine, and D-isoleucine did not promote transcription. Therefore, the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon. The concentration of L-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkd operon by BkdR during enzyme induction which incorporates these results is presented. PMID:10217783

  10. Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

    PubMed

    Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

    2014-09-26

    We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. PMID:25148941

  11. LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes.

    PubMed Central

    Friden, P; Schimmel, P

    1987-01-01

    Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine. We report here an analysis of LEU3, a putative leucine-specific regulatory locus. We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3. We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids. A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region. The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation. We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2. Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1. Images PMID:2823102

  12. Inflation Basics

    SciTech Connect

    Green, Dan

    2014-03-01

    metrical fluctuations, both scalar and tensor, are also produced in inflationary models. Thus, the time appears to be appropriate for a very basic and simple exposition of the inflationary model written from a particle physics perspective. Only the simplest scalar model will be explored because it is easy to understand and contains all the basic elements of the inflationary model.

  13. Escherichia coli Lrp (Leucine-Responsive Regulatory Protein) Does Not Directly Regulate Expression of the leu Operon Promoter

    PubMed Central

    Landgraf, Jeffrey R.; Boxer, Jonathan A.; Calvo, Joseph M.

    1999-01-01

    Studies by R. Lin et al. (J. Bacteriol. 174:1948–1955, 1992) suggested that the Escherichia coli leu operon might be a member of the Lrp regulon. Their results were obtained with a leucine auxotroph; in leucine prototrophs grown in a medium lacking leucine, there was little difference in leu operon expression between lrp+ and lrp strains. Furthermore, when leuP-lacZ transcriptional fusions that lacked the leu attenuator were used, expression from the leu promoter varied less than twofold between lrp+ and lrp strains, irrespective of whether or not excess leucine was added to the medium. The simplest explanation of the observations of Lin et al. is that the known elevated leucine transport capacity of lrp strains (S. A. Haney et al., J. Bacteriol. 174:108–115, 1992) leads to very high intracellular levels of leucine for strains grown with leucine, resulting in the superattenuation of leu operon expression. PMID:10515950

  14. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    SciTech Connect

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-04-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA.

  15. Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

    PubMed

    Miras, Isabelle; Saul, Frederick; Nowakowski, Mireille; Weber, Patrick; Haouz, Ahmed; Shepard, William; Picardeau, Mathieu

    2015-06-01

    Pathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria. PMID:26057675

  16. Characterization of a novel anther-specific gene encoding a leucine-rich repeat protein in petunia.

    PubMed

    Yue, Y Z; Sun, J; Huang, X; Peng, H; Liu, G F; Hu, H R

    2014-01-01

    In Petunia x hybrida 'Fantasy Red', a leucine-rich repeat (LRR) gene referred to as PhLRR, was identified in a flower bud cDNA library. The open reading frame sequence of PhLRR was 1251 bp, encoding a putative 46.2-kDa protein of 416 amino acids. The PhLRR protein showed high similarity to members of polygalacturonase inhibitor proteins (PGIPs), contained 11 conserved LRR domains, and was an extracellular localization protein. Phylogenetic analysis showed that PhLRR belonged to the same PGIPs subfamily as SHY, indicating that PhLRR may be involved in the development of pollen-like SHY. Expression analysis revealed that PhLRR was abundantly expressed during early stages of flower bud and anther development, while it was not detected in any other examined organs, such as sepals, petals, pistils, roots, stems, leaves, or open flowers. Furthermore, many cis-acting elements (such as AGAAA and GTGA) related to anther-specific gene expression were identified in the PhLRR gene promoter region, indicating that the promoter is also anther-specific. These results suggested that PhLRR is a novel anther-specific gene that may be essential for the early development of anthers. PMID:25501199

  17. The Protein Synthesis Inhibitor Blasticidin S Enters Mammalian Cells via Leucine-rich Repeat-containing Protein 8D

    PubMed Central

    Lee, Clarissa C.; Freinkman, Elizaveta; Sabatini, David M.; Ploegh, Hidde L.

    2014-01-01

    Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized, and no specific function has been assigned to them. There is no consensus on how this family of proteins might function because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes. PMID:24782309

  18. Leptin Signaling Is Required for Leucine Deprivation-enhanced Energy Expenditure*

    PubMed Central

    Zhang, Qian; Liu, Bin; Cheng, Ying; Meng, Qingshu; Xia, Tingting; Jiang, Lei; Chen, Shanghai; Liu, Yong; Guo, Feifan

    2014-01-01

    Leptin signaling in the hypothalamus is crucial in energy homeostasis. We have previously shown that dietary deprivation of the essential amino acid leucine in mice stimulates fat loss by increasing energy expenditure. The involvement of leptin signaling in this regulation, however, has not been reported. Here, we show that leucine deprivation promotes leptin signaling in mice maintained on an otherwise normal diet and restores leptin responses in mice maintained on a high fat diet, a regimen known to induce leptin resistance. In addition, we found that leucine deprivation stimulated energy expenditure, and fat loss was largely blocked in db/db mice homozygous for a mutation in leptin receptor and a knock-in mouse line Y3F with abrogation of leptin receptor Tyr1138-mediated signal transducer and activator transcript 3 signaling. Overall, our studies describe a novel link between hypothalamic leptin signaling and stimulation of energy expenditure under leucine deprivation. PMID:24302741

  19. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  20. Differential Assimilation of Inorganic Carbon and Leucine by Prochlorococcus in the Oligotrophic North Pacific Subtropical Gyre.

    PubMed

    Björkman, Karin M; Church, Matthew J; Doggett, Joseph K; Karl, David M

    2015-01-01

    The light effect on photoheterotrophic processes in Prochlorococcus, and primary and bacterial productivity in the oligotrophic North Pacific Subtropical Gyre was investigated using (14)C-bicarbonate and (3)H-leucine. Light and dark incubation experiments were conducted in situ throughout the euphotic zone (0-175 m) on nine expeditions to Station ALOHA over a 3-year period. Photosynthetrons were also used to elucidate rate responses in leucine and inorganic carbon assimilation as a function of light intensity. Taxonomic group and cell-specific rates were assessed using flow cytometric sorting. The light:dark assimilation rate ratios of leucine in the top 150 m were ∼7:1 for Prochlorococcus, whereas the light:dark ratios for the non-pigmented bacteria (NPB) were not significant different from 1:1. Prochlorococcus assimilated leucine in the dark at per cell rates similar to the NPB, with a contribution to the total community bacterial production, integrated over the euphotic zone, of approximately 20% in the dark and 60% in the light. Depth-resolved primary productivity and leucine incorporation showed that the ratio of Prochlorococcus leucine:primary production peaked at 100 m then declined steeply below the deep chlorophyll maximum (DCM). The photosynthetron experiments revealed that, for Prochlorococcus at the DCM, the saturating irradiance (E k) for leucine incorporation was reached at approximately half the light intensity required for light saturation of (14)C-bicarbonate assimilation. Additionally, high and low red fluorescing Prochlorococcus populations (HRF and LRF), co-occurring at the DCM, had similar E k values for their respective substrates, however, maximum assimilation rates, for both leucine and inorganic carbon, were two times greater for HRF cells. Our results show that Prochlorococcus contributes significantly to bacterial production estimates using (3)H-leucine, whether or not the incubations are conducted in the dark or light, and this should

  1. Differential Assimilation of Inorganic Carbon and Leucine by Prochlorococcus in the Oligotrophic North Pacific Subtropical Gyre

    PubMed Central

    Björkman, Karin M.; Church, Matthew J.; Doggett, Joseph K.; Karl, David M.

    2015-01-01

    The light effect on photoheterotrophic processes in Prochlorococcus, and primary and bacterial productivity in the oligotrophic North Pacific Subtropical Gyre was investigated using 14C-bicarbonate and 3H-leucine. Light and dark incubation experiments were conducted in situ throughout the euphotic zone (0–175 m) on nine expeditions to Station ALOHA over a 3-year period. Photosynthetrons were also used to elucidate rate responses in leucine and inorganic carbon assimilation as a function of light intensity. Taxonomic group and cell-specific rates were assessed using flow cytometric sorting. The light:dark assimilation rate ratios of leucine in the top 150 m were ∼7:1 for Prochlorococcus, whereas the light:dark ratios for the non-pigmented bacteria (NPB) were not significant different from 1:1. Prochlorococcus assimilated leucine in the dark at per cell rates similar to the NPB, with a contribution to the total community bacterial production, integrated over the euphotic zone, of approximately 20% in the dark and 60% in the light. Depth-resolved primary productivity and leucine incorporation showed that the ratio of Prochlorococcus leucine:primary production peaked at 100 m then declined steeply below the deep chlorophyll maximum (DCM). The photosynthetron experiments revealed that, for Prochlorococcus at the DCM, the saturating irradiance (Ek) for leucine incorporation was reached at approximately half the light intensity required for light saturation of 14C-bicarbonate assimilation. Additionally, high and low red fluorescing Prochlorococcus populations (HRF and LRF), co-occurring at the DCM, had similar Ek values for their respective substrates, however, maximum assimilation rates, for both leucine and inorganic carbon, were two times greater for HRF cells. Our results show that Prochlorococcus contributes significantly to bacterial production estimates using 3H-leucine, whether or not the incubations are conducted in the dark or light, and this should be

  2. Leucine metabolism in rat liver after a bolus injection of endotoxin.

    PubMed

    Holecek, M; Sprongl, L; Tichý, M; Pecka, M

    1998-06-01

    To evaluate the contribution of hepatic tissue to alterations in the metabolism of proteins and the branched-chain amino acids (BCAA) leucine, isoleucine, and valine in systemic inflammatory response syndrome, we studied the changes of leucine metabolism in isolated perfused liver (IPL) of endotoxin-treated rats. Male albino rats were injected with the endotoxin of Salmonella enteritidis (5 mg x kg(-1)) or saline (control). Four hours later, leucine and ketoisocaproate (KIC) oxidation and incorporation into liver proteins were determined in IPL using the single-pass liver perfusion technique. L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproic acid were used as a tracer in two separate experiments. Endotoxin treatment resulted in a decrease of plasma BCAA levels, an increase of leucine oxidation, and a decrease of KIC oxidation by IPL. Leucine incorporation into liver proteins was lower in endotoxin-treated rats, and we did not find measurable incorporation of the labeled carbon of KIC in liver proteins in either group of animals. The sum of individual amino acid concentrations in the effluent perfusate was higher in endotoxin-treated animals, although only leucine and phenylalanine increased significantly. The decrease in KIC oxidation indicates a decreased capacity of hepatic tissue to oxidize branched-chain ketoacids (BCKA). The increase in leucine oxidation by IPL of endotoxin-treated rats indicates an increase in BCAA aminotransferase activity. These changes demonstrate an important response of the body that enables the resynthesis of essential BCAA from their ketoanalogs delivered to the liver from peripheral tissues, particularly muscle. PMID:9627366

  3. Relationships between leucine and the pancreatic exocrine function for improving starch digestibility in ruminants.

    PubMed

    Liu, K; Liu, Y; Liu, S M; Xu, M; Yu, Z P; Wang, X; Cao, Y C; Yao, J H

    2015-04-01

    Four Holstein heifers (215 ± 7 kg; means ± SD), fitted with one pancreatic pouch, duodenal re-entrant cannulas, and duodenal infusion catheters, were used in this experiment. In phase 1, the 24-h profile of pancreatic fluid was determined. Pancreatic fluid flow peaked 1h after feeding, but peaks of similar magnitude also occurred before the morning feed, necessitating 24-h collection of pancreatic fluid to estimate daily excretion. In phase 2, the effects of duodenal infusions of 0, 10, 20, or 30 g of leucine on pancreatic fluid flow were determined in a 4 × 4 Latin square design. The leucine was infused for 12h in 2,500 mL of the infusate, and samples of pancreatic fluid and jugular blood were collected in 1-h intervals from the beginning of the infusion for 36 h. The results showed that the secretion rate of pancreatic fluid (mL/h) was significantly higher in 10-g leucine group than the other groups (mL/h). Protein concentration (mg/mL) in pancreatic fluid was elevated proportional to the amount of leucine infused. Leucine infusions increased both the concentration (U/mL) and secretion rate (U/h) of α-amylase. Infusion of 10 g of leucine also increased the secretion rates (U/h) of trypsin, chymotrypsin, and lipase, but did not change their concentrations. No significant effects of leucine infusions on plasma glucose and insulin concentrations were found. The results indicate that leucine could act as a nutrient signal to stimulate α-amylase production and pancreatic exocrine function in dairy heifers. PMID:25648818

  4. Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.

    PubMed

    Kimball, Scot R; Gordon, Bradley S; Moyer, Jenna E; Dennis, Michael D; Jefferson, Leonard S

    2016-08-01

    The studies described herein were designed to explore the role of Sestrin2 in mediating the selective action of leucine to activate mTORC1. The results demonstrate that Sestrin2 is a phosphoprotein and that its phosphorylation state is responsive to the availability of leucine, but not other essential amino acids. Moreover, leucine availability-induced alterations in Sestrin2 phosphorylation correlated temporally and dose dependently with the activation state of mTORC1, there being a reciprocal relationship between the degree of phosphorylation of Sestrin2 and the extent of repression of mTORC1. With leucine deprivation, Sestrin2 became more highly phosphorylated and interacted more strongly with proteins of the GATOR2 complex. Notably, in cells lacking the protein kinase ULK1, the activation state of mTORC1 was elevated in leucine-deficient medium, such that the effect of re-addition of the amino acid was blunted. In contrast, overexpression of ULK1 led to hyperphosphorylation of Sestrin2 and enhanced its interaction with GATOR2. Neither rapamycin nor Torin2 had any effect on Sestrin2 phosphorylation, suggesting that leucine deprivation-induced repression of mTORC1 was not responsible for the action of ULK1 on Sestrin2. Mass spectrometry analysis of Sestrin2 revealed three phosphorylation sites that are conserved across mammalian species. Mutation of the three sites to phospho-mimetic amino acids in exogenously expressed Sestrin2 promoted its interaction with GATOR2 and dramatically repressed mTORC1 even in the presence of leucine. Overall, the results support a model in which leucine selectively promotes dephosphorylation of Sestrin2, causing it to dissociate from and thereby activate GATOR2, leading to activation of mTORC1. PMID:27010498

  5. Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol.

    PubMed

    Takagi, Hiroshi; Hashida, Keisuke; Watanabe, Daisuke; Nasuno, Ryo; Ohashi, Masataka; Iha, Tomoya; Nezuo, Maiko; Tsukahara, Masatoshi

    2015-02-01

    Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor. PMID:25060730

  6. Sestrin2 is a leucine sensor for the mTORC1 pathway.

    PubMed

    Wolfson, Rachel L; Chantranupong, Lynne; Saxton, Robert A; Shen, Kuang; Scaria, Sonia M; Cantor, Jason R; Sabatini, David M

    2016-01-01

    Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, aGTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. PMID:26449471

  7. Sestrin2 is a leucine sensor for the mTORC1 pathway

    PubMed Central

    Wolfson, Rachel L.; Chantranupong, Lynne; Saxton, Robert A.; Shen, Kuang; Scaria, Sonia M.; Cantor, Jason R.; Sabatini, David M.

    2015-01-01

    Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, a GTPase activating protein (GAP); GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a Kd of 20 µM, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. PMID:26449471

  8. Diurnal variation in glucose and leucine metabolism in non-insulin-dependent diabetes.

    PubMed

    Umpleby, A M; Scobie, I N; Boroujerdi, M A; Carson, E R; Sonksen, P H

    1990-04-01

    Glucose and leucine metabolism were investigated in 5 poorly controlled non-insulin-dependent diabetics (NIDDM) following an i.v. injection of 3-[3H]glucose and 1-[14C]leucine in the morning and evening. In the morning glucose concentration (11.2 +/- 0.8 mmol/l) (mean +/- SEM) and production rate (14.2 +/- 1.3 mumol/min/kg) were significantly greater (P less than 0.001, P less than 0.05) and glucose metabolic clearance rate (MCR) (1.3 +/- 0.2 ml/min/kg) significantly lower (P less than 0.05) than in a group of control subjects. Glucose concentration was lower in the evening (P less than 0.05) as a result of a decrease in glucose production rate (P less than 0.05). Leucine concentration and production rate were not significantly different from normal but leucine oxidation rate was increased (P less than 0.05). There was no diurnal variation in leucine metabolism. Since leucine production is a measure of protein breakdown, the higher morning glucose production rate was not due to an increased supply of gluconeogenic precursors from protein catabolism. PMID:2190784

  9. Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins.

    PubMed

    Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P; Zhao, Zhendong; Wang, Yejun

    2016-08-01

    Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

  10. Magnetism and the putative early Martian life

    NASA Astrophysics Data System (ADS)

    Rochette, P.

    2001-08-01

    A short critical review is provided on three questions linking magnetism and the putative early Mars life. Was there a large internal Martian magnetic field, during which period, and is it a requisite for life? What is the origin of the paleomagnetic signal of Martian meteorites, including ALH84001? What is the present credibility of the case for fossil bacterial magnetite grains in ALH84001?

  11. Protein Ingestion Induces Muscle Insulin Resistance Independent of Leucine-Mediated mTOR Activation

    PubMed Central

    Smith, Gordon I.; Yoshino, Jun; Stromsdorfer, Kelly L.; Klein, Seth J.; Magkos, Faidon; Reeds, Dominic N.; Klein, Samuel

    2015-01-01

    Increased plasma branched-chain amino acid concentrations are associated with insulin resistance, and intravenous amino acid infusion blunts insulin-mediated glucose disposal. We tested the hypothesis that protein ingestion impairs insulin-mediated glucose disposal by leucine-mediated mTOR signaling, which can inhibit AKT. We measured glucose disposal and muscle p-mTORSer2448, p-AKTSer473, and p-AKTThr308 in 22 women during a hyperinsulinemic-euglycemic clamp procedure with and without concomitant ingestion of whey protein (0.6 g/kg fat-free mass; n = 11) or leucine that matched the amount given with whey protein (n = 11). Both whey protein and leucine ingestion raised plasma leucine concentration by approximately twofold and muscle p-mTORSer2448 by ∼30% above the values observed in the control (no amino acid ingestion) studies; p-AKTSer473 and p-AKTThr308 were not affected by whey protein or leucine ingestion. Whey protein ingestion decreased insulin-mediated glucose disposal (median 38.8 [quartiles 30.8, 61.8] vs. 51.9 [41.0, 77.3] µmol glucose/µU insulin · mL−1 · min−1; P < 0.01), whereas ingestion of leucine did not (52.3 [43.3, 65.4] vs. 52.3 [43.9, 73.2]). These results indicate that 1) protein ingestion causes insulin resistance and could be an important regulator of postprandial glucose homeostasis and 2) the insulin-desensitizing effect of protein ingestion is not due to inhibition of AKT by leucine-mediated mTOR signaling. PMID:25475435

  12. The mechanism of transfer for L-leucine into the vascular bed of the Anuran small intestine.

    PubMed Central

    Cheeseman, C I

    1981-01-01

    1. The vascularly perfused small intestine of Rana pipiens was used to investigate the movement of the amino acid L-leucine from the epithelium into the vascular bed. It was found that only a few amino acids when present in the lumen inhibited the wash-out leucine into the vascular bed. The series of amino acids which had this effect belonged to the group previously shown to be transported by 'L-type' carrier systems. 2. Nearly all amino acids when present in the lumen accelerated the flux of leucine from the vascular bed to the lumen and there was little correlation between the amino acids which caused this effect and those which inhibited leucine wash-out into the vascular bed. Replacement of luminal sodium also promoted serosal-to-mucosal leucine flux. 3. The effect of the presence of amino acids in the lumen on the uptake of leucine from the vascular bed was measured using a fractional extraction technique; sucrose was the extracellular marker. There was complete correlation between the amino acids which promoted the extraction of leucine from the vascular bed and those which inhibited leucine wash-out into the vascular bed. 4. In contrast, the wash-out of leucine into the vascular bed was not accelerated by the addition of amino acids to the vascular perfusate, and the presence of 10 mM-leucine in the vascular bed had very little effect upon the mucosal-to-serosal flux of leucine. 5. These results are discussed with regard to the specificity of an exit system for leucine, in the intestinal epithelium, which appears to have an energy requirement. PMID:6975822

  13. Phosphorylation of 4EBP by oral leucine administration was suppressed in the skeletal muscle of PGC-1α knockout mice.

    PubMed

    Yoshimura, Ryoji; Minami, Kimiko; Matsuda, Junichiro; Sawada, Naoki; Miura, Shinji; Kamei, Yasutomi

    2016-01-01

    Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis. PMID:26745679

  14. Reviewing the Effects of l-Leucine Supplementation in the Regulation of Food Intake, Energy Balance, and Glucose Homeostasis

    PubMed Central

    Pedroso, João A.B.; Zampieri, Thais T.; Donato, Jose

    2015-01-01

    Leucine is a well-known activator of the mammalian target of rapamycin (mTOR). Because mTOR signaling regulates several aspects of metabolism, the potential of leucine as a dietary supplement for treating obesity and diabetes mellitus has been investigated. The objective of the present review was to summarize and discuss the available evidence regarding the mechanisms and the effects of leucine supplementation on the regulation of food intake, energy balance, and glucose homeostasis. Based on the available evidence, we conclude that although central leucine injection decreases food intake, this effect is not well reproduced when leucine is provided as a dietary supplement. Consequently, no robust evidence indicates that oral leucine supplementation significantly affects food intake, although several studies have shown that leucine supplementation may help to decrease body adiposity in specific conditions. However, more studies are necessary to assess the effects of leucine supplementation in already-obese subjects. Finally, although several studies have found that leucine supplementation improves glucose homeostasis, the underlying mechanisms involved in these potential beneficial effects remain unknown and may be partially dependent on weight loss. PMID:26007339

  15. IGFBP-1 hyperphosphorylation in response to leucine deprivation is mediated by the AAR pathway.

    PubMed

    Malkani, Niyati; Jansson, Thomas; Gupta, Madhulika B

    2015-09-01

    Insulin-like growth factor-1 (IGF-I) is the key regulator of fetal growth. IGF-I bioavailability is markedly diminished by IGF binding protein-1 (IGFBP-1) phosphorylation. Leucine deprivation strongly induces IGFBP-1 hyperphosphorylation, and plays an important role in fetal growth restriction (FGR). FGR is characterized by decreased amino acid availability, which activates the amino acid response (AAR) and inhibits the mechanistic target of rapamycin (mTOR) pathway. We investigated the role of AAR and mTOR in mediating IGFBP-1 secretion and phosphorylation in HepG2 cells in leucine deprivation. mTOR inhibition (rapamycin or raptor + rictor siRNA), or activation (DEPTOR siRNA) demonstrated a role of mTOR in leucine deprivation-induced IGFBP-1 secretion but not phosphorylation. When the AAR was blocked (U0126, or ERK/GCN2 siRNA), both IGFBP-1 secretion and hyperphosphorylation (pSer101/pSer119/pSer169) due to leucine deprivation were prevented. CK2 inhibition by TBB also attenuated IGFBP-1 phosphorylation in leucine deprivation. These results suggest that the AAR and mTOR independently regulate IGFBP-1 secretion and phosphorylation in response to decreased amino acid availability. PMID:25957086

  16. Leucine Loading Test is Only Discriminative for 3-Methylglutaconic Aciduria Due to AUH Defect.

    PubMed

    Wortmann, Saskia B; Kluijtmans, Leo A J; Sequeira, Silvia; Wevers, Ron A; Morava, Eva

    2014-01-01

    Currently, six inborn errors of metabolism with 3-methylglutaconic aciduria as discriminative feature are known. The "Primary 3-methylglutaconic aciduria," 3-methylglutaconyl-CoA hydratase deficiency or AUH defect, is a disorder of leucine catabolism. For all other subtypes, also denoted "Secondary 3-methylglutaconic acidurias" (TAZ defect or Barth syndrome, SERAC1 defect or MEGDEL syndrome, OPA3 defect or Costeff syndrome, DNAJC19 defect or DCMA syndrome, TMEM70 defect, "not otherwise specified (NOS) 3-MGA-uria"), the origin of 3-methylglutaconic aciduria remains enigmatic but is hypothesized to be independent from leucine catabolism. Here we show the results of leucine loading test in 21 patients with different inborn errors of metabolism who present with 3-methylglutaconic aciduria. After leucine loading urinary 3-methylglutaconic acid levels increased only in the patients with an AUH defect. This strongly supports the hypothesis that 3-methylglutaconic aciduria is independent from leucine breakdown in other inborn errors of metabolism with 3-methylglutaconic aciduria and also provides a simple test to discriminate between primary and secondary 3-methylglutaconic aciduria in regular patient care. PMID:24757000

  17. Reduction in plasma leucine after sprint exercise is greater in males than in females.

    PubMed

    Esbjörnsson, M; Rooyackers, O; Norman, B; Rundqvist, H C; Nowak, J; Bülow, J; Simonsen, L; Jansson, E

    2012-06-01

    There is a pronounced gender difference in the accumulation of plasma ammonia after sprint exercise. Ammonia is a key intermediate in amino acid metabolism, which implies that gender-related differences in plasma and muscle amino acid concentrations after sprint exercise exist. To study this, three bouts of 30-s sprint exercise were performed by healthy females (n=8) and males (n=6). Blood leucine and muscle leucine were collected over the exercise period. Basal arterial plasma and skeletal muscle leucine were 40% higher in males than females (P<0.010 and P<0.020). Plasma, but not muscle, leucine decreased by sprint exercise and more so in males than females (g × t: P=0.025). Increase in ammonia was higher in males than females in both plasma and muscle (g × t: P<0.001 and P=0.003). An opposite pattern was shown for plasma glutamine, where an increase was found in females (P<0.001), but not in males. In conclusion, the lower plasma ammonia after sprint exercise in females seems to be explained by a lower accumulation of ammonia in skeletal muscle and by a buffering of ammonia in the form of glutamine in females. The greater reduction in plasma leucine in males seems to be related to their greater increase in muscle ammonia after sprint exercise. PMID:22612362

  18. Incorporation and release of tritiated leucine in rat prostate during castration induced involution

    SciTech Connect

    Bockrath, J.M.; Lee, C.; Grayhack, J.T.

    1981-11-01

    Rates of 3H-leucine incorporation into and release from the ventral prostate during castration induced involution were studied in adult male rats. We measured the rate of 3H-leucine incorporation by incubating the prostatic tissue in medium 199 containing 3H-leucine at 37 C for 1 hour. The rate of radioactivity incorporated into the protein fraction was expressed as cpm mg of protein. This rate reduced linearly from Day 0 to Day 6 post castration. Subcutaneous implantation of a silastic capsule containing crystalline testosterone to castrated rats restored the rate of incorporation to that of sham operated rats. To study the rate of 3H-leucine release, 3H-leucine prepared in 0.9 per cent Na C1 solution was injected intravenously into rats 1 day before castration. The amount of radioactivity remaining in the protein fraction of the prostate, expressed as cpm per prostate, was measured at different intervals after castration or after sham operation. Radioactivity disappeared at a significantly faster rate in the prostate of castrated rats than in sham operated controls. Testosterone replacement to castrated rats delayed the rate of loss of radioactivity to a degree similar to that of sham operated rats. These findings indicate that the rapid rate of protein loss in the regressing prostate is the result of a combined action of an accelerated rate of protein degradation and a rate of protein synthesis. Testosterone administration reversed these patterns of protein metabolism.

  19. Functional Profiling Discovers the Dieldrin Organochlorinated Pesticide Affects Leucine Availability in Yeast

    PubMed Central

    Vulpe, Chris D.

    2013-01-01

    Exposure to organochlorinated pesticides such as dieldrin has been linked to Parkinson’s and Alzheimer’s diseases, endocrine disruption, and cancer, but the cellular and molecular mechanisms of toxicity behind these effects remain largely unknown. Here we demonstrate, using a functional genomics approach in the model eukaryote Saccharomyces cerevisiae, that dieldrin alters leucine availability. This model is supported by multiple lines of congruent evidence: (1) mutants defective in amino acid signaling or transport are sensitive to dieldrin, which is reversed by the addition of exogenous leucine; (2) dieldrin sensitivity of wild-type or mutant strains is dependent upon leucine concentration in the media; (3) overexpression of proteins that increase intracellular leucine confer resistance to dieldrin; (4) leucine uptake is inhibited in the presence of dieldrin; and (5) dieldrin induces the amino acid starvation response. Additionally, we demonstrate that appropriate negative regulation of the Ras/protein kinase A pathway, along with an intact pyruvate dehydrogenase complex, is required for dieldrin tolerance. Many yeast genes described in this study have human orthologs that may modulate dieldrin toxicity in humans. PMID:23358190

  20. Amperometric bienzyme screen-printed biosensor for the determination of leucine.

    PubMed

    Labroo, Pratima; Cui, Yue

    2014-01-01

    Leucine plays an important role in protein synthesis, brain functions, building muscle mass, and helping the body when it undergoes stress. Here, we report a new amperometric bienzyme screen-printed biosensor for the determination of leucine, by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and leucine dehydrogenase (LDH) on a screen-printed electrode with NADP(+) and p-hydroxybenzoate as the cofactors. The detection principle of the sensor is that LDH catalyzes the specific dehydrogenation of leucine by using NADP(+) as a cofactor. The product, NADPH, triggers the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a change in electron concentration at the working carbon electrode, which is detected by the potentiostat. The sensor shows a linear detection range between 10 and 600 μM with a detection limit of 2 μM. The response is reproducible and has a fast measuring time of 5-10 s after the addition of a given concentration of leucine. PMID:24220759

  1. Putative Excitatory and Putative Inhibitory Inputs Localize to Different Dendritic Domains in a Drosophila Flight Motoneuron

    PubMed Central

    Kuehn, Claudia; Duch, Carsten

    2012-01-01

    Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the targeting of input synapses to specific parts of their dendrites. However, only few examples exist where dendritic architecture can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical, and confocal laser scanning methods this study estimates the location of the spike initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with more than 4000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provided only an estimate of putative input synapse distributions, the data indicated that inhibitory and excitatory synapses were targeted preferentially to different dendritic domains of MN5, and thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies. PMID:23279094

  2. Asymmetric photolysis of /RS/-leucine with circularly polarized ultraviolet light

    NASA Technical Reports Server (NTRS)

    Flores, J. J.; Bonner, W. A.; Massey, G. A.

    1977-01-01

    (RS)-leucine in 0.1 M HCl solution has been subjected to photolysis with 212.8-nm right (R-) and left circularly polarized light (LCPL) obtained from a laser source. RCPL preferentially photolyzed the (R)-leucine component and LCPL the (S)-leucine component of the RS substrate. The enantiomeric excess produced were 1.98% for the 59% conversion with RCPL and 2.50% for the 75% conversion with LCPL. These 'equal and opposite' effects represent the second highest enantiomeric enrichments yet reported for an asymmetric photolysis and the first ever reported for a prebiotically important substrate - an amino acid. Implications regarding the origin of optical activity are briefly discussed.

  3. Incorporation of fucose and leucine into PNS myelin proteins in nerves undergoing early Wallerian degeneration

    SciTech Connect

    Peterson, R.G.; Baughman, S.; Scheidler, D.M.

    1981-02-01

    The simultaneous incorporation of (/sup 3/H)fucose and (1-/sup 14/C)leucine into normal rat sciatic nerve was examined using an in vitro incubation model. A linear rate of protein precursor uptake was found in purified myelin protein over 1/2-6 hr of incubation utilizing a supplemented medium containing amino acids. This model was then used to examine myelin protein synthesis in nerves undergoing degeneration at 1-4 days following a crush injury. Data showed a statistically significant decrease in the ratio of fucose to leucine at 2, 3, and 4 days of degeneration, which was the consequence of a significant increase in leucine uptake. These results, plus substantial protein recovery in axotomized nerves, are indicative of active synthesis of proteins that purify with myelin during early Wallerian degeneration.

  4. Leucine metabolism in TNF-alpha- and endotoxin-treated rats: contribution of hepatic tissue.

    PubMed

    Holecek, M; Sprongl, L; Skopec, F; Andrýs, C; Pecka, M

    1997-12-01

    The effects of tumor necrosis factor-alpha (TNF-alpha; cachectin) and lipopolysaccharide of Salmonella enteritidis (LPS; endotoxin) on leucine metabolism in rats were evaluated in the whole body using intravenous infusion of L-[1-14C]leucine and in isolated perfused liver (IPL) using the single-pass perfusion technique with alpha-keto[1-14C]isocaproate as a tracer for measurement of ketoisocaproic acid (KIC) oxidation, and the recirculation technique for measurement of hepatic amino acid exchanges. The data obtained in TNF-alpha and LPS groups were compared with those obtained in controls. Both TNF-alpha and LPS treatment induced an increase of whole body leucine turnover, oxidation, and clearance. As the result of a higher increase of leucine oxidation than of incorporation into the pool of body proteins, the fractional oxidation of leucine was increased. The fractional rate of protein synthesis increased significantly in the spleen (both in TNF-alpha and LPS rats), in blood plasma, liver, colon, kidneys, gastrocnemius muscle (in LPS rats), and in lungs (TNF-alpha-treated rats), whereas it decreased in the jejunum (LPS rats). In IPL of TNF-alpha- and LPS-treated rats a decrease of KIC oxidation and higher uptake of branched-chain amino acids (BCAA; valine, leucine, and isoleucine) were observed when compared with control animals. We hypothesize that the negative consequences of increased whole body proteolysis and of increased oxidation of BCAA induced by TNF-alpha and/or LPS are reduced by decreased activity of hepatic branched-chain ketoacid dehydrogenase that can help resupply BCAA to the body. PMID:9435518

  5. Plasma arginine and leucine kinetics and urea production rates in burn patients.

    PubMed

    Yu, Y M; Young, V R; Castillo, L; Chapman, T E; Tompkins, R G; Ryan, C M; Burke, J F

    1995-05-01

    We measured plasma arginine and leucine kinetics and rates of urea production (appearance) in 12 severely burned patients (mean body surface burn area, 48%) during a basal state (low-dose intravenous glucose) and while receiving routine, total parenteral nutrition ([TPN] fed state) including an L-amino acid mixture, supplying a generous level of nitrogen (mean, 0.36 g N.kg-1.d-1). The two nutritional states were studied in random order using a primed 4-hour constant intravenous tracer infusion protocol. Stable-nuclide-labeled tracers were L-[guanidino-13C]arginine, L-[1-13C]leucine, [18O]urea, and NaH13CO3 (prime only), with blood and expired air samples drawn at intervals to determine isotopic abundance of arginine, citrulline, ornithine, alpha-ketoisocaproate ([KIC] for leucine), and urea in plasma and 13CO2 in breath. Results are compared with data obtained in these laboratories in healthy adults. Leucine kinetics (flux and disappearance into protein synthesis) indicated the expected higher turnover in burn patients than in healthy controls. Mean leucine oxidation rates are also higher and compared well with values predicted from urea production rates, provided that urea nitrogen recycling via intestinal hydrolysis is taken into account. The plasma urea flux was also higher than for normal subjects. Arginine fluxes as measured in the systemic whole body, via the plasma pool, were correspondingly higher in burned patients than in healthy controls and were in good agreement with values predicted from leucine-KIC kinetics. However, systemic whole-body arginine flux measured via the plasma pool was only 20% of the arginine flux estimated from the urea flux plus the rate of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7752916

  6. T Cell Fates Zipped Up: How the Bach2 Basic Leucine Zipper Transcriptional Repressor Directs T Cell Differentiation and Function.

    PubMed

    Richer, Martin J; Lang, Mark L; Butler, Noah S

    2016-08-15

    Recent data illustrate a key role for the transcriptional regulator bric-a-brac, tramtrack, and broad complex and cap'n'collar homology (Bach)2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super-enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation, and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulators such as B lymphocyte-induced maturation protein 1. In this review, we examine our present understanding of the role of Bach2 in T cell function and highlight the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation, and memory T cell generation. PMID:27496973

  7. Ten Putative Contributors to the Obesity Epidemic

    PubMed Central

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  8. Transport of leucine, isoleucine and valine by luminal membrane vesicles from rabbit proximal tubule.

    PubMed

    Jørgensen, K E; Kragh-Hansen, U; Sheikh, M I

    1990-03-01

    1. Transport of L- and D-isomers of leucine, isoleucine and valine by luminal membrane vesicles prepared from either the convoluted part (pars convoluta) or the straight part (pars recta) of rabbit proximal tubule was studied by a rapid filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. 2. Both types of renal membrane vesicle take up the amino acids in a Na(+)-dependent, H(+)-independent and electrogenic manner. The L-isomers are transported with higher affinities than their corresponding D-forms, of which only D-leucine is taken up to a significant extent. 3. Membrane vesicles prepared from pars convoluta take up the L-amino acids by a single and common system. Filtration studies showed that the Km values for L-leucine and L-valine transport are, on average, 0.23 and 0.83 mM, respectively. The values of KA (the concentration of amino acid producing a half-maximal optical response) are comparable to those of Km, namely 0.18 mM for L-leucine and 0.60 mM for L-valine. KA for L-isoleucine transport was found to be 0.19 mM. D-Leucine is taken up by the same system but with a much lower affinity (KA = 7.2 mM). 4. Membrane vesicles prepared from pars recta possess two, and probably common, transport systems for the L-isomers of the amino acids. The average Michaelis-Menten constants were as follows: L-leucine, K1m = 0.17 mM, K2m = 6.5 mM; L-valine, K1m = 0.19 mM, K2m = 11.5 mM. The KA values were: L-leucine, K1A = 0.12 mM, K2A = 7.4 mM; L-valine, K1A = 0.18 mM, K2A = 10.0 mM; L-isoleucine, K1A = 0.17 mM, K2A = 9.0 mM. D-Leucine is taken up by a low-affinity system only (KA = 6.5 mM), which seems to be the same as the low-affinity system transporting the L-forms of the amino acids. PMID:2352186

  9. Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway

    PubMed Central

    Dai, Jie-Min; Yu, Mu-Xue; Shen, Zhen-Yu; Guo, Chu-Yi; Zhuang, Si-Qi; Qiu, Xiao-Shan

    2015-01-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway. PMID:26007333

  10. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were tr...

  11. Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

    PubMed

    Yagasaki, Kazumi; Morisaki, Naoko; Kitahara, Yoshiro; Miura, Atsuhito; Funabiki, Ryuhei

    2003-11-01

    Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C. PMID:19003213

  12. Amino acid transport across the tonoplast of vacuoles isolated from barley mesophyll protoplasts: Uptake of alanine, leucine, and glutamine. [Hordeum vulgare L

    SciTech Connect

    Dietz, K.J.; Jaeger, R.; Kaiser, G.; Martinoia, E. )

    1990-01-01

    Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using {sup 14}C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.

  13. Decarboxylation of [1-(13)C]leucine by hydroxyl radicals.

    PubMed

    Guitton, J; Tinardon, F; Lamrini, R; Lacan, P; Desage, M; Francina, A

    1998-08-01

    The decarboxylation of [1-13C]leucine by hydroxyl radicals was studied by using gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to follow the production of 13CO2. A Fenton reaction between a (Fe2+)-porphyrin and hydrogen peroxide under aerobic conditions yielded hydroxyl radicals. The decarboxylation rates (VLeu) measured by GC-IRMS were dependent on [1-13C]leucine, porphyrin and hydrogen peroxide concentrations. The 13CO2 production was also dependent on bicarbonate or carbon dioxide added in the reaction medium. Bicarbonate facilitated 13CO2 production, whereas carbon dioxide decreased 13CO2 production. Proton effects on some decarboxylation intermediates could explain bicarbonate or carbon dioxide effects. No effect on the decarboxylation rates was observed in the presence of the classical hydroxyl radicals scavengers dimethyl sulfoxide, mannitol, and uric acid. By contrast, a competitive effect with a strong decrease of the decarboxylation rates was observed in the presence of various amino acids: unlabeled leucine, valine, phenylalanine, cysteine, lysine, and histidine. Two reaction products, methyl-4 oxo-2 pentanoate and methyl-3 butanoate were identified by gas chromatography-mass spectrometry in comparison with standards. The present results suggest that [1-13C]leucine can participate to the coordination sphere of (Fe2+)-porphyrin, with a caged process of the hydroxyl radicals which cannot get out of the coordination sphere. PMID:9680180

  14. Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

    PubMed Central

    Gore, Nigel R.; Wray, John L.

    1978-01-01

    Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-3H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [15N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [14N]nitrate and 100% H2O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation. PMID:16660229

  15. Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast

    PubMed Central

    Aris, John P.; Alvers, Ashley L.; Ferraiuolo, Roy A.; Fishwick, Laura K.; Hanvivatpong, Amanda; Hu, Doreen; Kirlew, Christine; Leonard, Michael T.; Losin, Kyle J.; Marraffini, Michelle; Seo, Arnold Y.; Swanberg, Veronica; Westcott, Jennifer L.; Wood, Michael S.; Leeuwenburgh, Christiaan; Dunn, William A.

    2013-01-01

    We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions. PMID:23337777

  16. 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

    PubMed

    Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. PMID:26902097

  17. Prolonged leucine infusion differentially affects tissue protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine (Leu) acutely stimulates protein synthesis by activating the mammalian target of rapamycin complex 1 (mTORC1) pathway. To determine whether Leu can stimulate protein synthesis in muscles of different fiber types and visceral tissues of the neonate for a prolonged period and to determine the ...

  18. Role of glucocorticoid-induced leucine zipper (GILZ) in bone acquisition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucocorticoids (GCs) have both anabolic and catabolic effects on bone. However, no GC anabolic effect mediator has been identified to date. In this report, we provide the first evidence that glucocorticoid-induced leucine zipper (GILZ), a GC anti-inflammatory effect mediator, can enhance bone forma...

  19. Identification and mapping of nucleotide binding site-leucine rich repeat resistance gene analogs in bermudagrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty-one bermudagrass (Cynodon spp.) disease resistance gene homologs (BRGH) were cloned and sequenced from diploid, triploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS- leucine rich repeat (LRR) resistance gene family. Alignment of ...

  20. Impact of prolonged leucine supplementation on protein synthesis and lean growth in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most low-birth weight infants experience extrauterine growth failure due to reduced nutrient intake as a result of feeding intolerance. The objective of this study was to determine whether prolonged enteral leucine supplementation improves lean growth in neonatal pigs fed a restricted protein diet. ...

  1. Selective single crystal complexation of L- or D-leucine by p-sulfonatocalix[6]arene.

    PubMed

    Atwood, Jerry L; Dalgarno, Scott J; Hardie, Michaele J; Raston, Colin L

    2005-01-21

    p-Sulfonatocalix[6]arene, organised in the 'double cone' conformation, has multi-guest capability binding either L- or D-leucine in a single crystal in a bi-layer type arrangement from a racemic mixture of the amino acid. PMID:15645029

  2. FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1

    PubMed Central

    Chen, Zhi; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

    2016-01-01

    The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle. PMID:27280402

  3. Leucine pulses enhance skeletal muscle protein synthesis during continuous feeding in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infants unable to maintain oral feeding can be nourished by orogastric tube. We have shown that orogastric continuous feeding restricts muscle protein synthesis compared with intermittent bolus feeding in neonatal pigs. To determine whether leucine leu infusion can be used to enhance protein synthes...

  4. Prolonged stimulation of muscle protein synthesis by leucine in neonates is dependent on amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rise in amino acids and insulin after a meal independently stimulate protein synthesis in skeletal muscle of neonates by activating the intracellular signalling pathways that regulate mRNA translation. Leucine, in particular, is important in mediating the response to amino acids. Previously, w...

  5. FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1.

    PubMed

    Wu, Xiaochun; Zhao, Lingling; Chen, Zhi; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

    2016-01-01

    The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle. PMID:27280402

  6. Leucine minimizes denervation-induced skeletal muscle atrophy of rats through akt/mtor signaling pathways

    PubMed Central

    Ribeiro, Carolina B.; Christofoletti, Daiane C.; Pezolato, Vitor A.; de Cássia Marqueti Durigan, Rita; Prestes, Jonato; Tibana, Ramires A.; Pereira, Elaine C. L.; de Sousa Neto, Ivo V.; Durigan, João L. Q.; da Silva, Carlos A.

    2015-01-01

    The aim of the present study was to evaluate the effect of leucine treatment (0.30 mM) on muscle weight and signaling of myoproteins related to synthesis and degradation pathways of soleus muscle following seven days of complete sciatic nerve lesion. Wistar rats (n = 24) of 3–4 months of age (192 ± 23 g) were used. The animals were randomly distributed into four experimental groups (n = 6/group): control, treated with leucine (L), denervated (D) and denervated treated with leucine (DL). Dependent measures were proteins levels of AKT, AMPK, mTOR, and ACC performed by Western blot. Leucine induced a reduction in the phosphorylation of AMPK (p < 0.05) by 16% in the L and by 68% in the DL groups as compared with control group. Denervation increased AMPK by 24% in the D group as compared with the control group (p < 0.05). AKT was also modulated by denervation and leucine treatment, highlighted by the elevation of AKT phosphorylation in the D (65%), L (98%) and DL (146%) groups as compared with the control group (p < 0.05). AKT phosphorylation was 49% higher in the D group as compared with the DL group. Furthermore, denervation decreased mTOR phosphorylation by 29% in the D group as compared with the control group. However, leucine treatment induced an increase of 49% in the phosphorylation of mTOR in the L group as compared with the control group, and an increase of 154% in the DL as compared with the D group (p < 0.05). ACC phosphorylation was 20% greater in the D group than the control group. Furthermore, ACC in the soleus was 22% lower in the in the L group and 50% lower in the DL group than the respective control group (p < 0.05). In conclusion, leucine treatment minimized the deleterious effects of denervation on rat soleus muscle by increasing anabolic (AKT and mTOR) and decreasing catabolic (AMPK) pathways. These results may be interesting for muscle recovery following acute denervation, which may contribute to musculoskeletal rehabilitation after denervation

  7. Crystal Structure of a Super Leucine Zipper an Extended Two-Stranded Super Long Coiled Coil

    SciTech Connect

    J Diao

    2011-12-31

    Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 {angstrom} resolution. The peptide monomer shows a helix trunk with short curved N- and C-termini. In the crystal, two monomers cross in 35{sup o} and form an X-shaped dimer, and each X-shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two-stranded, parallel, super long coiled coil rather than a discrete, two-helix coiled coil of the wild-type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild-type leucine zipper, the N-terminus of the mutant has a dramatic conformational change and the C-terminus has one more residue Glu 32 determined. The mutant X-shaped dimer has a large crossing angle of 35{sup o} instead of 18{sup o} in the wild-type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self-assembling protein fibers.

  8. Glycine restores the anabolic response to leucine in a mouse model of acute inflammation.

    PubMed

    Ham, Daniel J; Caldow, Marissa K; Chhen, Victoria; Chee, Annabel; Wang, Xuemin; Proud, Christopher G; Lynch, Gordon S; Koopman, René

    2016-06-01

    Amino acids, especially leucine, potently stimulate protein synthesis and reduce protein breakdown in healthy skeletal muscle and as a result have received considerable attention as potential treatments for muscle wasting. However, the normal anabolic response to amino acids is impaired during muscle-wasting conditions. Although the exact mechanisms of this anabolic resistance are unclear, inflammation and ROS are believed to play a central role. The nonessential amino acid glycine has anti-inflammatory and antioxidant properties and preserves muscle mass in calorie-restricted and tumor-bearing mice. We hypothesized that glycine would restore the normal muscle anabolic response to amino acids under inflammatory conditions. Relative rates of basal and leucine-stimulated protein synthesis were measured using SUnSET methodology 4 h after an injection of 1 mg/kg lipopolysaccharide (LPS). Whereas leucine failed to stimulate muscle protein synthesis in LPS-treated mice pretreated with l-alanine (isonitrogenous control), leucine robustly stimulated protein synthesis (+51%) in mice pretreated with 1 g/kg glycine. The improvement in leucine-stimulated protein synthesis was accompanied by a higher phosphorylation status of mTOR, S6, and 4E-BP1 compared with l-alanine-treated controls. Despite its known anti-inflammatory action in inflammatory cells, glycine did not alter the skeletal muscle inflammatory response to LPS in vivo or in vitro but markedly reduced DHE staining intensity, a marker of oxidative stress, in muscle cross-sections and attenuated LPS-induced wasting in C2C12 myotubes. Our observations in male C57BL/6 mice suggest that glycine may represent a promising nutritional intervention for the attenuation of skeletal muscle wasting. PMID:27094036

  9. Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction.

    PubMed

    Vogt, Michael; Haas, Sabine; Klaffl, Simon; Polen, Tino; Eggeling, Lothar; van Ooyen, Jan; Bott, Michael

    2014-03-01

    Using metabolic engineering, an efficient L-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a K(i) of 0.4 mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuA(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application. PMID:24333966

  10. Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine.

    PubMed Central

    Grandoni, J A; Fulmer, S B; Brizzio, V; Zahler, S A; Calvo, J M

    1993-01-01

    The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation. Images PMID:8244927

  11. Adult Basic Education Basic Computer Literacy Handbook.

    ERIC Educational Resources Information Center

    Manini, Catalina M.; Cervantes, Juan

    This handbook, in both English and Spanish versions, is intended for use with adult basic education (ABE) students. It contains five sections of basic computer literacy activities and information about the ABE computer literacy course offered at Dona Ana Community College (DACC) in New Mexico. The handbook begins with forewords by the handbook's…

  12. A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper.

    PubMed Central

    Thiem, S M; Miller, L K

    1989-01-01

    An Autographa californica nuclear polyhedrosis virus gene encoding a 30-kilodalton polypeptide with two different sequence motifs characteristic of DNA-binding proteins was identified immediately downstream of the major capsid protein gene (vp39). The gene, CG30, was characterized by sequencing, transcriptional mapping, in vitro translation of hybrid-selected RNA, and comparison of the derived polypeptide sequence with published data bases. The initial ATG of the 792-base-pair CG30 open reading frame is two nucleotides downstream of the vp39 terminal TAA codon. Early transcripts of CG30 initiate within the vp39 coding sequence. At late times, bicistronic transcripts initiate from the vp39 promoter, continue through CG30, and terminate at the same site as the early transcripts. In vitro translation of hybrid-selected early CG30 RNA yields a polypeptide of 30 kilodaltons. The predicted CG30 polypeptide sequence has characteristics of a eucaryotic transcriptional activator and is novel in having two potential DNA-binding domains. A stretch of acidic residues bridges a zinc finger at the amino terminus and a leucine zipper with a flanking basic region at the carboxyl terminus. Images PMID:2507791

  13. Nonmyofilament-Associated Troponin T3 Nuclear and Nucleolar Localization Sequence and Leucine Zipper Domain Mediate Muscle Cell Apoptosis

    PubMed Central

    Zhang, Tan; Birbrair, Alexander; Delbono, Osvaldo

    2013-01-01

    Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK) and the basic lysine and arginine residues might play an important role in the nuclear retention and nucleolar enrichment of KLKRQK-DsRed fusion proteins. Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized in this region. Additionally, we identified a classical DNA-binding Leucine Zipper Domain (LZD) which is conserved among TnT isoforms and species. Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. We conclude that TnT3 contains both a nuclear localization signal and a DNA binding domain, which may mediate nuclear/nucleolar signaling and muscle cell apoptosis. PMID:23378072

  14. Mutations associated with retinopathies alter mitogen-activated protein kinase-induced phosphorylation of neural retina leucine-zipper

    PubMed Central

    Kumar, Sandeep; Patel, Dharmesh; Richong, Sushmita; Oberoi, Pranav; Ghosh, Madhumita; Swaroop, Anand

    2007-01-01

    Purpose Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations. Methods Metabolic labeling with 33P-orthophosphate was used to study phosphorylation of NRL in transfected COS-1 cells. NRL or NRL mutants were expressed as glutathione S-transferase (GST)-fusion proteins and used as substrate to screen various kinases by in vitro phosphorylation assays. CV-1 cells were co-transfected with rhodopsin promoter-reporter construct and expression plasmids, with or without specific mitogen-activated protein kinase (MAPK) inhibitors, to examine their effect on NRL-mediated transactivation. Expression of activated MAPKs in postnatal mice retina was determined by immunoblot analysis. Results Metabolic labeling of NRL produces multiple phosphorylated protein bands in transfected COS-1 cells. Fewer but more intense radiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutants compared to wild-type NRL. We show that MAPK2 and p38 induce specific phosphorylation of NRL, but this pattern is altered in NRL mutants. Immunoblot analysis of extracts from developing mouse retina reveals enhanced expression of activated MAPK2 at postnatal day 0-3, concordant with the reported phosphorylation pattern of NRL in vivo. Inhibition of MAPK signaling pathways decreases NRL and CRX -mediated synergistic activation of rhodopsin promoter in transfected CV-1 cells. Conclusions Our results suggest that multiple MAPKs can phosphorylate NRL and this phosphorylation pattern is altered by

  15. Effect of strength training session on plasma amino acid concentration following oral ingestion of leucine, BCAAs or glutamine in men.

    PubMed

    Mero, Antti; Leikas, Anne; Knuutinen, Juha; Hulmi, Juha J; Kovanen, Vuokko

    2009-01-01

    We examined the acute effects of a 1-h strength training session (STS) on plasma amino acid concentration following orally ingestion of leucine, branched-chain amino acids (BCAAs) or glutamine in nine physically active men who participated in double-blinded and randomised experiments. The subjects took placebo, leucine, BCAAs, or glutamine capsules (50 mg/kg) in either rest (REST) or STS condition. Blood samples were taken before and at 30, 60, 90, and 120 min after the beginning of the treatment and they were assayed for plasma amino acids with HPLC. Following both leucine and BCAA ingestion the peak concentration of leucine was similar at rest (524 +/- 46 and 530 +/- 29 nmol/ml, respectively) and similar after STS (398 +/- 43 and 387 +/- 46 nmol/ml, respectively) but the rest and STS concentrations differed from each other (P < 0.01-0.001). The modelled polynomial data for the leucine treatment showed that the peak concentration of leucine occurred at 67 min at rest and at 90 min in STS (difference between REST and STS: P = 0.012). For the BCAA treatment the polynomial data showed that the peak concentration of leucine occurred at 72 min at rest and at 78 min in STS (P = 0.067). The peak concentration of glutamine was similar in both rest and STS condition and occurred at 60 min at rest and at 57 min in STS. In conclusion, 1-h of STS slows the increase in the peak concentration of plasma leucine similarly after oral ingestion of leucine or BCAAs but after oral ingestion of glutamine it has no slowing effect on glutamine concentration. PMID:19015870

  16. Excess leucine intake enhances muscle anabolic signaling but not net protein anabolism in young men and women.

    PubMed

    Glynn, Erin L; Fry, Christopher S; Drummond, Micah J; Timmerman, Kyle L; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2010-11-01

    Essential amino acids (EAA) stimulate skeletal muscle protein synthesis (MPS) in humans. Leucine may have a greater stimulatory effect on MPS than other EAA and/or decrease muscle protein breakdown (MPB). To determine the effect of 2 different leucine concentrations on muscle protein turnover and associated signaling, young men (n = 6) and women (n = 8) ingested 10 g EAA in 1 of 2 groups: composition typical of high quality proteins (CTRL; 1.8 g leucine) or increased leucine concentration (LEU; 3.5 g leucine). Participants were studied for 180 min postingestion. Fractional synthetic rate and leg phenylalanine and leucine kinetics were assessed on muscle biopsies using stable isotopic techniques. Signaling was determined by immunoblotting. Arterial leucine concentration and delivery to the leg increased in both groups and was significantly higher in LEU than in CTRL; however, transport into the muscle and intracellular availability did not differ between groups. MPS increased similarly in both groups 60 min postingestion. MPB decreased at 60 min only in LEU, but net muscle protein balance improved similarly. Components of mammalian target of rapamycin (mTOR) signaling were improved in LEU, but no changes were observed in ubiquitin-proteasome system signaling. Changes in light chain 3 and mTOR association with Unc-51-like kinase 1 indicate autophagy decreased more in LEU. We conclude that in 10 g of EAA, the leucine content typical of high quality proteins (~1.8 g) is sufficient to induce a maximal skeletal muscle protein anabolic response in young adults, but leucine may play a role in autophagy regulation. PMID:20844186

  17. Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

    PubMed Central

    Molitoris, B A; Wilson, P D; Schrier, R W; Simon, F R

    1985-01-01

    To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically

  18. Magnetic Pulse Affects a Putative Magnetoreceptor Mechanism

    PubMed Central

    Davila, Alfonso F.; Winklhofer, Michael; Shcherbakov, Valera P.; Petersen, Nikolai

    2005-01-01

    Clusters of superparamagnetic (SP) magnetite crystals have recently been identified in free nerve endings in the upper-beak skin of homing pigeons and are interpreted as being part of a putative magnetoreceptor system. Motivated by these findings, we developed a physical model that accurately predicts the dynamics of interacting SP clusters in a magnetic field. The main predictions are: 1), under a magnetic field, a group of SP clusters self-assembles into a chain-like structure that behaves like a compass needle under slowly rotating fields; 2), in a frequently changing field as encountered by a moving bird, a stacked chain is a structurally more stable configuration than a single chain; 3), chain-like structures of SP clusters disrupt under strong fields applied at oblique angles; and 4), reassemble on a timescale of hours to days (assuming a viscosity of the cell plasma η ∼ 1 P). Our results offer a novel mechanism for magnetic field perception and are in agreement with the response of birds observed after magnetic-pulse treatments, which have been conducted in the past to specifically test if ferrimagnetic material is involved in magnetoreception, but which have defied explanation so far. Our theoretical results are supported by experiments on a technical SP model system using a high-speed camera. We also offer new predictions that can be tested experimentally. PMID:15863473

  19. Toddlers’ Duration of Attention towards Putative Threat

    PubMed Central

    Kiel, Elizabeth J.; Buss, Kristin A.

    2010-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk for developing anxious behavior, toddlers’ attention towards a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined how attention towards an angry-looking gorilla mask in a room with alternative opportunities for play in 24-month-old toddlers predicted social inhibition when children entered kindergarten. Analyses examined attention to threat above and beyond and in interaction with both proximity to the mask and fear of novelty observed in other situations. Attention to threat interacted with proximity to the mask to predict social inhibition, such that attention to threat most strongly predicted social inhibition when toddlers stayed furthest from the mask. This relation occurred above and beyond the predictive relation between fear of novelty and social inhibition. Results are discussed within the broader literature of anxiety development and attentional processes in young children. PMID:21373365

  20. Biogenic Origin for Earth's Oldest Putative Microfossils

    SciTech Connect

    De Gregorio, B.; Sharp, T; Flynn, G; Wirick, S; Hervig, R

    2009-01-01

    Carbonaceous microbe-like features preserved within a local chert unit of the 3.5 Ga old Apex Basalt in Western Australia may represent some of the oldest evidence of life on Earth. However, the biogenicity of these putative microfossils has been called into question, primarily because the sample collection locality is a black, carbon-rich, brecciated chert dike representing an Archean submarine hydrothermal spring, suggesting a formation via an abiotic organic synthesis mechanism. Here we describe the macromolecular hydrocarbon structure, carbon bonding, functional group chemistry, and biotic element abundance of carbonaceous matter associated with these filamentous features. These characteristics are similar to those of biogenic kerogen from the ca. 1.9 Ga old Gunflint Formation. Although an abiotic origin cannot be entirely ruled out, it is unlikely that known abiotic synthesis mechanisms could recreate both the structural and compositional complexity of this ancient carbonaceous matter. Thus, we find that a biogenic origin for this material is more likely, implying that the Apex microbe-like features represent authentic biogenic organic matter.

  1. Leucine Supplementation Improves Acquired Growth Hormone Resistance in Rats with Protein-Energy Malnutrition

    PubMed Central

    Wang, Xinying; Zhao, Jie; Wan, Xiao; Zhang, Li; Wu, Chao; Li, Ning; Li, Jieshou

    2015-01-01

    Background Protein-energy malnutrition (PEM) can lead to growth hormone (GH) resistance. Leucine supplementation diets have been shown to increase protein synthesis in muscles. Our study aimed at investigating if long-term leucine supplementation could modulate GH-insulin-like growth factor (IGF)-1 system function and mammalian target of rapamycin (mTOR)-related signal transduction in skeletal muscles in a rat model of severe malnutrition. Methodology/Principal Findings Male Sprague-Dawley rats (n = 50; weight, 302 ± 5 g) were divided into 5 treatment groups, including 2 control groups (a normal control group that was fed chow and ad libitum water [CON, n = 10] and a malnourished control group [MC, n = 10] that was fed a 50% chow diet). After undergoing a weight loss stage for 4 weeks, rats received either the chow diet (MC-CON, n = 10), the chow diet supplemented with low-dose leucine (MC-L, n = 10), or the chow diet supplemented with high-dose leucine (MC-H, n = 10) for 2 weeks. The muscle masses of the gastrocnemius, soleus, and extensor digitorum longus were significantly reduced in the MC group. Re-feeding increased muscle mass, especially in the MC-L and MC-H groups. In the MC group, serum IGF-1, IGF-binding protein (IGFBP)-3, and hepatic growth hormone receptor (GHR) levels were significantly decreased and phosphorylation of the downstream anabolic signaling effectors protein kinase B (Akt), mTOR, and ribosomal protein S6 kinase 1 (S6K1) were significantly lower than in other groups. However, serum IGF-1 and IGF binding protein (IGFBP)-3 concentrations and hepatic growth hormone receptor (GHR) levels were significantly higher in the MC-L and MC-H groups than in the MC-CON group, and serum IGFBP-1 levels was significantly reduced in the MC-L and MC-H groups. These changes were consistent with those observed for hepatic mRNA expression levels. Phosphorylation of the downstream anabolic signaling effectors Akt, mTOR, and S6K1 were also significantly higher in

  2. N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebellum and thalamus.

    PubMed

    Günther, Lisa; Beck, Roswitha; Xiong, Guoming; Potschka, Heidrun; Jahn, Klaus; Bartenstein, Peter; Brandt, Thomas; Dutia, Mayank; Dieterich, Marianne; Strupp, Michael; la Fougère, Christian; Zwergal, Andreas

    2015-01-01

    An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by

  3. N-Acetyl-L-Leucine Accelerates Vestibular Compensation after Unilateral Labyrinthectomy by Action in the Cerebellum and Thalamus

    PubMed Central

    Xiong, Guoming; Potschka, Heidrun; Jahn, Klaus; Bartenstein, Peter; Brandt, Thomas; Dutia, Mayank; Dieterich, Marianne; Strupp, Michael; la Fougère, Christian; Zwergal, Andreas

    2015-01-01

    An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-μPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-μPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of μPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by

  4. Putative Lineage of Novel African Usutu Virus, Central Europe

    PubMed Central

    Cadar, Daniel; Bosch, Stefan; Jöst, Hanna; Börstler, Jessica; Garigliany, Mutien-Marie; Becker, Norbert

    2015-01-01

    We characterized the complete genome of a putative novel Usutu virus (USUV) strain (Usutu-BONN) detected in a dead blackbird from Germany. Genomic analysis revealed several unique amino acid substitutions among the polyprotein gene. Phylogenetic analyses demonstrated that Usutu-BONN constitutes a putative novel African USUV lineage, which was probably recently introduced to central Europe. PMID:26291923

  5. Basic BASIC; An Introduction to Computer Programming in BASIC Language.

    ERIC Educational Resources Information Center

    Coan, James S.

    With the increasing availability of computer access through remote terminals and time sharing, more and more schools and colleges are able to introduce programing to substantial numbers of students. This book is an attempt to incorporate computer programming, using BASIC language, and the teaching of mathematics. The general approach of the book…

  6. PASCAL vs BASIC

    ERIC Educational Resources Information Center

    Mundie, David A.

    1978-01-01

    A comparison between PASCAL and BASIC as general purpose microprocessor languages rates PASCAL above BASIC in such points as program structure, data types, structuring methods, control structures, procedures and functions, and ease in learning. (CMV)

  7. Health Insurance Basics

    MedlinePlus

    ... How Can I Help a Friend Who Cuts? Health Insurance Basics KidsHealth > For Teens > Health Insurance Basics Print ... thought advanced calculus was confusing. What Exactly Is Health Insurance? Health insurance is a plan that people buy ...

  8. Identification of Putative Fallopian Tube Stem Cells

    PubMed Central

    Snegovskikh, Victoria; Mutlu, Levent; Massasa, Effi

    2014-01-01

    Stem cells are used to repair and regenerate multiple tissues in the adult. We have previously shown that stem cells play a significant role in mediating endometrial repair and tissue regeneration. We hypothesized that the oviduct may possess a similar population of stem cells that contribute to the maintenance of this tissue. Here we identify label-retaining cells (LRCs) in the murine oviduct which indicate the presence of a stem/progenitor cell population in this tissue as well. Two-day-old CD-1 mice were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) or vehicle control. Female animals (n = 36 for each group) were killed at 6 weeks post injection. Reproductive tracts were removed, specimens were embedded in paraffin, and 5-µ sections were prepared. Oviduct was identified by hematoxylin and eosin staining and morphology. Immunofluorescence studies were performed on serial sections tissues (n = 12 per animal) using antibodies against BrdU. Confocal microscopy was used to identify 4′,6-diamidino-2-phenylindole (DAPI)- and BrdU-stained nuclei. In the group of mice exposed to BrdU, we identified a population of LRCs in all specimens and not in controls. The putative stem cells are located at the base of each villi, suggesting the location of the stem cell niche. The number of DAPI-stained nuclei divided by the number of LRCs; LRCs constituted 0.5% of all nucleated cells. The oviduct contains a population of progenitor cells, likely used in the repair and regeneration of fallopian tube. Defective or insufficient stem cell reserve may underlie common tubal diseases, including hydrosalpinx and ectopic pregnancy. PMID:25305130

  9. CSF myelin basic protein

    MedlinePlus

    CSF myelin basic protein is a test to measure the level of myelin basic protein (MBP) in the cerebrospinal fluid (CSF). The CSF ... less than 4 ng/mL of myelin basic protein in the CSF. Normal value ranges may vary ...

  10. Basic Cake Decorating Workbook.

    ERIC Educational Resources Information Center

    Bogdany, Mel

    Included in this student workbook for basic cake decorating are the following: (1) Drawings of steps in a basic way to ice a layer cake, how to make a paper cone, various sizes of flower nails, various sizes and types of tin pastry tubes, and special rose tubes; (2) recipes for basic decorating icings (buttercream, rose paste, and royal icing);…

  11. Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

    PubMed Central

    Rodrigues, G A; Park, M

    1993-01-01

    Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed. Images PMID:8413267

  12. Anatomical localization of leucine-rich repeat kinase 2 in mouse brain.

    PubMed

    Melrose, H; Lincoln, S; Tyndall, G; Dickson, D; Farrer, M

    2006-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been identified in autosomal dominant late-onset Parkinson's disease. Expression of LRRK2 has previously been reported in brain; however, no precise anatomical information is yet available. We have performed in situ hybridization and quantitative reverse transcription polymerase chain reaction to map LRRK2 mRNA expression in mouse brain. We find LRRK2 is highly expressed in the striatum, cortex and olfactory tubercle; however, little or no expression is found in the substantia nigra, where dopaminergic neurons preferentially degenerate in Parkinson's disease. These findings suggest that LRRK2 mRNA is expressed in dopamine-receptive areas rather than in the dopamine-synthesizing neurons. Consistent with a role LRRK2 in Parkinson's disease, dysfunction of leucine-rich repeat kinase 2 protein in dopamine-innervated areas may to lead to altered dopaminergic neurotransmission and degeneration of the nigro-striatal pathway. PMID:16504409

  13. Papain-Catalyzed Chemoenzymatic Synthesis of Telechelic Polypeptides Using Bis(Leucine Ethyl Ester) Initiator.

    PubMed

    Tsuchiya, Kousuke; Numata, Keiji

    2016-07-01

    In order to construct unique polypeptide architectures, a novel telechelic-type initiator with two leucine ethyl ester units is designed for chemoenzymatic polymerization. Glycine or alanine ethyl ester is chemoenzymatically polymerized using papain in the presence of the initiator, and the propagation occurs at each leucine ethyl ester unit to produce the telechelic polypeptide. The formation of the telechelic polypeptides is confirmed by (1) H NMR and MALDI-TOF mass spectroscopies. It is revealed by AFM observation that long nanofibrils are formed from the telechelic polyalanine, whereas a conventional linear polyalanine with a similar degree of polymerization shows granule-like structures. The telechelic polyglycine and polyalanine show the crystalline structures of Polyglycine II and antiparallel β-sheet, respectively. It is demonstrated that this method to synthesize telechelic-type polypeptides potentially opens up a pathway to construct novel hierarchical structures by self-assembly. PMID:26947148

  14. The radiolysis of tryptophan and leucine with P-32 beta-radiation

    NASA Technical Reports Server (NTRS)

    Blair, N. E.; Bonner, W. A.

    1980-01-01

    The paper extends earlier experiments on the radiolysis of DL-tryptophan using P-32 beta-radiation to longer reaction times, observing complete destruction of tryptophan by secondary, nonradiolytic processes. In addition DL-leucine is irradiated with P-32 beta-irradiation at -196 C, leading to radiolyses to the extents of about 20-30%, but observing no concomitant asymmetric bias. The complete absence of asymmetric bias in the present and earlier (Bonner et al., 1979) radiolyses of aqueous tryptophan at -25 C and the present radiolyses of water-free leucine at -196 C using P-32 beta-radiation and its accompanying bremsstrahlung leave it an open question whether or not the Vester-Ulbricht beta-decay/bremsstrahlung mechanism for the origin of optical activity is a viable one.

  15. Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis.

    PubMed Central

    Pan, Q; Liu, Y S; Budai-Hadrian, O; Sela, M; Carmel-Goren, L; Zamir, D; Fluhr, R

    2000-01-01

    The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families. PMID:10790405

  16. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  17. Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

    PubMed

    Myers, K A; Rahi-Saund, V; Davison, M D; Young, J A; Cheater, A J; Stern, P L

    1994-03-25

    The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed. PMID:8132670

  18. Impact of prolonged leucine supplementation on protein synthesis and lean growth in neonatal pigs.

    PubMed

    Columbus, Daniel A; Steinhoff-Wagner, Julia; Suryawan, Agus; Nguyen, Hanh V; Hernandez-Garcia, Adriana; Fiorotto, Marta L; Davis, Teresa A

    2015-09-15

    Most low-birth weight infants experience extrauterine growth failure due to reduced nutrient intake as a result of feeding intolerance. The objective of this study was to determine whether prolonged enteral leucine supplementation improves lean growth in neonatal pigs fed a restricted protein diet. Neonatal pigs (n = 14-16/diet, 5 days old, 1.8 ± 0.3 kg) were fed by gastric catheter a whey-based milk replacement diet with either a high protein (HP) or restricted protein (RP) content or RP supplemented with leucine to the same level as in the HP diet (RPL). Pigs were fed 40 ml·kg body wt(-1)·meal(-1) every 4 h for 21 days. Feeding the HP diet resulted in greater total body weight and lean body mass compared with RP-fed pigs (P < 0.05). Masses of the longissimus dorsi muscle, heart, and kidneys were greater in the HP- than RP-fed pigs (P < 0.05). Body weight, lean body mass, and masses of the longissimus dorsi, heart, and kidneys in pigs fed the RPL diet were intermediate to RP- and HP-fed pigs. Protein synthesis and mTOR signaling were increased in all muscles with feeding (P < 0.05); leucine supplementation increased mTOR signaling and protein synthesis rate in the longissimus dorsi (P < 0.05). There was no effect of diet on indices of protein degradation signaling in any tissue (P > 0.05). Thus, when protein intake is chronically restricted, the capacity for leucine supplementation to enhance muscle protein accretion in neonatal pigs that are meal-fed milk protein-based diets is limited. PMID:26374843

  19. Fragment-based discovery of type I inhibitors of maternal embryonic leucine zipper kinase.

    PubMed

    Johnson, Christopher N; Berdini, Valerio; Beke, Lijs; Bonnet, Pascal; Brehmer, Dirk; Coyle, Joseph E; Day, Phillip J; Frederickson, Martyn; Freyne, Eddy J E; Gilissen, Ron A H J; Hamlett, Christopher C F; Howard, Steven; Meerpoel, Lieven; McMenamin, Rachel; Patel, Sahil; Rees, David C; Sharff, Andrew; Sommen, François; Wu, Tongfei; Linders, Joannes T M

    2015-01-01

    Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology. PMID:25589925

  20. Regulation of transaminase C synthesis in Escherichia coli: conditional leucine auxotrophy.

    PubMed

    McGilvray, D; Umbarger, H E

    1974-11-01

    The regulation of synthesis of the valine-alanine-alpha-aminobutyrate transaminase (transaminase C) was studied in Escherichia coli mutants lacking the branched-chain amino acid transaminase (transaminase B). An investigation was made of two strains, CU2 and CU2002, each carrying the same transaminase B lesion but exhibiting different growth responses on a medium supplemented with branched-chain amino acids. Both had the absolute isoleucine requirement characteristic of ilvE auxotrophs, but growth of strain CU2 was stimulated by valine, whereas that of strain CU2002 was markedly inhibited by valine. Strain CU2002 behaved like a conditional leucine auxotroph in that the inhibition by valine was reversed by leucine. Results of enzymatic studies showed that synthesis of transaminase C was repressed by valine in strain CU2002 but not in strain CU2. Inhibition by valine in strain CU2002 appears to be the combined effect of repression on transaminase C synthesis and valine-dependent feedback inhibition of alpha-acetohydroxy acid synthase activity, causing alpha-ketoisovalerate (and hence leucine) limitation. The ilvE markers of strains CU2 and CU2002 were each transferred by transduction to a wild-type genetical background. All ilvE recombinants from both crosses resembled strain CU2002 and were inhibited by valine in the presence of isoleucine. Thus, strain CU2 carries an additional lesion that allows it to grow on a medium containing isoleucine plus valine. It is concluded that conditional leucine auxotrophy is characteristic of mutants carrying an ilvE lesion alone. PMID:4616947

  1. Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata)

    PubMed Central

    Azizi, Sheida; Nematollahi, Mohammad Ali; Mojazi Amiri, Bagher; Vélez, Emilio J.; Lutfi, Esmail; Navarro, Isabel; Capilla, Encarnación; Gutiérrez, Joaquim

    2016-01-01

    Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of

  2. Relations among arginine, citrulline, ornithine, and leucine kinetics in adult burn patients.

    PubMed

    Yu, Y M; Ryan, C M; Burke, J F; Tompkins, R G; Young, V R

    1995-11-01

    Plasma fluxes of arginine, citrulline, and leucine, and the rate of conversion of labeled citrulline to arginine (Qcit-->arg) were determined in nine severely burned patients (mean: 56% body surface burn area, mean 10 d postinjury) while they received total parenteral nutrition (TPN) including an L-amino acid mixture that supplied a generous amount of nitrogen (mean: 0.39 +/- 0.02 g.kg-1.d-1). Plasma fluxes were also studied in these patients during a basal state (low-dose intravenous glucose) by using a primed, 4-h constant intravenous tracer-infusion protocol. Stable-nuclide labeled tracers were L-[15N-15N-guanidino,5,5,2H2]arginine; L-[13C-ureido]citrulline; L-[1-13C]leucine; and NaH13CO3 (prime only), with blood and expired air samples drawn at intervals to determine isotopic abundance of arginine, citrulline, ornithine, and alpha-ketoisocaproate (KIC; for leucine) in plasma and 13CO2 in breath. Leucine kinetics (flux and disappearance into protein synthesis) confirmed the anticipated higher protein turnover in these burn patients compared with healthy control subjects. The plasma arginine fluxes were correspondingly higher in burn patients than in healthy control subjects. However, the citrulline flux and rate of conversion of citrulline to arginine were not higher than values obtained in our laboratories in healthy adult subjects. We hypothesize that the higher rates of arginine loss from the body after burn injury would need to be balanced by an appropriate exogenous intake of preformed arginine to maintain protein homeostasis and promote recovery from this catabolic condition. PMID:7572742

  3. Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana

    SciTech Connect

    He, Yan; Galant, Ashley; Pang, Qiuying; Strul, Johanna M.; Balogun, Sherifat F.; Jez, Joseph M.; Chen, Sixue

    2012-10-24

    The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 {angstrom} resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2'-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2'-methylthio)ethylmalate {approx}100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.

  4. Site reactivity in the free radicals induced damage to leucine residues: a theoretical study.

    PubMed

    Medina, M E; Galano, A; Alvarez-Idaboy, J R

    2015-02-21

    Several recent computational studies have tried to explain the observed selectivity in radical damage to proteins. In this work we use Density Functional Theory and Transition State Theory including tunnelling corrections, reaction path degeneracy, the effect of diffusion, and the role of free radicals to get further insights into this important topic. The reaction between a leucine derivative and free radicals of biological significance, in aqueous and lipid media, has been investigated. Both thermochemical and kinetic analyses, in both hydrophilic and hydrophobic environments, have been carried out. DPPH, ˙OOH, ˙OOCH3, ˙OOCH2Cl, ˙OOCHCl2 and ˙OOCHCH2 radicals do not react with the target molecule. The reactions are proposed to be kinetically controlled. The leucine gamma site was the most reactive for the reactions with ˙N3, ˙OOCCl3, ˙OCH3, ˙OCH2Cl, and ˙OCHCl2 radicals, with rate constants equal to 1.97 × 10(5), 3.24 × 10(4), 6.68 × 10(5), 5.98 × 10(6) and 8.87 × 10(8) M(-1) s(-1), respectively, in aqueous solution. The ˙Cl, ˙OH and ˙OCCl3 radicals react with leucine at the beta, gamma, and delta positions at rates close to the diffusion limit with the alpha position which is the slowest path and the most thermodynamically favored. The presented results confirm that the Bell-Evans-Polanyi principle does not apply for the reactions between amino acid residues and free radicals. Regarding the influence of the environment on the reactivity of the studied series of free radicals towards leucine residues, it is concluded that hydrophilic media slightly lower the reactivity of the studied radicals, compared to hydrophobic ones, albeit the trends in reactivity are very similar. PMID:25592549

  5. Fragment-Based Discovery of Type I Inhibitors of Maternal Embryonic Leucine Zipper Kinase

    PubMed Central

    2014-01-01

    Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology. PMID:25589925

  6. Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary

    PubMed Central

    Stegman, Monica R; Cottrell, Matthew T; Kirchman, David L

    2014-01-01

    Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell 3H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware coast from August through December and on transects through the Delaware estuary in August and November 2011. The percent of active AAP bacteria was up to twofold higher than the percentage of active cells in the rest of the bacterial community in the estuary. Likewise, the silver grain area around active AAP bacteria in microautoradiography preparations was larger than the area around cells in the rest of the bacterial community, indicating higher rates of leucine consumption by AAP bacteria. The cell size of AAP bacteria was 50% bigger than the size of other bacteria, about the same difference on average as measured for activity. The abundance of AAP bacteria was negatively correlated and their activity positively correlated with light availability in the water column, although light did not affect 3H-leucine incorporation in light–dark experiments. Our results suggest that AAP bacteria are bigger and more active than other bacteria, and likely contribute more to organic carbon fluxes than indicated by their abundance. PMID:24824666

  7. Phase Shift of the Circadian Rhythm of Lemna Caused by Pulses of a Leucine Analog, Trifluoroleucine

    PubMed Central

    Kondo, Takao

    1988-01-01

    Pulses of a fluorinated analog of leucine, 5′,5′,5′-trifluoroleucine, reset the phase of the circadian rhythm of K+ uptake in Lemna gibba G3 under continuous light conditions. The trifluoroleucine pulse caused the largest delay phase-shifts during the early subjective phase but it caused only small phase advances. The action of trifluoroleucine was investigated and the following results were obtained. (a) The uptake of trifluoroleucine was essentially the same at all circadian phases, even though phase shifting was dramatically different at different phases. At effective phases, the magnitude of phase shifting was well correlated with the amount of trifluoroleucine taken up by the duckweed. (b) The trifluoroleucine pulse lowered the endogenous content of valine and leucine but these decreases did not correlate with phase shifting. (c) Protein synthesis was not affected by trifluoroleucine pulses which caused large phase shifts. (d) Pulses of 4-azaleucine, a different structural analog of leucine, also caused phase shifting. However, neither the direction nor the effective times of phase shifting were similar to those of trifluoroleucine. Taken together, these results negate the proposition that trifluoroleucine and azaleucine caused phase shift by disturbing amino acid metabolism and/or inhibiting protein synthesis, but they suggest instead that these analogs are incorporated into some protein(s) which are necessary for normal clock operation. PMID:16666410

  8. Leucine metabolism regulates TRI6 expression and affects deoxynivalenol production and virulence in Fusarium graminearum.

    PubMed

    Subramaniam, Rajagopal; Narayanan, Swara; Walkowiak, Sean; Wang, Li; Joshi, Manisha; Rocheleau, Hélène; Ouellet, Thérèse; Harris, Linda J

    2015-11-01

    TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum. PMID:26248604

  9. Conversion of L-leucine to isovaleric acid by Propionibacterium freudenreichii TL 34 and ITGP23.

    PubMed

    Thierry, Anne; Maillard, Marie-Bernadette; Yvon, Mireille

    2002-02-01

    Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of L-leucine, alpha-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced alpha-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal noralpha-hydroxyisocaproic acid was detected. L-Isoleucine and L-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24 degrees C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening. PMID:11823198

  10. Specific regulatory interconnection between the leucine and histidine pathways of Neurospora crassa.

    PubMed Central

    Kidd, G L; Gross, S R

    1984-01-01

    Leucine auxotrophs of Neurospora fall into two discrete categories with respect to sensitivity to the herbicide, 3-amino-1,2,4-triazole. The pattern of resistance corresponds exactly to the ability to produce the leucine pathway control elements, alpha-isopropylmalate and the leu-3 product. An analysis of the regulatory response of the production of enzymes of histidine biosynthesis to alpha-isopropylmalate implicates the control elements of the leucine pathway as important components of the mechanism governing the production of the target enzyme of aminotriazole inhibition, imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19). The evidence suggests that the regulatory interconnection between the two pathways is direct and is independent of other general integrating regulatory mechanisms which appear to be operative in both pathways. A general method for isolating leu-1 and leu-2, as well as other regulatory mutants, is described, which takes advantage of the specificity of the resistance to the inhibitor. Use of analogous systems is prescribed for the analysis of other regulatory interconnections which, like this one, might not be anticipated directly from structural or biosynthetic considerations. PMID:6325383

  11. Estimation of whole body protein synthesis from oxidation of infused [1-14C]leucine.

    PubMed

    Yagi, M; Walser, M

    1990-01-01

    In rats infused with NaH14CO3, 14CO2 exhalation approached an asymptotic rate equal to 96.0 +/- 1.3 (SD) % of the infusion rate of 14C in a monoexponential manner with a half-time of 20 min. In rats infused with [1-14C]leucine, 14CO2 exhalation (/0.96) approached an asymptotic value, (1-F), of 16-25% of 14C infusion rate (mean 20.4%) in a monoexponential manner with a half-time of approximately 31 min. Whole body protein synthesis (S) was calculated from 1-F and urea N plus ammonia N excretion (C) as S = CF/(1-F). S was a uniform function of body weight in these rats, in six additional rats in which S was measured 6 h after single intravenous injection of [1-14C]leucine and also in previously reported rats given single injections. The relationship was S (mg.100 g-1.h-1) = 11-0.143 body wt (g) +/- 8.3. In six of these rats, S was also estimated from the plateau specific activity of plasma leucine or plasma 2-ketoisocaproate (KIC); the former estimate of S was significantly lower (by an average of 17%), but S was the same when specific activity of KIC was employed. These results support the validity of the expired 14CO2 technique for measuring S. PMID:2154116

  12. Impact of Leucine Supplementation on Exercise Training Induced Anti-Cardiac Remodeling Effect in Heart Failure Mice

    PubMed Central

    de Moraes, Wilson Max Almeida Monteiro; Melara, Thaís Plasti; de Souza, Pamella Ramona Moraes; de Salvi Guimarães, Fabiana; Bozi, Luiz Henrique Marchesi; Brum, Patricia Chakur; Medeiros, Alessandra

    2015-01-01

    Leucine supplementation potentiates the effects of aerobic exercise training (AET) on skeletal muscle; however, its potential effects associated with AET on cardiac muscle have not been clarified yet. We tested whether leucine supplementation would potentiate the anti-cardiac remodeling effect of AET in a genetic model of sympathetic hyperactivity-induced heart failure in mice (α2A/α2CARKO). Mice were assigned to five groups: wild type mice treated with placebo and sedentary (WT, n = 11), α2A/α2CARKO treated with placebo and sedentary (KO, n = 9), α2A/α2CARKO treated with leucine and sedentary (KOL, n = 11), α2A/α2CARKO treated with placebo and AET (KOT, n = 12) or α2A/α2CARKO treated with leucine and AET (KOLT, n = 12). AET consisted of four weeks on a treadmill with 60 min sessions (six days/week, 60% of maximal speed) and administration by gavage of leucine (1.35 g/kg/day) or placebo (distilled water). The AET significantly improved exercise capacity, fractional shortening and re-established cardiomyocytes’ diameter and collagen fraction in KOT. Additionally, AET significantly prevented the proteasome hyperactivity, increased misfolded proteins and HSP27 expression. Isolated leucine supplementation displayed no effect on cardiac function and structure (KOL), however, when associated with AET (KOLT), it increased exercise tolerance to a higher degree than isolated AET (KOT) despite no additional effects on AET induced anti-cardiac remodeling. Our results provide evidence for the modest impact of leucine supplementation on cardiac structure and function in exercised heart failure mice. Leucine supplementation potentiated AET effects on exercise tolerance, which might be related to its recognized impact on skeletal muscle. PMID:25988767

  13. Lean body mass change over 6 years is associated with dietary leucine intake in an older Danish population.

    PubMed

    McDonald, Cameron Keith; Ankarfeldt, Mikkel Z; Capra, Sandra; Bauer, Judy; Raymond, Kyle; Heitmann, Berit Lilienthal

    2016-05-01

    Higher protein intake, and particularly higher leucine intake, is associated with attenuated loss of lean body mass (LBM) over time in older individuals. Dietary leucine is thought to be a key mediator of anabolism. This study aimed to assess this relationship over 6 years among younger and older adult Danes. Dietary leucine intake was assessed at baseline and after 6 years in men and women, aged 35-65 years, participating in the Danish cohort of the WHO-MONICA (Multinational MONItoring of trends and determinants in CArdiovascular disease) study (n 368). Changes in LBM over the 6 years were measured by bioelectrical impedance using equations developed for this Danish population. The association between leucine and LBM changes was examined using multivariate linear regression and ANCOVA analyses adjusted for potential confounders. After adjustment for baseline LBM, sex, age, energy intake and physical activity, leucine intake was associated with LBM change in those older than 65 years (n 79), with no effect seen in those younger than 65 years. Older participants in the highest quartile of leucine intake (7·1 g/d) experienced LBM maintenance, whereas lower intakes were associated with LBM loss over 6 years (for trend: β=0·434, P=0·03). Sensitivity analysis indicated no effect modification of sex or the presence of CVD. Greater leucine intake in conjunction with adequate total protein intake was associated with long-term LBM retention in a healthy older Danish population. This study corroborates findings from laboratory investigations in relation to protein and leucine intakes and LBM change. A more diverse and larger sample is needed for confirmation of these results. PMID:26979049

  14. Biosynthesis of 4-methyleneglutamic acid by peanut seedlings: Evidence for the involvement of a distinct source of leucine

    SciTech Connect

    Winter, H.C.; Dekker, E.E. )

    1989-04-01

    Germinating peanut seeds accumulate 4-methyleneglutamic acid its {gamma}-amide(MeGlx), as well as 4-methylglutamic acid(MGlu) for which leucine has been implicated as a precursor. When we incubated detached peanut cotyledons with {sup 14}C-leucine for 24-96 hr, most of the label was found in non-extractable components, while small but significant amounts were present in MeGlx, MGlu, and free leucine. The level of leucine in storage protein of ungerminated seeds is similar to the maximum level of MeGlx found in germinated seeds; further correlations were observed in various peanut tissues between rapid accumulation of MeGlx and the presence of high levels of glyoxysomal enzymes (catalase and isocitrate lyase). These results suggest that during germination, most of the leucine in the seed storage protein is converted to MeGlx, possibly by a glyoxysomal oxidase system in cotyledons, whereas most of the free leucine for protein synthesis is formed de novo.

  15. Hypertrophy-Promoting Effects of Leucine Supplementation and Moderate Intensity Aerobic Exercise in Pre-Senescent Mice

    PubMed Central

    Xia, Zhi; Cholewa, Jason; Zhao, Yan; Yang, Yue-Qin; Shang, Hua-Yu; Guimarães-Ferreira, Lucas; Naimo, Marshall Alan; Su, Quan-Sheng; Zanchi, Nelo Eidy

    2016-01-01

    Several studies have indicated a positive influence of leucine supplementation and aerobic training on the aging skeletal muscle signaling pathways that control muscle protein balance and muscle remodeling. However, the effect of a combined intervention requires further clarification. Thirteen month old CD-1® mice were subjected to moderate aerobic exercise (45 min swimming per day with 3% body weight workload) and fed a chow diet with 5% leucine or 3.4% alanine for 8 weeks. Serum and plasma were prepared for glucose, urea nitrogen, insulin and amino acid profile analysis. The white gastrocnemius muscles were used for determination of muscle size and signaling proteins involved in protein synthesis and degradation. The results show that both 8 weeks of leucine supplementation and aerobic training elevated the activity of mTOR (mammalian target of rapamycin) and its downstream target p70S6K and 4E-BP1, inhibited the ubiquitin-proteasome system, and increased fiber cross-sectional area (CSA) in white gastrocnemius muscle. Moreover, leucine supplementation in combination with exercise demonstrated more significant effects, such as greater CSA, protein content and altered phosphorylation (suggestive of increased activity) of protein synthesis signaling proteins, in addition to lower expression of proteins involved in protein degradation compared to leucine or exercise alone. The current study shows moderate aerobic training combined with 5% leucine supplementation has the potential to increase muscle size in fast-twitch skeletal muscle during aging, potentially through increased protein synthesis and decreased protein breakdown. PMID:27144582

  16. Discovery and application of new bacterial strains for asymmetric synthesis of L-tert-butyl leucine in high enantioselectivity.

    PubMed

    Jin, Jian-Zhong; Chang, Dong-Liang; Zhang, Jie

    2011-06-01

    Discovery of new bacterial strains with fast identification in a miniaturized system was performed for the synthesis of optically active L-tert-butyl leucine. With tert-butyl leucine amide as nitrogen source, one bacterial strain with high conversion and high enantioselectivity was discovered among 120 isolated microorganisms from local soils and identified as Mycobacterium sp. JX009. Glucose and ammonium chloride were examined as the good carbon source and nitrogen source for the cells' growth separately. The cells grew better at 30 °C and at pH 7.5 with higher activity of 2,650 U/l in comparison with other conditions. Cells' stability was improved by immobilization on synthetic resin 0730 without pretreatment. Tert-butyl leucine amide (30 mM) was successfully hydrolyzed by immobilized cells and examined as the highest chemical concentration that cells could endure. After six reaction cycles, the immobilized cells retained 90% activity with production of L-tert-butyl leucine in 98% ee. The results firstly reported the application of new bacterial strain in the hydrolysis of tert-butyl leucine amide to produce optically active L-tert-butyl leucine in an efficient way with investigation in detail. PMID:21153891

  17. Physical exercise and a leucine-rich diet modulate the muscle protein metabolism in Walker tumor-bearing rats.

    PubMed

    Salomão, Emilianne M; Toneto, Aline T; Silva, Gisele O; Gomes-Marcondes, Maria Cristina C

    2010-01-01

    Leucine-supplemented diet can recover lean body mass and preserve muscle protein mass. Additionally, physical exercise can be an excellent alternative to improve the rehabilitation of cancer patients. Knowing these facts, we examined the effects of a leucine-rich diet with or without physical aerobic exercise on muscle protein metabolism in Walker tumor-bearing rats. Young rats were divided into 4 groups that did or did not perform light aerobic exercise (swim training) and were on a leucine-rich diet or a control diet for 2 mo. After this time, these animals were implanted or not with tumors (subcutaneously) following groups for either control diet or leucine-rich diet fed rats: control, trained, tumor-bearing, and trained tumor-bearing. Twenty-one days after implantation, the tumor growth induced a decrease in the muscle protein synthesis and increased the catabolic process, which was associated with an increase in the expression of the ubiquitin-proteasome subunits (20S, 19S, and 11S). In contrast, the exercise program minimized the muscle degradation process and increased muscle myosin content. Additionally, leucine supplementation also modulated proteasome subunits, especially the 19S and 11S. In summary, the exercise has beneficial effects by reducing tumor growth, leading to an improvement in protein turnover especially when in conjunction with a leucine-rich diet. PMID:21058197

  18. Hypertrophy-Promoting Effects of Leucine Supplementation and Moderate Intensity Aerobic Exercise in Pre-Senescent Mice.

    PubMed

    Xia, Zhi; Cholewa, Jason; Zhao, Yan; Yang, Yue-Qin; Shang, Hua-Yu; Guimarães-Ferreira, Lucas; Naimo, Marshall Alan; Su, Quan-Sheng; Zanchi, Nelo Eidy

    2016-01-01

    Several studies have indicated a positive influence of leucine supplementation and aerobic training on the aging skeletal muscle signaling pathways that control muscle protein balance and muscle remodeling. However, the effect of a combined intervention requires further clarification. Thirteen month old CD-1(®) mice were subjected to moderate aerobic exercise (45 min swimming per day with 3% body weight workload) and fed a chow diet with 5% leucine or 3.4% alanine for 8 weeks. Serum and plasma were prepared for glucose, urea nitrogen, insulin and amino acid profile analysis. The white gastrocnemius muscles were used for determination of muscle size and signaling proteins involved in protein synthesis and degradation. The results show that both 8 weeks of leucine supplementation and aerobic training elevated the activity of mTOR (mammalian target of rapamycin) and its downstream target p70S6K and 4E-BP1, inhibited the ubiquitin-proteasome system, and increased fiber cross-sectional area (CSA) in white gastrocnemius muscle. Moreover, leucine supplementation in combination with exercise demonstrated more significant effects, such as greater CSA, protein content and altered phosphorylation (suggestive of increased activity) of protein synthesis signaling proteins, in addition to lower expression of proteins involved in protein degradation compared to leucine or exercise alone. The current study shows moderate aerobic training combined with 5% leucine supplementation has the potential to increase muscle size in fast-twitch skeletal muscle during aging, potentially through increased protein synthesis and decreased protein breakdown. PMID:27144582

  19. Leucine supplementation is anti-atrophic during paradoxical sleep deprivation in rats.

    PubMed

    de Sá Souza, Helton; Antunes, Hanna Karen Moreira; Dáttilo, Murilo; Lee, Kil Sun; Mônico-Neto, Marcos; de Campos Giampa, Sara Quaglia; Phillips, Stuart M; Tufik, Sergio; de Mello, Marco Túlio

    2016-04-01

    The purpose of this study was to identify sleep deprivation-induced atrophy and the muscle-specific fiber types affected and to determine the effects of leucine supplementation on atrophy and pertinent portions of the pathways of muscle protein synthesis and degradation in rats. A total of 46 Wistar rats were distributed in four groups: control (CTL), leucine supplementation (LEU), sleep deprivation (SD), and leucine supplementation + sleep deprivation (LEU + SD). Leucine supplementation was by gavage (1.35 g/kg/daily), and the animals were subjected to SD for 96 h. Testosterone and corticosterone concentrations, along with proteins involved in protein synthesis and degradation and proteasome activity levels, were measured in the gastrocnemius (GA) muscle. Myosin ATPase staining was used to evaluate the different muscle fibers. After sleep deprivation, GA muscle and body masses decreased in the SD group compared to the CTL, LEU, and LEU + SD groups. There was no difference between groups in type I fiber cross-sectional area (CSA). The CSAs for type IIa fibers were lower in the SD and LEU + SD groups vs. the CTL and LEU groups, while the IIb fiber CSA was lower in the SD group vs. the CSAs in all other groups. The phospho (p)-Akt levels were lower in the SD and LEU + SD groups vs. the CTL and LEU groups. The p-mTORC1 levels were higher in the LEU, SD, and LEU + SD groups vs. the CTL group. The p-p70S6k levels were higher in the LEU and LEU + SD groups; the 4E-BP1 levels were higher in the SD and LEU + SD groups compared to those in the CTL and LEU groups, and the p-4E-BP1 levels were higher in the LEU and SD groups compared to those in the CTL group and even higher in the LEU + SD group compared to those in the LEU and SD groups. Ubiquitinated proteins, LC3, and p62/SQSTM, and proteasome activity levels were higher in the SD and LEU + SD groups vs. the LEU and CTL groups. Sleep deprivation led to the atrophy of IIa and IIb muscle fibers; however, leucine

  20. The Basic and Semi-Basic

    ERIC Educational Resources Information Center

    Spitler, Gail

    1978-01-01

    Presented is a paradigm for teaching basic and semibasic arithmetic facts to children with arithmetic difficulties, in which the student progresses from the use of concrete materials such as blocks, to the use of diagrams such as tally marks, to a reasoning process, to responding in an automatic manner. (DLS)

  1. "Back to Basics" or "Forward to Basics"?

    ERIC Educational Resources Information Center

    Perso, Thelma

    2007-01-01

    Politicians have used the promise of "back to basics in our schools" as an educational platform for some time now, possibly in recognition that this is something the general population perceives as an issue they might just vote for. In the various positions the author has held, both professional and in community service, she has been required to…

  2. Leucine modulates peptide transport system-1 across the blood-brain barrier at the stereospecific site within the central nervous system.

    PubMed

    Banks, W A; Kastin, A J

    1991-04-01

    Previous results have shown that leucine injected into a cerebral ventricle (i.c.v.) can act as an allosteric regulator of peptide transport system-1 (PTS-1), the system that transports Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1) and the enkephalins out of the central nervous system (CNS). D-Leucine appeared more potent than L-leucine. In the current study, dose-response curves were constructed for each compound after both intravenous (i.v.) and i.c.v. injection. Based on ED50 values after i.c.v. injection, D-leucine was about 200 times more potent than L-leucine in its inhibition of PTS-1, thereby confirming stereospecificity of the allosteric site. D- and L-Leucine were also more potent when given i.c.v. than when given i.v., suggesting that the site is located on the CNS side of the blood-brain barrier (BBB). The finding that D-leucine was less potent than L-leucine when given i.v. is also consistent with a CNS site of action because the L-isomer of leucine has been shown to be preferentially transported into the brain. These findings agree with the previous suggestion that some of the neurotoxic effects of leucine may be mediated through PTS-1 and could help explain how D-amino acids can exert opiate-related effects on the CNS. PMID:1676737

  3. Blood-brain barrier permeability to leucine-enkephalin, D-alanine2-D-leucine5-enkephalin and their N-terminal amino acid (tyrosine).

    PubMed

    Zlokovic, B V; Begley, D J; Chain-Eliash, D G

    1985-06-10

    The permeability of the blood-brain barrier to [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) (abbreviated to Leu-Enk) and of its synthetic analogue D-alanine2-[tyrosyl-3,5-3H]enkephalin-(5-D-leucine) (abbreviated to D-Ala2-D-Leu5-Enk) was studied, in the adult rat, by means of Oldendorf's27 intracarotid injection technique. The brain uptake index (BUI) corrected for residual vascular radioactivity was about the same for both peptides, indicating a low extraction from the blood during a 5- or 15-s period of exposure to the peptides. Transport of Leu-Enk was not saturated by unlabelled Enk at a concentration as high as 5 mM but was completely abolished by 5mM tyrosine and by the inhibitor of aminopeptidase activity, bacitracin (2 mM). Also the typical L-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2 carboxylic acid (BCH)9 at 10 mM concentration markedly reduced (by 80%) Leu-Enk uptake by the brain. In contrast, brain uptake of D-Ala2-D-Leu5-Enk was reduced only to about one-half of its control value by bacitracin or by 25% by BCH. Brain uptake for L-tyrosine was typically large and markedly inhibited by BCH but not inhibited by 5 mM unlabelled Leu-Enk. These results show that the measurable but low first-pass extractions for enkephalins are not representative of the uptake of these peptides into the brain, but rather reflect their extreme sensitivity to enzymatic degradation with a release of the N-terminal tyrosine residue. The results also suggest that small amounts of D-Ala2-D-Leu5-Enk might cross the blood-brain barrier in an intact form.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3891014

  4. Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

    PubMed

    Coffelt, Seth B; Tomchuck, Suzanne L; Zwezdaryk, Kevin J; Danka, Elizabeth S; Scandurro, Aline B

    2009-06-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  5. Leucine Leucine-37 Uses Formyl Peptide Receptor–Like 1 to Activate Signal Transduction Pathways, Stimulate Oncogenic Gene Expression, and Enhance the Invasiveness of Ovarian Cancer Cells

    PubMed Central

    Coffelt, Seth B.; Tomchuck, Suzanne L.; Zwezdaryk, Kevin J.; Danka, Elizabeth S.; Scandurro, Aline B.

    2009-01-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor–like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein–coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37–induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37–stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37–treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  6. Prolonged leucine supplementation does not augment muscle mass or affect glycemic control in elderly type 2 diabetic men.

    PubMed

    Leenders, Marika; Verdijk, Lex B; van der Hoeven, Letty; van Kranenburg, Janneau; Hartgens, Fred; Wodzig, Will K W H; Saris, Wim H M; van Loon, Luc J C

    2011-06-01

    The loss of muscle mass with aging has been, at least partly, attributed to a blunted muscle protein synthetic response to food intake. Leucine coingestion has been reported to stimulate postprandial insulin release and augment postprandial muscle protein accretion. We assessed the clinical benefits of 6 mo of leucine supplementation in elderly, type 2 diabetes patients. Sixty elderly males with type 2 diabetes (age, 71 ± 1 y; BMI, 27.3 ± 0.4 kg/m(2)) were administered 2.5 g L-leucine (n = 30) or a placebo (n = 30) with each main meal during 6 mo of nutritional intervention (7.5 g/d leucine or placebo). Body composition, muscle fiber characteristics, muscle strength, glucose homeostasis, and basal plasma amino acid and lipid concentrations were assessed prior to, during, and after intervention. Lean tissue mass did not change or differ between groups and at 0, 3, and 6 mo were 61.9 ± 1.1, 62.2 ± 1.1, and 62.0 ± 1.0 kg, respectively, in the leucine group and 62.2 ± 1.3, 62.2 ± 1.3, and 62.2 ± 1.3 kg in the placebo group. There also were no changes in body fat percentage, muscle strength, and muscle fiber type characteristics. Blood glycosylated hemoglobin did not change or differ between groups and was 7.1 ± 0.1% in the leucine group and 7.2 ± 0.2% in the placebo group. Consistent with this, oral glucose insulin sensitivity and plasma lipid concentrations did not change or differ between groups. We conclude that prolonged leucine supplementation (7.5 g/d) does not modulate body composition, muscle mass, strength, glycemic control, and/or lipidemia in elderly, type 2 diabetes patients who habitually consume adequate dietary protein. PMID:21525248

  7. Basic Science Training Program.

    ERIC Educational Resources Information Center

    Brummel, Clete

    These six learning modules were developed for Lake Michigan College's Basic Science Training Program, a workshop to develop good study skills while reviewing basic science. The first module, which was designed to provide students with the necessary skills to study efficiently, covers the following topics: time management; an overview of a study…

  8. HEBREW BASIC COURSE.

    ERIC Educational Resources Information Center

    LEVINSON, HANNA; REIF, JOSEPH A.

    THIS HEBREW BASIC COURSE IS INTENDED AS A TRAINING MANUAL TO TEACH STUDENTS THE INFORMAL SPEECH OF EDUCATED NATIVE ISRAELIS AND IS DESIGNED TO BE USED WITH A NATIVE INSTRUCTOR AND TAPE RECORDINGS PREPARED FOR THIS COURSE. THE UNITS CONSIST OF BASIC CONVERSATION, ADDITIONAL VOCABULARY, GRAMMAR NOTES, REVIEW CONVERSATIONS, AND DRILLS ON VOCABULARY,…

  9. BASIC Beats PASCAL.

    ERIC Educational Resources Information Center

    Ever, Jacob

    1981-01-01

    Features of two versions of the BASIC programing language are compared with the features of the PASCAL programing language. The application chosen for comparison was a word processor. The conclusion was that PASCAL had the best language features, but BASIC had better systems capabilities. (MP)

  10. Exponentiation: A New Basic?

    ERIC Educational Resources Information Center

    Davis, Brent

    2015-01-01

    For centuries, the basic operations of school mathematics have been identified as addition, subtraction, multiplication, and division. Notably, these operations are "basic," not because they are foundational to mathematics knowledge, but because they were vital to a newly industrialized and market-driven economy several hundred years…

  11. TOOLS AND BASIC MACHINES.

    ERIC Educational Resources Information Center

    George Washington Univ., Washington, DC. School of Education.

    THIS BASIC READER IS A PART OF AN EXPERIMENTAL CURRICULUM DEVELOPMENT PROJECT DESCRIBED IN VT 004 454, TO DEVELOP AND EVALUATE SPECIAL NEW TRAINING MATERIALS TO TEACH BASIC VOCATIONAL TALENT SKILLS TO DISADVANTAGED STUDENTS WHICH WERE TESTED ON APPROXIMATELY 2,500 EIGHTH AND NINTH GRADERS IN EIGHT SCHOOL SYSTEMS ACROSS THE NATION. THIS READER WAS…

  12. Romanian Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    The "Romanian Basic Course," consisting of 89 lesson units in eight volumes, is designed to train native English language speakers to Level 3 proficiency in comprehension, speaking, reading, and writing Romanian (based on a 1-5 scale in which Level 5 is native speaker proficiency). Volume 1, which introduces basic sentences in dialog form with…

  13. Construction & Basic Skills.

    ERIC Educational Resources Information Center

    BCEL Newsletter for the Business and Literacy Communities, 1991

    1991-01-01

    Basic skills education has become a pressing need in the construction industry as jobs become more complex and fewer workers have needed skills. However, the construction industry lags in spending on training for entry-level workers. The Home Builders Institute (HBI) is testing a pilot basic skills program that it hopes will prove useful to the…

  14. Fluency with Basic Addition

    ERIC Educational Resources Information Center

    Garza-Kling, Gina

    2011-01-01

    Traditionally, learning basic facts has focused on rote memorization of isolated facts, typically through the use of flash cards, repeated drilling, and timed testing. However, as many experienced teachers have seen, "drill alone does not develop mastery of single-digit combinations." In contrast, a fluency approach to learning basic addition…

  15. Basic Electronics I.

    ERIC Educational Resources Information Center

    Robertson, L. Paul

    Designed for use in basic electronics programs, this curriculum guide is comprised of twenty-nine units of instruction in five major content areas: Orientation, Basic Principles of Electricity/Electronics, Fundamentals of Direct Current, Fundamentals of Alternating Current, and Applying for a Job. Each instructional unit includes some or all of…

  16. Bracken Basic Concept Scale.

    ERIC Educational Resources Information Center

    Naglieri, Jack A.; Bardos, Achilles N.

    1990-01-01

    The Bracken Basic Concept Scale, for use with preschool and primary-aged children, determines a child's school readiness and knowledge of English-language verbal concepts. The instrument measures 258 basic concepts in such categories as comparisons, time, quantity, and letter identification. This paper describes test administration, scoring and…

  17. The leucine twenty homeobox (LEUTX) gene, which lacks a histone acetyltransferase domain, is fused to KAT6A in therapy-related acute myeloid leukemia with t(8;19)(p11;q13).

    PubMed

    Chinen, Yoshiaki; Taki, Tomohiko; Tsutsumi, Yasuhiko; Kobayashi, Satoru; Matsumoto, Yosuke; Sakamoto, Natsumi; Kuroda, Junya; Horiike, Shigeo; Nishida, Kazuhiro; Ohno, Hirofumi; Uike, Naokuni; Taniwaki, Masafumi

    2014-04-01

    The monocytic leukemia zinc finger protein KAT6A (formerly MOZ) gene is recurrently rearranged by chromosomal translocations in acute myeloid leukemia (AML). KAT6A is known to be fused to several genes, all of which have histone acetyltransferase (HAT) activity and interact with a number of transcription factors as a transcriptional coactivator. The present study shows that the leucine twenty homeobox (LEUTX) gene on 19q13 is fused to the KAT6A gene on 8p11 in a therapy-related AML with t(8;19)(p11;q13) using the cDNA bubble PCR method. The fusion transcripts contained 83 nucleotides upstream of the first ATG of LEUTX and are presumed to create in-frame fusion proteins. LEUTX is known to have a homeobox domain. Expression of the LEUTX gene was only detected in placenta RNA by RT-PCR, but not in any tissues by Northern blot analysis. The putative LEUTX protein does not contain any HAT domain, and this is the first study to report that KAT6A can fuse to the homeobox gene. The current study, with identification of a new partner gene to KAT6A in a therapy-related AML, does not elucidate the mechanisms of leukemogenesis in KAT6A-related AML but describes a new gene with a different putative function. PMID:24446090

  18. Leucine disposal rate for assessment of amino acid metabolism in maintenance hemodialysis patients

    PubMed Central

    Denny, Gerald B.; Deger, Serpil M.; Chen, Guanhua; Bian, Aihua; Sha, Feng; Booker, Cindy; Kesler, Jaclyn T.; David, Sthuthi; Ellis, Charles D.; Ikizler, T. Alp

    2016-01-01

    Background Protein energy wasting (PEW) is common in patients undergoing maintenance hemodialysis (MHD) and closely associated with poor outcomes. Insulin resistance and associated alterations in amino acid metabolism are potential pathways leading to PEW. We hypothesized that the measurement of leucine disposal during a hyperinsulinemic- euglycemic-euaminoacidemic clamp (HEAC) procedure would accurately measure the sensitivity to insulin for its actions on concomitant carbohydrate and protein metabolism in MHD patients. Methods We examined 35 MHD patients and 17 control subjects with normal kidney function by hyperinsulinemic-euglycemic clamp (HEGC) followed by HEAC clamp procedure to obtain leucine disposal rate (LDR) along with isotope tracer methodology to assess whole body protein turnover. Results The glucose disposal rate (GDR) by HEGC was 5.1 ± 2.1 mg/kg/min for the MHD patients compared to 6.3 ± 3.9 mg/kg/min for the controls (p = 0.38). The LDR during HEAC was 0.09 ± 0.03 mg/kg/min for the MHD patients compared to 0.11 ± 0.05 mg/kg/min for the controls (p = 0.009). The LDR level was correlated with whole body protein synthesis (r = 0.25; p = 0.08), with whole body protein breakdown (r = −0.38 p = 0.01) and net protein balance (r = 0.85; p < 0.001) in the overall study population. Correlations remained significant in subgroup analysis. The GDR derived by HEGC and LDR correlated well in the controls (r = 0.79, p < 0.001), but less so in the MHD patients (r = 0.58, p < 0.001). Conclusions Leucine disposal rate reliably measures amino acid utilization in MHD patients and controls in response to high dose insulin. PMID:27413537

  19. A Membrane Leucine Heptad Contributes to Trafficking, Signaling, and Transformation by Latent Membrane Protein 1▿

    PubMed Central

    Lee, Jisook; Sugden, Bill

    2007-01-01

    Latent membrane protein 1 (LMP1) of Epstein Barr virus (EBV) is important for maintaining proliferation of EBV-infected B cells. LMP1, unlike its cellular counterpart, CD40, signals without a ligand and is largely internal to the plasma membrane. In order to understand how LMP1 initiates its ligand-independent signaling, we focused on a leucine heptad in LMP1's first membrane-spanning domain that was shown to be necessary for LMP1's signaling through NF-κB. LZ1EBV, a recombinant EBV genetically altered to express LZ1, a derivative of LMP1 in which a leucine heptad was replaced with alanines, transformed B cells with 56% of wild-type (wt) EBV's efficiency, demonstrating the importance of this heptad. To elucidate the mechanism by which this domain contributes to the functions of LMP1, the properties of the wt and LZ1 were compared in transfected cells. LZ1 failed to home to lipid rafts as efficiently as did wt LMP1. The distribution of tagged derivatives of LZ1 also differed from that of wt LMP1 in transfected cells. LZ1's defect in homing to lipid rafts and altered trafficking likely underlie the defect in transformation of LZ1EBV. While the third and fourth membrane-spanning domains of LMP1 foster its trafficking to the Golgi, the leucine heptad within the first membrane-spanning domain contributes to its trafficking, particularly to internal rafts. B cells that are successfully transformed by LZ1EBV have the same average number of viral genomes and the same fraction of cells with capped LZ1 at the cell surface but express 50% more of the LZ1 allele than wt infected cells. PMID:17581993

  20. Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway.

    PubMed

    Suryawan, Agus; Nguyen, Hanh V; Almonaci, Rosemarie D; Davis, Teresa A

    2012-02-28

    Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine's action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg(-1)·h(-1)) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2α and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation. PMID:22675606

  1. Long-term remission with rituximab in refractory leucine-rich glioma inactivated 1 antibody encephalitis.

    PubMed

    Brown, J William L; Martin, Peter J; Thorpe, John W; Michell, Andrew W; Coles, Alasdair J; Cox, Amanda L; Vincent, Angela; Zandi, Michael S

    2014-06-15

    Autoimmune encephalitis associated with antibodies to leucine-rich glioma inactivated 1 (LGI1) is recently described and there is a lack of detailed reports on the treatment of relapsing or refractory cases and long-term outcomes. Two case reports are presented. Both cases had faciobrachial dystonic seizures (FBDS) and received rituximab after relapsing or refractory disease. Both cases achieved sustained clinical remission of up to 15 and 56 months respectively. Rituximab use allowed withdrawal of corticosteroids and was well tolerated. Randomized clinical trials are needed in LGI1 encephalitis and other autoimmune encephalitides. PMID:24703099

  2. Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase.

    PubMed

    Johnson, Christopher N; Adelinet, Christophe; Berdini, Valerio; Beke, Lijs; Bonnet, Pascal; Brehmer, Dirk; Calo, Frederick; Coyle, Joseph E; Day, Phillip J; Frederickson, Martyn; Freyne, Eddy J E; Gilissen, Ron A H J; Hamlett, Christopher C F; Howard, Steven; Meerpoel, Lieven; Mevellec, Laurence; McMenamin, Rachel; Pasquier, Elisabeth; Patel, Sahil; Rees, David C; Linders, Joannes T M

    2015-01-01

    A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK. PMID:25589926

  3. Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors.

    PubMed

    Greshock, Thomas J; Sanders, John M; Drolet, Robert E; Rajapakse, Hemaka A; Chang, Ronald K; Kim, Boyoung; Rada, Vanessa L; Tiscia, Heather E; Su, Hua; Lai, Ming-Tain; Sur, Sylvie M; Sanchez, Rosa I; Bilodeau, Mark T; Renger, John J; Kern, Jonathan T; McCauley, John A

    2016-06-01

    Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules. PMID:27106707

  4. Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase

    PubMed Central

    2014-01-01

    A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein–ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK. PMID:25589926

  5. Additive insulinogenic action of Opuntia ficus-indica cladode and fruit skin extract and leucine after exercise in healthy males

    PubMed Central

    2013-01-01

    Background Oral intake of a specific extract of Opuntia ficus-indica cladode and fruit skin (OpunDia™) (OFI) has been shown to increase serum insulin concentration while reducing blood glucose level for a given amount of glucose ingestion after an endurance exercise bout in healthy young volunteers. However, it is unknown whether OFI-induced insulin stimulation after exercise is of the same magnitude than the stimulation by other insulinogenic agents like leucine as well as whether OFI can interact with those agents. Therefore, the aims of the present study were: 1) to compare the degree of insulin stimulation by OFI with the effect of leucine administration; 2) to determine whether OFI and leucine have an additive action on insulin stimulation post-exercise. Methods Eleven subjects participated in a randomized double-blind cross-over study involving four experimental sessions. In each session the subjects successively underwent a 2-h oral glucose tolerance test (OGTT) after a 30-min cycling bout at ~70% VO2max. At t0 and t60 during the OGTT, subjects ingested 75 g glucose and capsules containing either 1) a placebo; 2) 1000 mg OFI; 3) 3 g leucine; 4) 1000 mg OFI + 3 g leucine. Blood samples were collected before and at 30-min intervals during the OGTT for determination of blood glucose and serum insulin. Results Whereas no effect of leucine was measured, OFI reduced blood glucose at t90 by ~7% and the area under the glucose curve by ~15% and increased serum insulin concentration at t90 by ~35% compared to placebo (P<0.05). From t60 to the end of the OGTT, serum insulin concentration was higher in OFI+leucine than in placebo which resulted in a higher area under the insulin curve (+40%, P<0.05). Conclusion Carbohydrate-induced insulin stimulation post-exercise can be further increased by the combination of OFI with leucine. OFI and leucine could be interesting ingredients to include together in recovery drinks to resynthesize muscle glycogen faster post

  6. Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

    PubMed

    Kathuria, Sagar V; Chan, Yvonne H; Nobrega, R Paul; Özen, Ayşegül; Matthews, C Robert

    2016-03-01

    Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates. PMID:26660714

  7. CREST - a large and diverse superfamily of putative transmembrane hydrolases

    PubMed Central

    2011-01-01

    Background A number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs) were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes. Results Using sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8). The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs. Conclusions Universal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are metal-dependent hydrolases

  8. Functional characterization of leucine transport induced in Xenopus laevis oocytes injected with mRNA isolated from midguts of lepidopteran larvae (Philosamia cynthia).

    PubMed

    Sacchi, V F; Perego, C; Magagnin, S

    1995-04-01

    The injection of poly(A)+ mRNA prepared from Philosamia cynthia midgut caused time- and dose-dependent increases of leucine transport in Xenopus laevis oocytes, with an increase in leucine uptake 1.5-3 times that of oocytes injected with water. When the NaCl concentration was reduced from 100 to 5 mmol l-1, the difference between mRNA- and water-injected oocytes was greater and a fourfold increase of L-leucine uptake was measured. D-Leucine (10 mmol l-1) completely inhibited the induced uptake of 0.1 mmol l-1 L-leucine. The newly expressed component of L-leucine uptake increased at alkaline pH and was abolished by incubation for 15 min with 15 mmol l-1 phenylglyoxal. The mean Km values, calculated using Na+ activation curves of leucine uptake, were 23.3 +/- 6.1 mmol l-1 in water-injected oocytes and 0.4 +/- 0.2 mmol l-1 for the newly expressed component of leucine uptake in mRNA-injected oocytes. On the basis of these results, we conclude that the increase of L-leucine uptake in mRNA-injected oocytes was due to the expression of a new transport system, which differs from the endogenous ones and shares many features with that found previously in Philosamia cynthia midgut. PMID:7730757

  9. The role of the N-terminal leucine residue in snake venom cardiotoxin II (Naja naja atra).

    PubMed

    Wu, C Y; Chen, W C; Ho, C L; Chen, S T; Wang, K T

    1997-04-28

    The N-terminal leucine residue of snake venom cardiotoxin II (CTX II) (Naja naja atra) was systematically replaced with D-leucine (CTXII-L1-D-L), glycine (CTXII-L1G) or deleted [CTXII-(2-60)] to study the role of leucine residue in CTX II molecule. CTX II, CTXL1-D-L, CTXL1G and CTX(2-60) were produced by chemical synthesis method and purified by high performance liquid chromatography. Owing to folding problem in CTXII-(2-60), only CTX II, CTXII-L1-D-L and CTXII-L1G were produced in a pure form and characterized by amino acid analysis, mass spectrometry and peptide mapping. In the structural aspect, changing the Leu-1 by D-Leu or Gly causes a drastic alteration in the whole CTX II structure as detected by circular dichroism, 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence assay. In the functional aspect, both CTXII-L1-D-L and CTXII-L1G are still retained substantial biological activity of CTX II. Therefore, the results indicate that both the chirality and the side-chain of the N-terminal leucine residue of CTX II are important elements in maintaining the whole CTX II structure. In addition, this study is the first report in elucidating the reason why the first N-terminal residue of most CTXs (90.3%) is leucine residue. PMID:9168920

  10. Investigations on particle surface characteristics vs. dispersion behaviour of L-leucine coated carrier-free inhalable powders.

    PubMed

    Raula, Janne; Thielmann, Frank; Naderi, Majid; Lehto, Vesa-Pekka; Kauppinen, Esko I

    2010-01-29

    Aerosol microparticles of salbutamol sulphate are gas-phase coated with an amino acid L-leucine. Depending of the saturated state of L-leucine, the coating is formed by the surface diffusion of L-leucine molecules within a droplet or by the physical vapour deposition (PVD) of L-leucine or by the combination thereof. The PVD coated particles showed excellent aerosolization characteristics in a carrier-free powder delivery from an inhaler. The aerosolization of the fine powders is compared with surface energy parameters analysed by inverse gas chromatography (IGC). The dispersion testing is conducted by a Inhalation Simulator using a fast inhalation profile with inhalation flow rate of 67 l min(-1). It is found that the powder emission is affected by the morphology, surface roughness (asperity size and density) of the particles and acidity of particle surface. The latter affects the dispersion and dose repeatability of fine powder in a case if L-leucine content is high enough. However, there is no direct correlation between dispersive surface energies and aerosolization performances of the powders. Crucial factors for the improved aerosolization rely weakly on surface acid-base properties but strongly on particle morphology and fine-scale surface roughness. PMID:19879344

  11. Effect of hyperglucagonemia on whole-body leucine metabolism in immature pigs before and during a meal

    SciTech Connect

    Ostaszewski, P.; Nissen, S. )

    1988-03-01

    Leucine metabolism was measured isotopically in 12 immature female pigs to assess the effect of acute hyperglucagonemia on leucine kinetics in both the fed and fasting states. After an overnight fast, immature pigs were infused with {alpha}-({sup 3}H)ketoisocaproate and ({sup 14}C)leucine. After a 2-h equilibration period, an infusion of either saline or 7 pg {center dot} kg{sup {minus}1} {center dot} min{sup {minus}1} of glucagon was begun, which increased plasma glucagon from {approximately}140 to {approximately}640 pg/ml and doubled the insulin concentrations. Two hours later, pigs were fed small meals to which (5,5,5-{sup 2}H{sub 3})leucine was added to trace absorption. By subtracting absorption from total leucine flux, an estimate of endogenous proteolysis during the meal was made. In the fasting state, glucagon increased proteolysis and increased oxidation. No significant glucagon-related changes in any other flux parameters occurred in the fasting state. Ingestion of the meals caused oxidation to increase 41% in control animals, whereas in glucagon-infused animals, oxidation increased 84%. Additional, animals infused with glucagon suppressed endogenous proteolysis 43% after the meal compared with 55% decrease in control animals. These data indicate that glucagon stimulates whole-body proteolysis in both the fasting and fed states.

  12. The DNA binding specificity of the basic region of the yeast transcriptional activator GCN4 can be changed by substitution of a single amino acid.

    PubMed Central

    Suckow, M; von Wilcken-Bergmann, B; Müller-Hill, B

    1993-01-01

    The X-ray structure of a GCN4 DNA complex (1) shows, that specific DNA binding of the GCN4 basic region is mediated by a complicated network of base pair and DNA backbone contacts. According to the X-ray structure, alanine -14 of the basic region of GCN4 (we define the first leucine of the leucine zipper as +1) makes a hydrophobic contact to the methyl group of the thymine next to the center of the GCN4 binding site 5' ATGACTCAT 3'. We tested the DNA binding properties of the nineteen derivatives of GCN4, which carry all possible amino acids in position -14 of the basic region. Substitution of alanine -14 of GCN4 by either asparagine or cysteine changes the DNA binding specificity. Serine in this position broadens the specificity for position 1 of the target, whereas other amino acids either retain or decrease GCN4 specificity. Images PMID:8502548

  13. Video Screen Capture Basics

    ERIC Educational Resources Information Center

    Dunbar, Laura

    2014-01-01

    This article is an introduction to video screen capture. Basic information of two software programs, QuickTime for Mac and BlueBerry Flashback Express for PC, are also discussed. Practical applications for video screen capture are given.

  14. Kidney Disease Basics

    MedlinePlus

    ... Links Take the first step Alternate Language URL Kidney Disease Basics Page Content Your kidneys filter extra water ... blood pressure are the most common causes of kidney disease. ​These conditions can slowly damage the kidneys over ...

  15. HIV Treatment: The Basics

    MedlinePlus

    HIV Treatment HIV Treatment: The Basics (Last updated 3/1/2016; last reviewed 3/1/2016) Key Points Antiretroviral therapy (ART) ... reduces the risk of HIV transmission . How do HIV medicines work? HIV attacks and destroys the infection- ...

  16. E-Mail Basics.

    ERIC Educational Resources Information Center

    Cohen, Sacha

    1996-01-01

    Offers electronic mail basics, mail etiquette and tips, interesting World Wide Web sites, and how to do a Web search. Includes Web sites that offer beginner tutorials and a glossary of Internet terms. (JOW)

  17. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    USGS Publications Warehouse

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  18. The Role of Leucine-Rich Repeat Containing Protein 10 (LRRC10) in Dilated Cardiomyopathy

    PubMed Central

    Brody, Matthew J.; Lee, Youngsook

    2016-01-01

    Leucine-rich repeat containing protein 10 (LRRC10) is a cardiomyocyte-specific member of the Leucine-rich repeat containing (LRRC) protein superfamily with critical roles in cardiac function and disease pathogenesis. Recent studies have identified LRRC10 mutations in human idiopathic dilated cardiomyopathy (DCM) and Lrrc10 homozygous knockout mice develop DCM, strongly linking LRRC10 to the molecular etiology of DCM. LRRC10 localizes to the dyad region in cardiomyocytes where it can interact with actin and α-actinin at the Z-disc and associate with T-tubule components. Indeed, this region is becoming increasingly recognized as a signaling center in cardiomyocytes, not only for calcium cycling, excitation-contraction coupling, and calcium-sensitive hypertrophic signaling, but also as a nodal signaling hub where the myocyte can sense and respond to mechanical stress. Disruption of a wide range of critical structural and signaling molecules in cardiomyocytes confers susceptibility to cardiomyopathies in addition to the more classically studied mutations in sarcomeric proteins. However, the molecular mechanisms underlying DCM remain unclear. Here, we review what is known about the cardiomyocyte functions of LRRC10, lessons learned about LRRC10 and DCM from the Lrrc10 knockout mouse model, and discuss ongoing efforts to elucidate molecular mechanisms whereby mutation or absence of LRRC10 mediates cardiac disease. PMID:27536250

  19. Positive selection in the leucine-rich repeat domain of Gro1 genes in Solanum species.

    PubMed

    Ruggieri, Valentino; Nunziata, Angelina; Barone, Amalia

    2014-12-01

    In pathogen resistant plants, solvent-exposed residues in the leucine-rich repeat (LRR) proteins are thought to mediate resistance by recognizing plant pathogen elicitors. In potato, the gene Gro1-4 confers resistance to Globodera rostochiensis. The investigation of variability in different copies of this gene represents a good model for the verification of positive selection mechanisms. Two datasets of Gro1 LRR sequences were constructed, one derived from the Gro1-4 gene, belonging to different cultivated and wild Solanum species, and the other belonging to paralogues of a resistant genotype. Analysis of nonsynonymous to synonymous substitution rates (K(a)/K(s)) highlighted 14 and six amino acids with K(a)/K(s) >1 in orthologue and paralogue datasets, respectively. Selection analysis revealed that the leucine-rich regions accumulate variability in a very specific way, and we found that some combinations of amino acids in these sites might be involved in pathogen recognition. The results confirm previous studies on positive selection in the LRR domain of R protein in Arabidopsis and other model plants and extend these to wild Solanum species. Moreover, positively selected sites in the Gro1 LRR domain show that coevolution mainly occurred in two regions on the internal surface of the three-dimensional horseshoe structure of the domain, albeit with different evolutionary forces between paralogues and orthologues. PMID:25572234

  20. Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: INSIGHTS ON LEUCINE AND ALIPHATIC GLUCOSINOLATE BIOSYNTHESIS.

    PubMed

    Lee, Soon Goo; Nwumeh, Ronald; Jez, Joseph M

    2016-06-24

    Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different β-hydroxyacids in the leucine- and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here, we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg(2+) or NAD(+) These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys-232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys-232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the β-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions. PMID:27137927

  1. Complex coacervates obtained from peptide leucine and gum arabic: formation and characterization.

    PubMed

    Gulão, Eliana da S; de Souza, Clitor J F; Andrade, Cristina T; Garcia-Rojas, Edwin E

    2016-03-01

    In this study, interactions between polypeptide-leucine (0.2% w/w) and gum arabic (0.03, 0.06, 0.09, 0.12, and 0.15% w/w) were examined at concentrations of NaCl (0, 0.01, 0.25, 0.3, 0.5mol/l) and at different pH values (from 1.0 to 12.0). Formation of insoluble complex coacervates was highest at pH 4.0. At pH 2.0, which is the pKa of the gum Arabic, the dissociation of precipitate occurred. The pHØ2 positively shifted with the addition of higher concentrations of salt. Samples containing 0.2% PL and 0.03% GA and no salt had higher turbidity and increased formation of precipitates showing greater turbidity and particle sizes. The Fourier transform infrared spectroscopy confirms the complex coacervate formation of leucine and gum arabic, and rheological measurements suggest the elastic behavior of 0.2% PL and 0.03% GA complex. Overall, the study suggests that complex coacervates of PLs could be one feasible ways of incorporating amino acids in food products. PMID:26471607

  2. A novel D-leucine-containing Conus peptide: diverse conformational dynamics in the contryphan family.

    PubMed

    Jacobsen, R B; Jimenez, E C; De la Cruz, R G; Gray, W R; Cruz, L J; Olivera, B M

    1999-08-01

    A Conus peptide family (the contryphans) is noteworthy because of the presence of a post-translationally modified D-amino acid in all members of the family. A new contryphan peptide, Leu-contryphan-P, was isolated from the venom of Conus purpurascens; the sequence of this peptide is: Gly-Cys-Val-D-Leu-Leu-Pro-Trp-Cys-OH. This is the first known occurrence of D-leucine in a Conus peptide. The discovery of Leu-contryphan-P suggests that there may be branches of the contryphan peptide family that diverge much more in sequence than previously anticipated. Several natural contryphans provide dramatic examples of interconversion between multiple conformational states in small constrained peptides. The contryphans that have 4-trans-hydroxyproline and D-tryptophan in positions 3 and 4, respectively, exhibit two peaks under reverse-phase HPLC conditions, indicating interconversion between two discrete conformations. However, [L-Trp4]contryphan-Sm (with L-Trp substituted for D-Trp) exhibits a single, broad peak that elutes later than the natural peptide, suggesting that D-Trp stabilizes a conformation in which hydrophobic residues are buried. Leucontryphan-P which has valine and D-leucine instead of 4-trans-hydroxyproline and D-tryptophan shows only a single peak that elutes much later than the other contryphans. PMID:10461743

  3. Leucine Zipper-bearing Kinase promotes axon growth in mammalian central nervous system neurons

    PubMed Central

    Chen, Meifan; Geoffroy, Cédric G.; Wong, Hetty N.; Tress, Oliver; Nguyen, Mallorie T.; Holzman, Lawrence B.; Jin, Yishi; Zheng, Binhai

    2016-01-01

    Leucine Zipper-bearing Kinase (LZK/MAP3K13) is a member of the mixed lineage kinase family with high sequence identity to Dual Leucine Zipper Kinase (DLK/MAP3K12). While DLK is established as a key regulator of axonal responses to injury, the role of LZK in mammalian neurons is poorly understood. By gain- and loss-of-function analyses in neuronal cultures, we identify LZK as a novel positive regulator of axon growth. LZK signals specifically through MKK4 and JNKs among MAP2Ks and MAPKs respectively in neuronal cells, with JNK activity positively regulating LZK protein levels. Neuronal maturation or activity deprivation activates the LZK-MKK4-JNK pathway. LZK and DLK share commonalities in signaling, regulation, and effects on axon extension. Furthermore, LZK-dependent regulation of DLK protein expression and the lack of additive effects on axon growth upon co-manipulation suggest complex functional interaction and cross-regulation between these two kinases. Together, our data support the possibility for two structurally related MAP3Ks to work in concert to mediate axonal responses to external insult or injury in mammalian CNS neurons. PMID:27511108

  4. The Native GCN4 Leucine-Zipper Domain Does Not Uniquely Specify a Dimeric Oligomerization State

    PubMed Central

    Oshaben, Kaylyn M.; Salari, Reza; McCaslin, Darrell R.; Chong, Lillian T.; Horne, W. Seth

    2012-01-01

    The dimerization domain of the yeast transcription factor GCN4, one of the first coiled-coil proteins to be structurally characterized at high resolution, has served as the basis for numerous fundamental studies on α-helical folding. Mutations in the GCN4 leucine zipper are known to change its preferred oligomerization state from dimeric to trimeric or tetrameric; however, the wild-type sequence has been assumed to encode a two-chain assembly exclusively. Here we demonstrate that the GCN4 coiled-coil domain can populate either a dimer or trimer fold, depending on environment. We report high-resolution crystal structures of the wild-type sequence in dimeric and trimeric assemblies. Biophysical measurements suggest populations of both oligomerization states under certain experimental conditions in solution. We use parallel tempering molecular dynamics simulations on the microsecond timescale to compare the stability of the dimer and trimer folded states in isolation. In total, our results suggest that the folding behavior of the well-studied GCN4 leucine-zipper domain is more complex than was previously appreciated. Our results have implications in ongoing efforts to establish predictive algorithms for coiled-coil folds and the selection of coiled-coil model systems for design and mutational studies where oligomerization state specificity is an important consideration. PMID:23116373

  5. Leucine Zipper-bearing Kinase promotes axon growth in mammalian central nervous system neurons.

    PubMed

    Chen, Meifan; Geoffroy, Cédric G; Wong, Hetty N; Tress, Oliver; Nguyen, Mallorie T; Holzman, Lawrence B; Jin, Yishi; Zheng, Binhai

    2016-01-01

    Leucine Zipper-bearing Kinase (LZK/MAP3K13) is a member of the mixed lineage kinase family with high sequence identity to Dual Leucine Zipper Kinase (DLK/MAP3K12). While DLK is established as a key regulator of axonal responses to injury, the role of LZK in mammalian neurons is poorly understood. By gain- and loss-of-function analyses in neuronal cultures, we identify LZK as a novel positive regulator of axon growth. LZK signals specifically through MKK4 and JNKs among MAP2Ks and MAPKs respectively in neuronal cells, with JNK activity positively regulating LZK protein levels. Neuronal maturation or activity deprivation activates the LZK-MKK4-JNK pathway. LZK and DLK share commonalities in signaling, regulation, and effects on axon extension. Furthermore, LZK-dependent regulation of DLK protein expression and the lack of additive effects on axon growth upon co-manipulation suggest complex functional interaction and cross-regulation between these two kinases. Together, our data support the possibility for two structurally related MAP3Ks to work in concert to mediate axonal responses to external insult or injury in mammalian CNS neurons. PMID:27511108

  6. The Role of Leucine-Rich Repeat Containing Protein 10 (LRRC10) in Dilated Cardiomyopathy.

    PubMed

    Brody, Matthew J; Lee, Youngsook

    2016-01-01

    Leucine-rich repeat containing protein 10 (LRRC10) is a cardiomyocyte-specific member of the Leucine-rich repeat containing (LRRC) protein superfamily with critical roles in cardiac function and disease pathogenesis. Recent studies have identified LRRC10 mutations in human idiopathic dilated cardiomyopathy (DCM) and Lrrc10 homozygous knockout mice develop DCM, strongly linking LRRC10 to the molecular etiology of DCM. LRRC10 localizes to the dyad region in cardiomyocytes where it can interact with actin and α-actinin at the Z-disc and associate with T-tubule components. Indeed, this region is becoming increasingly recognized as a signaling center in cardiomyocytes, not only for calcium cycling, excitation-contraction coupling, and calcium-sensitive hypertrophic signaling, but also as a nodal signaling hub where the myocyte can sense and respond to mechanical stress. Disruption of a wide range of critical structural and signaling molecules in cardiomyocytes confers susceptibility to cardiomyopathies in addition to the more classically studied mutations in sarcomeric proteins. However, the molecular mechanisms underlying DCM remain unclear. Here, we review what is known about the cardiomyocyte functions of LRRC10, lessons learned about LRRC10 and DCM from the Lrrc10 knockout mouse model, and discuss ongoing efforts to elucidate molecular mechanisms whereby mutation or absence of LRRC10 mediates cardiac disease. PMID:27536250

  7. Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake

    PubMed Central

    Tsuboi, Shun; Yamamura, Shigeki; Imai, Akio; Iwasaki, Kazuhiro

    2016-01-01

    We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla—Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis. PMID:26936797

  8. Non-universal decoding of the leucine codon CUG in several Candida species.

    PubMed Central

    Ohama, T; Suzuki, T; Mori, M; Osawa, S; Ueda, T; Watanabe, K; Nakase, T

    1993-01-01

    It has been reported that CUG, a universal leucine codon, is read as serine in an asporogenic yeast, Candida cylindracea. The distribution of this non-universal genetic code in various yeast species was studied using an in vitro translation assay system with a synthetic messenger RNA containing CUG codons in-frame. It was found that CUG is used as a serine codon in six out of the fourteen species examined, while it is used for leucine in the remaining eight. The tRNA species responsible for the translation of codon CUG as serine was detected in all the six species in which CUG is translated as serine. The grouping according to the CUG codon assignments in these yeast species shows a good correlation with physiological classification by the chain lengths of the isoprenoid moiety of ubiquinone and the cell-wall sugar contained in the yeasts. The six Candida species examined in which CUG is used as serine belong to one distinct group in Hemiascomycetes. PMID:8371978

  9. Stimulating effects of inosine, uridine and glutamine on the tissue distribution of radioactive D-leucine in tumor bearing mice.

    PubMed

    Goto, R; Takeda, A; Tamemasa, O; Chaney, J E; Digenis, G A

    1984-02-01

    This experiment was carried out in search for stimulators of the in vivo uptake of D- and L-leucine by tumor and pancreas for the possible application to gamma-emitter labeled amino acids in nuclear medical diagnosis. Inosine, uridine, and glutamine which are stimulators of the in vitro incorporation of radioactive L-amino acids into some tumor cells significantly enhanced the uptake of D-leucine into the pancreas, while in Ehrlich solid tumor only a little if any increase was observed. Of the compounds tested inosine showed the highest stimulation of pancreas uptake in the range of doses used, resulting in the best pancreas-to-liver concentration ratio, a factor of significant consideration for pancreas imaging. The uptake of L-leucine by the tumor and pancreas was little affected by these compounds. PMID:6739860

  10. Leucine improves glucose and lipid status in offspring from obese dams, dependent on diet type, but not caloric intake.

    PubMed

    Chen, H; Simar, D; Ting, J H Y; Erkelens, J R S; Morris, M J

    2012-10-01

    Previously, we showed that offspring from obese rat dams were hyperphagic, with increased adiposity, hyperlipidaemia and glucose intolerance associated with increased orexigenic neuropeptide expression after fasting. Mammalian target of rapamycin (mTOR) can inhibit food intake through a hypothalamic action. As we previously showed that maternal obesity down-regulated hypothalamic mTOR, in the present study, we hypothesised that dietary leucine supplementation would activate hypothalamic mTOR to reduce food intake, thus limiting metabolic disorders in offspring from obese dams, regardless of postweaning diet. Obesity was induced in Sprague-Dawley females by high-fat diet (HFD) for 5 weeks before mating, throughout gestation and lactation. Male pups from HFD-fed mothers were weaned onto chow or HFD; within each dietary group, half were supplied with leucine via drinking water (1.5%) versus water control for 10 weeks. Those from chow-fed mothers were fed chow and water. Maternal obesity led to increased adiposity in chow-fed offspring. Postweaning HFD consumption exaggerated adiposity, hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. Supplementation with leucine doubled leucine intake and increased hypothalamic mTOR activation; however, appetite regulation was not affected. A reduction in blood lipid levels was observed in offspring regardless of diet, as well as improved glucose tolerance in HFD-fed rats. In HFD-fed rats, up-regulated carnitine palmitoyl-transferase-1 and peroxisome-proliferator-activated receptor-γ coactivator-1α in muscle and glucose transporter 4 in fat suggested that leucine improved peripheral fat oxidation and glucose transport. Leucine is able to improve peripheral glucose and lipid metabolism independent of appetite and weight regulation, suggesting its potential application in the management of metabolic disorders. PMID:22612562

  11. The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes.

    PubMed Central

    Milligan, S B; Bodeau, J; Yaghoobi, J; Kaloshian, I; Zabel, P; Williamson, V M

    1998-01-01

    The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes. PMID:9707531

  12. Basic science highlights.

    PubMed

    Stevenson, Mario

    2007-01-01

    The 14th Conference on Retroviruses and Opportunistic Infections generated a lot of excitement with the announcement of clinical studies employing the use of 2 new classes of antiretroviral drugs that target the viral integrase enzyme and the viral coreceptor CCR5. In addition, a number of presentations on cellular restriction factors provided surprises regarding the mechanism by which cellular restrictions antagonize viral infection. There was also much interest in studies presenting novel cellular cofactors of HIV-1 infection. The conference illustrated how basic science research is paying off. Essential steps in the viral life cycle, uncovered through basic research, are now being targeted by new classes of antiviral agents. In addition, basic science is unveiling potential new targets of antiretroviral therapy. PMID:17485783

  13. Putative excitatory and putative inhibitory inputs are localised in different dendritic domains in a Drosophila flight motoneuron.

    PubMed

    Kuehn, Claudia; Duch, Carsten

    2013-03-01

    Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the location of input synapses on specific parts of their dendrites. However, only a few examples exist of dendritic architecture which can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical and confocal laser scanning methods this study estimates the location of the spike-initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with > 4,000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provide only an estimate of putative input synapse distributions, the data indicate that inhibitory and excitatory synapses were located preferentially on different dendritic domains of MN5 and, thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that, in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies. PMID:23279094

  14. Differential effects of leucine on translation initiation factor activation and protein synthesis in skeletal muscle, renal and adipose tissues of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In adult rats, protein synthesis in skeletal muscle and adipose tissue increases in response to pharmacological doses of leucine (Leu) administered orally. In neonatal pigs, a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle without increasing hepatic protein...

  15. Leucine supplementation of a low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with amino acids. Leucine appears to be the most effective single amino acid to trigger these effects. To examine the response to enteral leucine supplementation...

  16. Chronic leucine supplementation of a low protein diet increases protein synthesis in skeletal muscle and visceral tissues of neonatal pigs through mTOR signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine acutely stimulates protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that leucine supplementation of a low protein diet will enhance protein synthesis and mTOR signaling in the neonate for prolonged periods. Fasted 5-d-old pigs (n=6–8...

  17. LEUCINE-NITROGEN METABOLISM IN THE BRAIN OF CONSCIOUS RATS: ITS ROLE AS A NITROGEN CARRIER IN GLUTAMATE SYNTHESIS IN GLIAL AND NEURONAL METABOLIC COMPARTMENTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine, and gamma-aminobutyrate (GABA)remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched-chain aminotransferase, we hypothesized that leucine is th...

  18. Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that a physiological increase in plasma leucine for 60 and 120 min increases translation initiation factor activation in muscle of neonatal pigs. Although muscle protein synthesis is increased by leucine at 60 min, it is not maintained at 120 min, perhaps because of the decr...

  19. Characterization of epiphycan, a small proteoglycan with a leucine-rich repeat core protein.

    PubMed

    Johnson, H J; Rosenberg, L; Choi, H U; Garza, S; Höök, M; Neame, P J

    1997-07-25

    The epiphysis of developing bones is a cartilaginous structure that is eventually replaced by bone during skeletal maturation. We have separated a dermatan sulfate proteoglycan, epiphycan, from decorin and biglycan by using dissociative extraction of bovine fetal epiphyseal cartilage, followed by sequential ion-exchange, gel permeation, hydrophobic, and Zn2+ chelate chromatographic steps. Epiphycan is a member of the small leucine-rich proteoglycan family, contains seven leucine-rich repeats (LRRs), is related to osteoglycin (osteoinductive factor) (Bentz, H., Nathan, R. M., Rosen, D. M., Armstrong, R. M., Thompson, A. Y., Segarini, P. R., Mathews, M. C., Dasch, J., Piez, K. A., and Seyedin, S. M. (1989) J. Biol. Chem. 264, 20805-20810), and appears to be the bovine equivalent of the chick proteoglycan PG-Lb (Shinomura, T., and Kimata, K. (1992) J. Biol. Chem. 267, 1265-1270). The intact proteoglycan had a median size of approximately 133 kDa. The core protein was 46 kDa by electrophoretic analysis, had a calculated size of 34,271 Da, and had two approximately equimolar N termini (APTLES ... and ETYDAT ... ) separated by 11 amino acids. There were at least three O-linked oligosaccharides in the N-terminal region of the protein, based on blank cycles in Edman degradation and corresponding serine or threonine residues in the translated cDNA sequence. The glycosaminoglycans ranged in size from 23 to 34 kDa were more heterogeneous than those in other dermatan sulfate small leucine-rich proteoglycans and were found in the acidic N-terminal region of the protein core, N-terminal to the LRRs. A four-cysteine cluster was present at the N terminus of the LRRs, and a disulfide-bonded cysteine pair was present at the C terminus of the protein core. The seventh LRR and an N-linked oligosaccharide were between the two C-terminal cysteines. An additional potential N-glycosylation site near the C terminus did not appear to be substituted at a significant level. PMID:9228042

  20. A Hamiltonian replica exchange method for building protein-protein interfaces applied to a leucine zipper

    NASA Astrophysics Data System (ADS)

    Cukier, Robert I.

    2011-01-01

    Leucine zippers consist of alpha helical monomers dimerized (or oligomerized) into alpha superhelical structures known as coiled coils. Forming the correct interface of a dimer from its monomers requires an exploration of configuration space focused on the side chains of one monomer that must interdigitate with sites on the other monomer. The aim of this work is to generate good interfaces in short simulations starting from separated monomers. Methods are developed to accomplish this goal based on an extension of a previously introduced [Su and Cukier, J. Phys. Chem. B 113, 9595, (2009)] Hamiltonian temperature replica exchange method (HTREM), which scales the Hamiltonian in both potential and kinetic energies that was used for the simulation of dimer melting curves. The new method, HTREM_MS (MS designates mean square), focused on interface formation, adds restraints to the Hamiltonians for all but the physical system, which is characterized by the normal molecular dynamics force field at the desired temperature. The restraints in the nonphysical systems serve to prevent the monomers from separating too far, and have the dual aims of enhancing the sampling of close in configurations and breaking unwanted correlations in the restrained systems. The method is applied to a 31-residue truncation of the 33-residue leucine zipper (GCN4-p1) of the yeast transcriptional activator GCN4. The monomers are initially separated by a distance that is beyond their capture length. HTREM simulations show that the monomers oscillate between dimerlike and monomerlike configurations, but do not form a stable interface. HTREM_MS simulations result in the dimer interface being faithfully reconstructed on a 2 ns time scale. A small number of systems (one physical and two restrained with modified potentials and higher effective temperatures) are sufficient. An in silico mutant that should not dimerize because it lacks charged residues that provide electrostatic stabilization of the dimer

  1. Decontamination: back to basics.

    PubMed

    Meredith, Susan J; Sjorgen, Geoff

    2008-07-01

    My invitation from this Journal's Editor, Felicia Cox, to provide a paper for this themed issue, included the sentence 'I was wondering if you or a colleague would like to contribute a back to basics article on the relevant standards and guidelines for decontamination, including what is compliance?'. The reason it is so interesting to me is that the term 'back to basics' implies reverting to a simpler time in life - when by just sticking to the rules, life became easier. However, with decontamination this is not actually true. PMID:18710126

  2. Functional analysis of putative genes encoding the PIP2 water channel subfamily in Populus trichocarpa.

    PubMed

    Secchi, Francesca; MacIver, Bryce; Zeidel, Mark L; Zwieniecki, Maciej A

    2009-11-01

    We located fully sequenced putative genes of the plasma membrane intrinsic proteins (PIPs) family in the Populus trichocarpa (Torr. Gray), genome. Of 23 gene candidates, we assigned eight genes to the PIP2 subfamily. All eight putative genes were expressed in vegetative tissues (roots, leaves, bark and wood), and all of them showed water channel activity after being expressed in Xenopus oocytes. Six of eight proteins were affected by mercury ions. No proteins were affected by the presence of nickel or tungsten ions, or by lowering the pH of bathing external solution from 7.4 to 6.5. The presence of copper ions caused seven of eight PIP2 proteins to increase their water transport capacity by as much as 50%. This systematic study of the PIP2 subfamily of proteins in P. trichocarpa provides a basic overview of their activity as water channels and will be a useful reference for future physiological studies of plant water relations that use P. trichocarpa as a model system. PMID:19808706

  3. Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

    SciTech Connect

    Sharma, Rohit B.; Wang, Qingde; Khillan, Jaspal S.

    2013-07-12

    Highlights: •Vitamin A supports self renewal of putative CSCs from mammary tumors. •These cells exhibit impaired retinol metabolism into retinoic acid. •CSCs from mammary tumors differentiate into mammary specific cell lineages. •The cells express mammary stem cell specific CD29 and CD49f markers. •Putative CSCs form highly metastatic tumors in NOD SCID mouse. -- Abstract: Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibit mammary stem cell specific CD29{sup hi}/CD49f{sup hi}/CD24{sup hi} markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.

  4. L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter.

    PubMed

    Shennan, D B; Thomson, J; Gow, I F; Travers, M T; Barber, M C

    2004-08-30

    The transport of L-leucine by two human breast cancer cell lines has been examined. L-leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na+ -independent pathway. L-leucine uptake by both cell lines was inhibited by L-alanine, D-leucine and to a lesser extent by L-lysine but not by L-proline. Estrogen (17beta-estradiol) stimulated L-leucine uptake by MCF-7 but not by MDA-MB-231 cells. L-leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on L-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 degrees C. There was, however, a significant efflux of L-leucine under zero-trans conditions which was also temperature-sensitive. L-glutamine, L-leucine, D-leucine, L-alanine, AIB and L-lysine all trans-stimulated L-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, D-alanine and L-proline had little or no effect. The anti-cancer agent melphalan inhibited L-leucine uptake by MDA-MB-231 cells but had no effect on L-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for L-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc. PMID:15328053

  5. Bartonella henselae AS A PUTATIVE CAUSE OF CONGENITAL CHOLESTASIS

    PubMed Central

    VELHO, Paulo Eduardo Neves Ferreira; BELLOMO-BRANDÃO, Maria Ângela; DRUMMOND, Marina Rovani; MAGALHÃES, Renata Ferreira; HESSEL, Gabriel; BARJAS-CASTRO, Maria de Lourdes; ESCANHOELA, Cecília Amélia Fazzio; NEGRO, Gilda Maria Barbaro DEL; OKAY, Thelma Suely

    2016-01-01

    SUMMARY Severe anemia and cholestatic hepatitis are associated with bartonella infections. A putative vertical Bartonella henselae infection was defined on the basis of ultrastructural and molecular analyses in a three-year-old child with anemia, jaundice and hepatosplenomegaly since birth. Physicians should consider bartonellosis in patients with anemia and hepatitis of unknown origin. PMID:27410916

  6. DIFFERENTIAL GENE EXPRESSION OF PUTATIVE VIRULENCE GENES IN Flavobacterium columnare

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A shot-gun genomic library of the Flavobacterium columnare ALG-530 virulent strain has been constructed and more than 3,000 clones have been sequenced to date (800 contigs). Based on sequence identity with putative known virulence genes from related species, seven genes were selected for differentia...

  7. Developing putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content data

    EPA Science Inventory

    Developing putative AOPs from high content data Shannon M. Bell1,2, Stephen W. Edwards2 1 Oak Ridge Institute for Science and Education 2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,...

  8. Sulfur Isotope Composition of Putative Primary Troilite in Chondrules

    NASA Technical Reports Server (NTRS)

    Tachibana, Shogo; Huss, Gary R.

    2002-01-01

    Sulfur isotope compositions of putative primary troilites in chondrules from Bishunpur were measured by ion probe. These primary troilites have the same S isotope compositions as matrix troilites and thus appear to be isotopically unfractionated. Additional information is contained in the original extended abstract.

  9. Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The completed genome sequences of Arabidopsis thaliana and rice have revealed very large multi-gene families encoding predicted proteins with an organization of functional domains similar to that of animal receptor kinases, including a putative extracellular ligand-binding domain, a single-pass tran...

  10. Luganda Basic Course.

    ERIC Educational Resources Information Center

    Kamoga, Frederick Katabazi; Stevick, Earl W.

    This "Luganda Basic Course" is not a course in the usual sense. Rather, it is a collection of materials which can be useful in the interaction between teachers and learners. It follows the method by which foreigners interact when they do not speak a common language: personal names and names of respective countries and cities are exchanged.…

  11. Czech Basic Course.

    ERIC Educational Resources Information Center

    Foreign Service (Dept. of State), Washington, DC. Foreign Service Inst.

    This introductory Czech text is based on principles emphasizing development of basic communication skills. Speech samples reflect practical language spoken in everyday situations. The text is designed to be used by American foreign service professionals in foreign countries and to be accompanied by videotapes (unavailable to the public). The text…

  12. MORE BASIC COURSE.

    ERIC Educational Resources Information Center

    LEHR, MARIANNE; AND OTHERS

    THIS BASIC COURSE IN MORE, AN AFRICAN TONE LANGUAGE SPOKEN BY THE MOSSI PEOPLE OF UPPER VOLTA, IS INTENDED TO PROVIDE THE STUDENT WITH DIALOGS THAT RELATE TO SOME OF THE FIRST SITUATIONS IN WHICH HE IS LIKELY TO USE THE LANGUAGE, AS WELL AS WITH SYSTEMATIC PRACTICE IN ALL MAJOR POINTS OF GRAMMAR. THE COURSE COMPRISES 48 UNITS DIVIDED INTO THREE…

  13. Twi Basic Course.

    ERIC Educational Resources Information Center

    Redden, J.E.; And Others

    This course is designed to provide basic structures and vocabulary in Twi in the context of situations commonly encountered by foreigners in Ghana. The dialect presented is Ashanti Twi and the transcription system used is the standard orthography of the Bureau of Ghana Languages with discritic marks added to indicate tone levels. After an…

  14. SHONA BASIC COURSE.

    ERIC Educational Resources Information Center

    STEVICK, EARL W.

    THIS BASIC COURSE IN SHONA, ONE OF THE TWO PRINCIPAL LANGUAGES OF RHODESIA AND PARTS OF ADJACENT MOZAMBIQUE, IS INTENDED TO PROVIDE THE STUDENT WITH DIALOG THAT RELATE TO SOME OF THE SITUATIONS IN WHICH HE IS LIKELY TO USE THE LANGUAGE, AS WELL AS PROVIDE HIM WITH SYSTEMATIC PRACTICE ON ALL MAJOR POINTS OF GRAMMAR. THE TEXT CONSISTS OF 49 UNITS OF…

  15. Microeconomic Analysis with BASIC.

    ERIC Educational Resources Information Center

    Tom, C. F. Joseph

    Computer programs written in BASIC for the study of microeconomic analysis with special emphasis in economic decisions on price, output, and profit of a business firm are described. A very brief overview of the content of each of the 28 computer programs comprising the course is provided; four of the programs are then discussed in greater detail.…

  16. Basics of Weight Control

    MedlinePlus

    ... to: • Eat and drink fewer calories • Increase physical activity • Combine the two for the best results The foods you eat and the beverages you drink provide energy and nutrients. The basic required nutrients are: water, carbohydrates, proteins, fats, dietary fibers, vitamins, and minerals. ...

  17. Basic Media in Education.

    ERIC Educational Resources Information Center

    Harrell, John

    Intended as a guide to the use of different media for use in the classroom, this document demonstrates alternative approaches that may be taken to depicting and communicating images and concepts to others. Some basic tools and materials--including a ruler, matte knife, rubber cement, stapler, felt-tip pens, paint brushes, and lettering pens--are…

  18. Basic Nuclear Physics.

    ERIC Educational Resources Information Center

    Bureau of Naval Personnel, Washington, DC.

    Basic concepts of nuclear structures, radiation, nuclear reactions, and health physics are presented in this text, prepared for naval officers. Applications to the area of nuclear power are described in connection with pressurized water reactors, experimental boiling water reactors, homogeneous reactor experiments, and experimental breeder…

  19. FULA BASIC COURSE.

    ERIC Educational Resources Information Center

    SWIFT, LLOYD B.; AND OTHERS

    THIS BEGINNING COURSE IS AN INTRODUCTION TO FULA (KNOWN VARIOUSLY AS FULANI, FUL, PEUL, OR PHEUL), A NIGER-CONGO LANGUAGE SPOKEN THROUGHOUT THE GRASSLAND AREAS OF WEST AFRICA FROM THE ATLANTIC TO CAMEROUN. THE TEXT IS ONE OF A SERIES OF SHORT BASIC COURSES IN SELECTED AFRICAN LANGUAGES BEING PREPARED BY THE FOREIGN SERVICE INSTITUTE. IT IS…

  20. Basic Drafting: Book One.

    ERIC Educational Resources Information Center

    Davis, Ronald; And Others

    The first of a two-book course in drafting, this manual consists of 13 topics in the following units: introduction to drafting, general safety, basic tools and lines, major equipment, applying for a job, media, lettering, reproduction, drawing sheet layout, architect's scale usage, civil engineer's scale usage, mechanical engineer's scale usage,…

  1. Basic Drafting: Book Two.

    ERIC Educational Resources Information Center

    Davis, Ronald; And Others

    The second of a two-book course in drafting, this manual consists of 12 topics in the following units: sketching techniques, geometric constructions, orthographic views, dimensioning procedures, basic tolerancing, auxiliary views, sectional views, inking tools and techniques, axonometrics, oblique, perspective, and computer-aided drafting.…

  2. Czech Basic Course: Folklore.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    This booklet is designed for use in the advanced phase of the Defense Language Institute's "Basic Course" in Czech. It is used in the advanced phase as a part of cultural background information. Reading selections, with vocabulary lists, include: (1) ethnography; (2) incantations and spells; (3) proverbs, sayings, and weather lore; (4) fairy tales…

  3. Adult Basic Education Curriculum.

    ERIC Educational Resources Information Center

    Massachusetts Career Development Inst., Springfield.

    This booklet, aimed at adult basic education students, pinpoints and summarizes a few common spelling rules to help make spelling easier, and includes a component on using the dictionary. In the text, each rule is presented with many examples. Exercises follow each spelling rule, allowing students the opportunity to apply the rule to specific…

  4. Focus on Basics, 1998.

    ERIC Educational Resources Information Center

    Focus on Basics, 1998

    1998-01-01

    This volume contains the four 1998 quarterly issues of this newsletter that present best practices, current research on adult learning and literacy, and information on how research is used by adult basic education teachers, counselors, program administrators, and policy makers. The following are among the major articles included: "Power, Literacy,…

  5. Hindi Basic Reader.

    ERIC Educational Resources Information Center

    Harter, J. Martin; And Others

    This reader is intended to accompany the Basic Course in Spoken Hindi. Following an outline of the Devanagari script, 20 lessons are presented. Each consists of a reading selection, several illustrative sentences in English and Hindi, and a series of questions. Most of the reading selections were adapted from the magazine "Bal-Bharati." (RM)

  6. Internet Training: The Basics.

    ERIC Educational Resources Information Center

    Gallo, Gail; Wichowski, Chester P.

    This paper outlines the basic information teachers need to know to use the World Wide Web for research and communication, using Netscape 3.04. Topics covered include the following: what is the World Wide Web?; what is a browser?; accessing the Web; moving around a web document; the Uniform Resource Locator (URL); Bookmarks; saving and printing a…

  7. Basic Pneumatics. Instructor's Guide.

    ERIC Educational Resources Information Center

    Fessehaye, Michael

    This instructor's guide is designed for use by industrial vocational teachers in teaching a course on basic pneumatics. Covered in the individual units are the following topics: an introduction to pneumatics (including the operation of a service station hoist); fundamentals and physical laws; air compressors (positive displacement compressors;…

  8. Reading for Basic Understanding.

    ERIC Educational Resources Information Center

    Mercer County Community Coll., Trenton, NJ.

    This document offers materials for a year-long course on general basic reading skills that was part of a workplace literacy project developed by Mercer County Community College (New Jersey), and its partners. The document contains the following: (1) outlines (each of which contains objectives, a topical outline, and list of textbooks) for two…

  9. Navajo Adult Basic Education.

    ERIC Educational Resources Information Center

    Navajo Community Coll., Tsaile, AZ.

    The objectives of this Special Experimental Demonstration Project in Adult Basic Education for the Navajo were: (1) to raise the educational and social level of Navajo adult students who are unable to read, write, and speak English; (2) to assist the Navajo adult students to take advantage of occupational and vocational training programs; (3) to…

  10. Basic Skills Assessment

    ERIC Educational Resources Information Center

    Yin, Alexander C.; Volkwein, J. Fredericks

    2010-01-01

    After surveying 1,827 students in their final year at eighty randomly selected two-year and four-year public and private institutions, American Institutes for Research (2006) reported that approximately 30 percent of students in two-year institutions and nearly 20 percent of students in four-year institutions have only basic quantitative…

  11. Focus on Basics, 1997.

    ERIC Educational Resources Information Center

    Focus on Basics, 1997

    1997-01-01

    Together, these four newsletters contain 36 articles devoted to adult literacy research and practice and the relationship between them. The following articles are included: "A Productive Partnership" (Richard J. Murnane, Bob Bickerton); "Welcome to 'Focus on Basics'" (Barbara Garner); "Applying Research on the Last Frontier" (Karen Backlund, Kathy…

  12. IGBO, BASIC COURSE.

    ERIC Educational Resources Information Center

    SWIFT, LLOYD B.; AND OTHERS

    A BASIC COURSE WAS PREPARED ON THE SPEECH OF TWO MEMBERS OF THE EZINEHITE GROUP OF IGBOS IN EASTERN NIGERIA. THE ESSENTIAL PHONOLOGICAL AND GRAMMATICAL STRUCTURES OF IGBO ARE PRESENTED WITHIN A SMALL VOCABULARY OF APPROXIMATELY 600 ITEMS. THE COURSE MATERIALS CONSIST OF (1) TONE DRILLS, (2) 24 UNITS OF DIALOGS, NOTES, AND DRILLS, (3) SIX UNITS OF…

  13. Swahili Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    This basic audiolingual course in standard Swahili appears in six volumes, Lesson Units 1-56. Units consist of a "blueprint" prefatory page outlining the phonological, morphological, and syntactic structures and new vocabulary to be presented; perception drills; Swahili dialog with cartoon guides and English translation; pattern and recombination…

  14. Turkish Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    These 14 volumes of the Defense Language Institute's basic course in Turkish consist of 112 lesson units designed to train native English language speakers to Level 3 proficiency in comprehending, speaking, reading, and writing Turkish. (Native-speaker fluency is Level 5.) An introduction to the sound system, vowel harmony, and syllable division…

  15. Korean Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    These 11 volumes of the Korean Basic Course comprise 112 lesson units designed to train native English language speakers to Level 3 proficiency in comprehension and speaking and Level 2 proficiency in reading and writing Korean. (Level 5 on this scale is native-speaker level.) Intended for classroom use in the Defense Language Institute intensive…

  16. Portuguese Basic Courses.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    This basic course in Brazilian Portuguese consists of 75 lessons in six volumes. Volume I is in two parts, with the dialogs, questions and exercises presented in Portuguese in the first part, and the intonation patterns and English translations presented in the second. The general format follows the Defense Language Institute format, employing…

  17. Assessing Basic Fact Fluency

    ERIC Educational Resources Information Center

    Kling, Gina; Bay-Williams, Jennifer M.

    2014-01-01

    In this article, the authors share a variety of ways to formatively assess basic fact fluency. The define fluency, raise some issues related to timed testing, and then share a collection of classroom-tested ideas for authentic fact fluency assessment. This article encourages teachers to try a variety of alternative assessments from this sampling,…

  18. Reflections on basic science.

    PubMed

    Piatigorsky, Joram

    2010-01-01

    After almost 50 years in science, I believe that there is an acceptable, often advantageous chasm between open-ended basic research-free exploration without a practical destination and in which the original ideas may fade into new concepts-and translational research or clinical research. My basic research on crystalline (proteins conferring the optical properties of the eye lens) led me down paths I never would have considered if I were conducting translational research. My investigations ranged from jellyfish to mice and resulted in the gene-sharing concept, which showed that the same protein can have distinct molecular functions depending upon its expression pattern and, conversely, that different proteins can serve similar functional roles. This essay portrays basic science as a creative narrative, comparable to literary and artistic endeavors. Preserving the autonomy of open-ended basic research and recognizing its artistic, narrative qualities will accelerate the development of innovative concepts, create a rich resource of information feeding translational research, and have a positive impact by attracting creative individuals to science. PMID:21037410

  19. Ethanol Basics (Fact Sheet)

    SciTech Connect

    Not Available

    2015-01-01

    Ethanol is a widely-used, domestically-produced renewable fuel made from corn and other plant materials. More than 96% of gasoline sold in the United States contains ethanol. Learn more about this alternative fuel in the Ethanol Basics Fact Sheet, produced by the U.S. Department of Energy's Clean Cities program.

  20. Basic Engineer Equipment Mechanic.

    ERIC Educational Resources Information Center

    Marine Corps Inst., Washington, DC.

    This student guide, one of a series of correspondence training courses designed to improve the job performance of members of the Marine Corps, deals with the skills needed by basic engineer equipment mechanics. Addressed in the four individual units of the course are the following topics: mechanics and their tools (mechanics, hand tools, and power…

  1. Basic Skills Program Standards.

    ERIC Educational Resources Information Center

    Page, Lenore; And Others

    This handbook for kindergarten through grade eight provides instructional objectives for student mastery in the basic skills of reading, mathematics, written communication, and oral communication. The section on reading is divided into the following strands: word identification skills; vocabulary skills; comprehension (literal, inferential, and…

  2. Canadian Adult Basic Education.

    ERIC Educational Resources Information Center

    Brooke, W. Michael, Comp.

    "Trends," a publication of the Canadian Association for Adult Education, is a collection of abstracts on selected subjects affecting adult education; this issue is on adult basic education (ABE). It covers teachers and teacher training, psychological factors relating to the ABE teacher and students, manuals for teachers, instructional materials,…

  3. Basic Electronics II.

    ERIC Educational Resources Information Center

    Willison, Neal A.; Shelton, James K.

    Designed for use in basic electronics programs, this curriculum guide is comprised of 15 units of instruction. Unit titles are Review of the Nature of Matter and the P-N Junction, Rectifiers, Filters, Special Semiconductor Diodes, Bipolar-Junction Diodes, Bipolar Transistor Circuits, Transistor Amplifiers, Operational Amplifiers, Logic Devices,…

  4. Basic Internet Software Toolkit.

    ERIC Educational Resources Information Center

    Buchanan, Larry

    1998-01-01

    Once schools are connected to the Internet, the next step is getting network workstations configured for Internet access. This article describes a basic toolkit comprising software currently available on the Internet for free or modest cost. Lists URLs for Web browser, Telnet, FTP, file decompression, portable document format (PDF) reader,…

  5. Basic Electricity. Part 1.

    ERIC Educational Resources Information Center

    Kilmer, Donald C.

    A primarily illustrated introduction to the basics of electricity is presented in this guide, the first of a set of four designed for the student interested in a vocation in electrical work. This guide is intended for the first-year student and provides mostly diagrams with accompanying defintions/information in three units, each covering one of…

  6. Networks: The Basics.

    ERIC Educational Resources Information Center

    Lomarcan, Diana L.

    1995-01-01

    Introduces the information superhighway (the Internet), and presents a guide to navigating it. Offers basic instruction on obtaining and learning to use network accounts; locating addresses using Archie and Wide Area Information Server; retrieving information using file transfer protocol; utilizing Gopher to find and retrieve; browsing the World…

  7. Projectable Basic Electronics Kit.

    ERIC Educational Resources Information Center

    H'ng, John; And Others

    1982-01-01

    Outlines advantages derived from constructing and using a Projectable Basic Electronics Kit and provides: (1) list of components; (2) diagrams of 10 finished components (resistor; capacitor; diode; switch; bulb; transistor; meter; variable capacitor; coil; connecting terminal); and (3) diode and transistor activities. (JN)

  8. Computer Programming: BASIC.

    ERIC Educational Resources Information Center

    Fisher, Patience; And Others

    This guide was prepared to help teachers of the Lincoln Public School's introductory computer programming course in BASIC to make the necessary adjustments for changes made in the course since the purchase of microcomputers and such peripheral devices as television monitors and disk drives, and the addition of graphics. Intended to teach a…

  9. Burmese Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    These five volumes, comprising 65 lesson units, follow the Defense Language Institute audiolingual approach and general format. New materials, introduced in "basic dialogs," are followed by colloquial and literal translations, word lists, and in later lessons, by a variety of drills and reading exercises. A consonant chart and a transcribed list…

  10. Czech Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    These eight volumes of the Defense Language Institute's audiolingual course in basic Czech are comprised of an introductory volume presenting the phonology with pronunciation dialogs, followed by seven volumes of Lesson Units 1-150. The Course is designed to train native English language speakers to Level 3 proficiency in understanding, speaking,…

  11. Basic Soils. Revision.

    ERIC Educational Resources Information Center

    Montana State Univ., Bozeman. Dept. of Agricultural and Industrial Education.

    This curriculum guide is designed for use in teaching a course in basic soils that is intended for college freshmen. Addressed in the individual lessons of the unit are the following topics: the way in which soil is formed, the physical properties of soil, the chemical properties of soil, the biotic properties of soil, plant-soil-water…

  12. Basic Skills Packet.

    ERIC Educational Resources Information Center

    Smith, Geoffrey

    The document describes the basic skills programs in reading and mathematics of the High School in the Community (HSC) in New Haven, Connecticut. HSC, designed to provide a choice of learning environments within the public school system, serves students dissatisfied with their previous school experience. Each student, on entering HSC, is screened…

  13. Basic Skills: Visual Arts.

    ERIC Educational Resources Information Center

    Kentucky State Dept. of Education, Frankfort.

    A curriculum guide for the visual arts is presented. The goal of elementary and middle school education in the four arts disciplines is the development of basic understanding and skills by every student. In secondary education the aim is to continue a sequential curriculum for those students who study the arts. This document is intended as a guide…

  14. Basic Publication Fundamentals.

    ERIC Educational Resources Information Center

    Savedge, Charles E., Ed.

    Designed for students who produce newspapers and newsmagazines in junior high, middle, and elementary schools, this booklet is both a scorebook and a fundamentals text. The scorebook provides realistic criteria for judging publication excellence at these educational levels. All the basics for good publications are included in the text of the…

  15. Ten Years and Counting: Moving Leucine-Rich Repeat Kinase 2 Inhibitors to the Clinic

    PubMed Central

    West, Andrew B

    2015-01-01

    The burden that Parkinson's disease (PD) exacts on the population continues to increase year after year. Though refinement of symptomatic treatments continues at a reasonable pace, no accepted therapies are available to slow or prevent disease progression. The leucine-rich repeat kinase 2 (LRRK2) gene was identified in PD genetic studies and offers new hope for novel therapeutic approaches. The evidence linking LRRK2 kinase activity to PD susceptibility is presented, as well as seminal discoveries relevant to the prosecution of LRRK2 kinase inhibition. Finally, suggestions are made for predictive preclinical modeling and successful first-in-human trials. © 2014 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. PMID:25448543

  16. LEUCINE-RICH AMELOGENIN PEPTIDE INDUCES OSTEOGENESIS IN MOUSE EMBRYONIC STEM CELLS

    PubMed Central

    Warotayanont, Rungnapa; Zhu, Danhong; Snead, Malcolm L.; Zhou, Yan

    2008-01-01

    Leucine-rich amelogenin peptide (LRAP), an alternatively spliced amelogenin protein, possesses a signaling property shown to induce osteogenic differentiation. In the current study, we detected LRAP expression during osteogenesis of wild-type (WT) embryonic stem (ES) cells and observed the absence of LRAP expression in amelogenin-null (KO) ES cells. We explored the signaling effect of LRAP on wild-type ES cells, and the ability of LRAP to rescue the impaired osteogenesis phenotype observed in KO ES cells. Our data indicate that LRAP treatment of WT and KO ES cells induces a significant increase in mineral matrix formation, and significant increases in bone sialoprotein and osterix gene expression. In addition, the amelogenin KO phenotype is partially rescued by the addition of exogenous LRAP. These data suggest a unique function of LRAP during ES cell differentiation along osteogenic lineage. PMID:18086559

  17. Studies on an L-leucine hydriodide semiorganic crystal for frequency conversion applications

    NASA Astrophysics Data System (ADS)

    Baskaran, P.; Vimalan, M.; Anandan, P.; Bakiyaraj, G.; Kirubavathi, K.; Praveen, S. G.; Selvaraju, K.

    2016-03-01

    An L-leucine hydriodide semiorganic crystal has been synthesized and grown by a slow evaporation technique. The lattice parameters of the grown crystal have been confirmed using single-crystal x-ray diffractometry. Various functional groups present in the crystal were identified by Fourier transform infrared (FTIR spectral) assessment. The optical transmission percentage of the crystal was ascertained by UV-vis-near-infrared (NIR) studies. The thermal stability of the crystal was determined by thermogravimetric and differential thermal analysis curves. The mechanical behavior of the crystal was studied using the Vicker’s microhardness analysis. The dielectric properties of the crystal have been investigated for varying temperatures. The second-harmonic generation efficiency was measured by the Kurtz and Perry powder technique and the efficiency is comparable to that of potassium dihydrogen orthophosphate.

  18. Regulation of Beta-Cell Function and Mass by the Dual Leucine Zipper Kinase.

    PubMed

    Oetjen, Elke

    2016-06-01

    Diabetes mellitus is one of the most rapidly increasing diseases worldwide, whereby approximately 90-95% of patients suffer from type 2 diabetes. Considering its micro- and macrovascular complications like blindness and myocardial infarction, a reliable anti-diabetic treatment is needed. Maintaining the function and the mass of the insulin producing beta-cells despite elevated levels of beta-cell-toxic prediabetic signals represents a desirable mechanism of action of anti-diabetic drugs. The dual leucine zipper kinase (DLK) inhibits the action of two transcription factors within the beta-cell, thereby interfering with insulin secretion and production and the conservation of beta-cell mass. Furthermore, DLK action is regulated by prediabetic signals. Hence, the inhibition of this kinase might protect beta-cells against beta-cell-toxic prediabetic signals and prevent the development of diabetes. DLK might thus present a novel drug target for the treatment of diabetes mellitus type 2. PMID:27100796

  19. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins.

    PubMed

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  20. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins

    PubMed Central

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  1. Characterization of the conglomerate form of acetyl-DL-leucine by thermal analysis and solubility measurements

    NASA Astrophysics Data System (ADS)

    Estime, N.; Pena, R.; Teychené, S.; Autret, J. M.; Biscans, B.

    2012-03-01

    Starting from a mixture of enantiomers in solution, crystallization can generate different types of crystals. In order to determine which type of crystal is obtained in the case of acetyl leucine, an active pharmaceutical ingredient (API), analytical methods have been used to partially elucidate the binary and ternary phase diagrams of the system composed of the two enantiomers and water. The melting temperature phase diagram of this compound has been obtained by using differential scanning calorimetry (DSC) analyzes. The results show that it is characteristic of a conglomerate. This mode of crystallization has also been confirmed by X-ray powder diffraction analysis. Solubility measurements of enantiomerical mixtures in water enabled the determination of the ternary diagram of solubility. The empiric Meyerhoffer double solubility rule has been modified, due to the characterization of interactions between enantiomers.

  2. Structural basis for the regulation of maternal embryonic leucine zipper kinase.

    PubMed

    Cao, Lu-Sha; Wang, Jue; Chen, Yuling; Deng, Haiteng; Wang, Zhi-Xin; Wu, Jia-Wei

    2013-01-01

    MELK (maternal embryonic leucine zipper kinase), which is a member of the AMPK (AMP-activated protein kinase)-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA) domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment. PMID:23922895

  3. Glucocorticoid-induced leucine zipper (GILZ) in immuno suppression: master regulator or bystander?

    PubMed Central

    Hoppstädter, Jessica; Kiemer, Alexandra K.

    2015-01-01

    Induction of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids has been reported to be essential for their anti-inflammatory actions. At the same time, GILZ is actively downregulated under inflammatory conditions, resulting in an enhanced pro-inflammatory response. Two papers published in the recent past showed elevated GILZ expression in the late stage of an inflammation. Still, the manuscripts suggest seemingly contradictory roles of endogenous GILZ: one of them suggested compensatory actions by elevated corticosterone levels in GILZ knockout mice, while our own manuscript showed a distinct phenotype upon GILZ knockout in vivo. Herein, we discuss the role of GILZ in inflammation with a special focus on the influence of endogenous GILZ on macrophage responses and suggest a cell-type specific action of GILZ as an explanation for the conflicting results as presented in recent reports. PMID:26498359

  4. Acute effects of enteral leucine supplementation of a low protein diet on muscle protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with insulin and amino acids (AA), particularly leucine. We hypothesized that enteral Leu supplementation of a low protein diets in neonatal pigs will acutely in...

  5. Rapamycin blocks leucine-induced protein synthesis by suppressing mTORC1 activation in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine (Leu). To elucidate the molecular mechanism by which Leu stimulates protein synthesis in neonatal muscle, overnight fasted 7-day-old piglets were...

  6. An alternative domain containing a leucine-rich sequence regulates nuclear cytoplasmic localization of protein 4.1R.

    PubMed

    Luque, Carlos M; Pérez-Ferreiro, Carmen M; Pérez-Gonzalez, Alicia; Englmeier, Ludwig; Koffa, Maria D; Correas, Isabel

    2003-01-24

    In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms. PMID:12427749

  7. Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase

    SciTech Connect

    Guo, Min; Shapiro, Ryan; Schimmel, Paul; Yang, Xiang-Lei

    2010-03-01

    E. coli alanyl-tRNA synthetase is recalcitrant to crystallization. A group of leucine substitutions has transformed the protein. Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an α-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered α-helix did not form a leucine zipper that interlocked with the same α-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization.

  8. L-leucine transport systems in Saccharomyces cerevisiae participation of GAP1, S1 and S2 transport systems.

    PubMed

    Kotliar, N; Stella, C A; Ramos, E H; Mattoon, J R

    1994-09-01

    L-leucine uptake in Saccharomyces cerevisiae is mediated by three different transport systems, S1, S2 and GAP1. Their activities are dependent on the nitrogen source of the culture media. Wild type cells grown in L-proline exhibit a single transport system with high affinity and high Vmax that is partially inhibited by L-citrulline. A gap1 mutant shows two transport systems with Km and Vmax values similar to those previously described as S1 and S2, this transport activity is not inhibited by D-leucine, D-isoleucine or D-valine. Two systems can be also determined in wild type cells grown in rich medium containing a mixed nitrogen source where decreased GAP1 function is observed. In either wild type or gap1 cells grown in medium containing ammonium ions as sole nitrogen source, L-leucine uptake kinetics shows two systems with lower Vmax and similar Km values to those of the S1 and S2 systems. These results show that in S. cerevisiae GAP1, S1 and S2 participate in L-leucine entrance in cells grown in a poor nitrogen source, and that S1 and S2 are two ammonia-sensitive permeases that mediate the uptake in cells grown in a rich nitrogen source. PMID:7812191

  9. Leucine-enriched essential amino acid supplementation during moderate steady state exercise enhances postexercise muscle protein synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The effects of essential amino acid (EAA) supplementation during moderate steady state (ie, endurance) exercise on postexercise skeletal muscle metabolism are not well described, and the potential role of supplemental leucine on muscle protein synthesis (MPS) and associated molecular re...

  10. Modeling leucine's metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis.

    PubMed

    Coutte, François; Niehren, Joachim; Dhali, Debarun; John, Mathias; Versari, Cristian; Jacques, Philippe

    2015-08-01

    A Bacillus subtilis mutant strain overexpressing surfactin biosynthetic genes was previously constructed. In order to further increase the production of this biosurfactant, our hypothesis is that the surfactin precursors, especially leucine, must be overproduced. We present a three step approach for leucine overproduction directed by methods from computational biology. Firstly, we develop a new algorithm for gene knockout prediction based on abstract interpretation, which applies to a recent modeling language for reaction networks with partial kinetic information. Secondly, we model the leucine metabolic pathway as a reaction network in this language, and apply the knockout prediction algorithm with the target of leucine overproduction. Out of the 21 reactions corresponding to potential gene knockouts, the prediction algorithm selects 12 reactions. Six knockouts were introduced in B. subtilis 168 derivatives strains to verify their effects on surfactin production. For all generated mutants, the specific surfactin production is increased from 1.6- to 20.9-fold during the exponential growth phase, depending on the medium composition. These results show the effectiveness of the knockout prediction approach based on formal models for metabolic reaction networks with partial kinetic information, and confirms our hypothesis that precursors supply is one of the main parameters to optimize surfactin overproduction. PMID:26220295

  11. Distinct Plasma Profile of Polar Neutral Amino Acids, Leucine, and Glutamate in Children with Autism Spectrum Disorders

    ERIC Educational Resources Information Center

    Tirouvanziam, Rabindra; Obukhanych, Tetyana V.; Laval, Julie; Aronov, Pavel A.; Libove, Robin; Banerjee, Arpita Goswami; Parker, Karen J.; O'Hara, Ruth; Herzenberg, Leonard A.; Herzenberg, Leonore A.; Hardan, Antonio Y.

    2012-01-01

    The goal of this investigation was to examine plasma amino acid (AA) levels in children with Autism Spectrum Disorders (ASD, N = 27) and neuro-typically developing controls (N = 20). We observed reduced plasma levels of most polar neutral AA and leucine in children with ASD. This AA profile conferred significant post hoc power for discriminating…

  12. Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have previously shown that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in...

  13. Mitochondrial and sarcoplasmic proteins, but not myosin heavy chain, are sensitive to leucine supplementation in old rat skeletal muscle.

    PubMed

    Guillet, Christelle; Zangarelli, Aude; Mishellany, Anne; Rousset, Paulette; Sornet, Claire; Dardevet, Dominique; Boirie, Yves

    2004-05-01

    Leucine has a major anabolic impact on muscle protein synthesis in young as in old animals. However, myosin heavy chain (MHC), sarcoplasmic and mitochondrial proteins may differently respond to anabolic factors, especially during aging. To test this hypothesis, fractional synthesis rates (FSR) of the three muscle protein fractions were measured using a flooding dose of [1-(13)C] phenylalanine, in gastrocnemius muscle of adult (8 months) and old (22 months) rats, either in postabsorptive state (PA), or 90-120 min after ingestion of a alanine-supplemented meal (PP+A) or a leucine-supplemented meal (PP+L). In adult and old rats, in comparison with PA, leucine stimulated mitochondrial (adult: 0.260+/-0.011 vs 0.238+/-0.012%h(-1); old: 0.289+/-0.010 vs 0.250+/-0.010%h(-1); PP+L vs PA, P<0.05) and sarcoplasmic (adult: 0.182+/-0.011 vs 0.143+/-0.006%h(-1); old: 0.195+/-0.010 vs 0.149+/-0.008%h(-1); PP+L vs PA, P<0.05) protein FSR, but not MHC synthesis in old rats (0.101+/-0.009 vs 0.137+/-0.018%h(-1); PP+L vs PA, P=NS). In conclusion, synthesis of specific muscle protein is activated by leucine supplementation, but MHC may be less sensitive to anabolic factors with aging. PMID:15130669

  14. Leucine and alpha-Ketoisocaproic acid, but not norleucine, stimulate skeletal muscle protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are ...

  15. CORRELATION BETWEEN CYTOPLASMIC DOMAIN SEQUENCE AND AUTOPHOSPHORYLATION AMONG ARABIDOPSIS LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The leucine-rich repeat receptor-like kinases (LRR-RLKs) are implicated in signaling roles during plant growth, development and defense. A paradigm for receptor kinase activation involves dimerization and auto- or trans-phosphorylation within the cytoplasmic domain. Our goals are to identify intrace...

  16. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs

    PubMed Central

    Rinaldi, Gabriel; Morales, Maria E.; Alrefaei, Yousef N.; Cancela, Martín; Castillo, Estela; Dalton, John P.; Tort, José F.; Brindley, Paul J.

    2009-01-01

    Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the Schistosoma mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP 1 and 2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP 2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate L-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by ≥ 80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases. PMID:19463860

  17. Characterization of an N-glycosylated Bacillus subtilis leucine aminopeptidase expressed in Pichia pastoris.

    PubMed

    Xi, Hongxing; Tian, Yaping; Zhou, Nandi; Zhou, Zhemin; Shen, Wei

    2015-02-01

    Aminopeptidase is an important flavorsome especially in protein hydrolysate debittering by removing hydrophobic amino acid residue at the N-terminal end. Besides, it is also applied to preparation of active peptides and analysis of protein sequence. In this study, leucine aminopeptidase from Bacillus subtilis was cloned and expressed in Pichia pastoris, a widely used heterologous protein expression host. Then it was purified and characterized. After methanol induction for 96 h, the aminopeptidase activity in culture supernatant reached 28.4 U ml(À1) , which was 7.1 times that of wild strain B. subtilis Zj016. The optimal temperature and pH of the purified recombinant enzyme were 60 °C and 8.5, respectively. The purified aminopeptidase was stable within 30-60 °C and pH 8.0-9.0. It was intensively inhibited by Ni(2β) , Ca(2β) , DL-dithiothreitol (DTT) and ethylene diamine tetraacetic acid (EDTA), but activated by Co(2β) . The Km toward leucine-p-nitroanilines (Leu-pNA) of the enzyme was 0.97 mM. The sequence analysis of aminopeptidase indicated three potential N-glycosylation sites and it was further verified via MALDI-TOF-MS analysis. Consequently, the N-glycosylated aminopeptidase exhibited higher thermostability and catalytic efficiency. The purified enzyme exhibited two bands through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) while a single band can be identified when the enzyme was deglycosylated. Circular dichroism spectroscopy indicated that the secondary structure of recombinant aminopeptidase was similar to the wild-type. PMID:25389014

  18. Mutational Analysis of the Arabidopsis Nucleotide Binding Site–Leucine-Rich Repeat Resistance Gene RPS2

    PubMed Central

    Tao, Yi; Yuan, Fenghua; Leister, R. Todd; Ausubel, Frederick M.; Katagiri, Fumiaki

    2000-01-01

    Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins. PMID:11148296

  19. Diurnal pattern of the interrelationships among leucine oxidation, urea production, and hydrolysis in humans.

    PubMed

    el-Khoury, A E; Ajami, A M; Fukagawa, N K; Chapman, T E; Young, V R

    1996-09-01

    We investigated in six healthy adult men, who received an adequate intake of protein (1 g.kg-1.day-1), the relationship among urea production, excretion, and hydrolysis. At the end of a 6-day diet-adjustment period, subjects were studied using a 24-h continuous intravenous [1-13C]leucine and [15N,15N]urea tracer protocol (A. E. El-Khoury, N. K. Fukagawa, M. Sánchez, R. H. Tsay, R. E. Gleason, T. E. Chapman, and V. R. Young. Am. J. Clin. Nutr. 59: 1000-1011, 1994) to determine rates of irreversible protein nitrogen loss and urea kinetics. By combining leucine and urea kinetic data, we found a significant degree of urea hydrolysis over the 24-h period but no evidence to support the thesis that there is a net retention or "salvage" of the urea nitrogen liberated. Our measurements revealed little or no urea hydrolysis during the fed 12-h period of the 24-h tracer protocol but substantial hydrolysis during the 12-h fasting phase. Furthermore, a mass balance model and calculations (APPENDIX) indicated that nitrogen salvage, if any, is quantitatively indistinguishable from insensible nitrogen losses and aggregate estimation errors, accounting for no more than 5% of the nitrogen intake. We conclude that urea hydrolysis, via the intestinal microflora, although representing a component of the overall cycles of nitrogen flow within the body, does not contribute via a net retention of amino nitrogen to the maintenance of body nitrogen homeostasis in healthy adults consuming an adequate diet. PMID:8843752

  20. Linkage Relationships of Genes Controlling Isoleucine, Valine, and Leucine Biosynthesis in Bacillus subtilis

    PubMed Central

    Barat, M.; Anagnostopoulos, C.; Schneider, A.-M.

    1965-01-01

    Barat, M. (Centre National de la Recherche Scientifique, Gif-sur-Yvette, Seine et Oise, France), C. Anagnostopoulos, and A.-M. Schneider. Linkage relationships of genes controlling isoleucine, valine, and leucine biosynthesis in Bacillus subtilis. J. Bacteriol.90:357–369. 1965.—In Bacillus subtilis, the genetic loci controlling isoleucine and valine biosynthesis are not all clustered. Some of them were located on two distinct transforming deoxyribonucleic acid “molecules.” One of these molecules (the “ileilva2–4-met segment”) carries the threonine deaminase and the dihydroxy acid dehydrase loci linked to methionine markers. The other (the “ilva1–3-leu segment”) bears the reductoisomerase locus and one or more loci involved in leucine synthesis. A phenylalanine marker was also shown to be weakly linked to this latter group. In transduction mediated by phage PBS-1, these groups are transferred jointly with other gene clusters. The phage appears to convey chromosome fragments considerably longer than the transforming “molecules.” The genetic maps of both the above segments were extended by transduction. Some groups previously studied by transformation can be placed in the following linear order: the ile-ilva2–4-met segment, the cluster of loci involved in aromatic amino acid synthesis (try segment), and a lysine locus. An arginine locus is cotransduced with the phe-ilva1–2-leu segment. Recombination frequencies between linked markers are much lower in transduction by this phage than in transformation. PMID:14329448

  1. Metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate.

    PubMed

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  2. Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics

    SciTech Connect

    Lichtenstein, A.H.; Cohn, J.S.; Hachey, D.L.; Millar, J.S.; Ordovas, J.M.; Schaefer, E.J. )

    1990-09-01

    The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of (5,5,5,-2H3)leucine, (4,4,4,-2H3)valine, and (6,6-2H2,1,2-13C2)lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine) mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine), and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.

  3. l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

    PubMed Central

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E.; Gerton, Jennifer L.

    2015-01-01

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  4. L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

    PubMed

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

    2015-03-15

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  5. Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus.

    PubMed

    Mao, Xiangbing; Liu, Minghui; Tang, Jun; Chen, Hao; Chen, Daiwen; Yu, Bing; He, Jun; Yu, Jie; Zheng, Ping

    2015-01-01

    The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling. PMID:26336074

  6. Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus

    PubMed Central

    Mao, Xiangbing; Liu, Minghui; Tang, Jun; Chen, Hao; Chen, Daiwen; Yu, Bing; He, Jun; Yu, Jie; Zheng, Ping

    2015-01-01

    The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling. PMID:26336074

  7. Establishing a framework for the Ad/abaxial regulatory network of Arabidopsis: ascertaining targets of class III homeodomain leucine zipper and KANADI regulation.

    PubMed

    Reinhart, Brenda J; Liu, Tie; Newell, Nicole R; Magnani, Enrico; Huang, Tengbo; Kerstetter, Randall; Michaels, Scott; Barton, M Kathryn

    2013-09-01

    The broadly conserved Class III homeodomain leucine zipper (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator-target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as indeterminate domain4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development. PMID:24076978

  8. Establishing a Framework for the Ad/Abaxial Regulatory Network of Arabidopsis: Ascertaining Targets of Class III HOMEODOMAIN LEUCINE ZIPPER and KANADI Regulation[W

    PubMed Central

    Reinhart, Brenda J.; Liu, Tie; Newell, Nicole R.; Magnani, Enrico; Huang, Tengbo; Kerstetter, Randall; Michaels, Scott; Barton, M. Kathryn

    2013-01-01

    The broadly conserved Class III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator–target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as INDETERMINATE DOMAIN4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development. PMID:24076978

  9. Basic Emotions: A Reconstruction

    PubMed Central

    Mason, William A.; Capitanio, John P.

    2016-01-01

    Emotionality is a basic feature of behavior. The argument over whether the expression of emotions is based primarily on culture (constructivism, nurture) or biology (natural forms, nature) will never be resolved because both alternatives are untenable. The evidence is overwhelming that at all ages and all levels of organization, the development of emotionality is epigenetic: The organism is an active participant in its own development. To ascribe these effects to “experience” was the best that could be done for many years. With the rapid acceleration of information on how changes in organization are actually brought about, it is a good time to review, update, and revitalize our views of experience in relation to the concept of basic emotion. PMID:27110280

  10. Basic lubrication equations

    NASA Technical Reports Server (NTRS)

    Hamrock, B. J.; Dowson, D.

    1981-01-01

    Lubricants, usually Newtonian fluids, are assumed to experience laminar flow. The basic equations used to describe the flow are the Navier-Stokes equation of motion. The study of hydrodynamic lubrication is, from a mathematical standpoint, the application of a reduced form of these Navier-Stokes equations in association with the continuity equation. The Reynolds equation can also be derived from first principles, provided of course that the same basic assumptions are adopted in each case. Both methods are used in deriving the Reynolds equation, and the assumptions inherent in reducing the Navier-Stokes equations are specified. Because the Reynolds equation contains viscosity and density terms and these properties depend on temperature and pressure, it is often necessary to couple the Reynolds with energy equation. The lubricant properties and the energy equation are presented. Film thickness, a parameter of the Reynolds equation, is a function of the elastic behavior of the bearing surface. The governing elasticity equation is therefore presented.

  11. Basics of Biosafety

    NASA Technical Reports Server (NTRS)

    Wong, Willy

    2009-01-01

    This slide presentation reviews the basics of biosafety and the importance of assuring proper biosafety practices. The objectives of the presentation are to review regulations about biosafety, and the different biosafety levels; the biosafety facilities at Johnson Space Center; the usage and maintenance of the biosafety cabinet, the proper methods to handle biologically hazardous materials upon exposure, and the methods of cleanup in the event of a spill, and the training requirements that are mandated for personnel handling biologically hazardous materials.

  12. The Basic Anaesthesia Machine

    PubMed Central

    Gurudatt, CL

    2013-01-01

    After WTG Morton's first public demonstration in 1846 of use of ether as an anaesthetic agent, for many years anaesthesiologists did not require a machine to deliver anaesthesia to the patients. After the introduction of oxygen and nitrous oxide in the form of compressed gases in cylinders, there was a necessity for mounting these cylinders on a metal frame. This stimulated many people to attempt to construct the anaesthesia machine. HEG Boyle in the year 1917 modified the Gwathmey's machine and this became popular as Boyle anaesthesia machine. Though a lot of changes have been made for the original Boyle machine still the basic structure remains the same. All the subsequent changes which have been brought are mainly to improve the safety of the patients. Knowing the details of the basic machine will make the trainee to understand the additional improvements. It is also important for every practicing anaesthesiologist to have a thorough knowledge of the basic anaesthesia machine for safe conduct of anaesthesia. PMID:24249876

  13. The basic anaesthesia machine.

    PubMed

    Gurudatt, Cl

    2013-09-01

    After WTG Morton's first public demonstration in 1846 of use of ether as an anaesthetic agent, for many years anaesthesiologists did not require a machine to deliver anaesthesia to the patients. After the introduction of oxygen and nitrous oxide in the form of compressed gases in cylinders, there was a necessity for mounting these cylinders on a metal frame. This stimulated many people to attempt to construct the anaesthesia machine. HEG Boyle in the year 1917 modified the Gwathmey's machine and this became popular as Boyle anaesthesia machine. Though a lot of changes have been made for the original Boyle machine still the basic structure remains the same. All the subsequent changes which have been brought are mainly to improve the safety of the patients. Knowing the details of the basic machine will make the trainee to understand the additional improvements. It is also important for every practicing anaesthesiologist to have a thorough knowledge of the basic anaesthesia machine for safe conduct of anaesthesia. PMID:24249876

  14. Human biliverdin reductase is a leucine zipper-like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress.

    PubMed

    Ahmad, Zulfiqar; Salim, Mohammad; Maines, Mahin D

    2002-03-15

    Human biliverdin reductase (hBVR) is a serine/threonine kinase that catalyzes reduction of the heme oxygenase (HO) activity product, biliverdin, to bilirubin. A domain of biliverdin reductase (BVR) has primary structural features that resemble leucine zipper proteins. A heptad repeat of five leucines (L(1)--L(5)), a basic domain, and a conserved alanine characterize the domain. In hBVR, a lysine replaces L(3). The secondary structure model of hBVR predicts an alpha-helix-turn-beta-sheet for this domain. hBVR translated by the rabbit reticulocyte lysate system appears on a nondenaturing gel as a single band with molecular mass of approximately 69 kDa. The protein on a denaturing gel separates into two anti-hBVR immunoreactive proteins of approximately 39.9 + 34.6 kDa. The dimeric form, but not purified hBVR, binds to a 100-mer DNA fragment corresponding to the mouse HO-1 (hsp32) promoter region encompassing two activator protein (AP-1) sites. The specificity of DNA binding is suggested by the following: (a) hBVR does not bind to the same DNA fragment with one or zero AP-1 sites; (b) a 56-bp random DNA with one AP-1 site does not form a complex with hBVR; (c) in vitro translated HO-1 does not interact with the 100-mer DNA fragment with two AP-1 sites; (d) mutation of Lys(143), Leu(150), or Leu(157) blocks both the formation of the approximately 69-kDa specimens and hBVR DNA complex formation; and (e) purified preparations of hBVR or hHO-1 do not bind to DNA with two AP-1 sites. The potential significance of the AP-1 binding is suggested by the finding that the response of HO-1, in COS cells stably transfected with antisense hBVR, with 66% reduced BVR activity, to superoxide anion (O(2)()) formed by menadione is attenuated, whereas induction by heme is not affected. We propose a role for BVR in the signaling cascade for AP-1 complex activation necessary for HO-1 oxidative stress response. PMID:11773068

  15. Putative melatonin receptors in a human biological clock

    SciTech Connect

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  16. A study of L-leucine, L-phenylalanine and L-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2.

    PubMed

    Shennan, D B; Calvert, D T; Travers, M T; Kudo, Y; Boyd, C A R

    2002-08-19

    The transport of L-leucine, L-phenylalanine and L-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of L-leucine was inhibited by BCH (65%) and D-leucine (58%) but not by L-proline. The inhibition of L-leucine clearance by BCH and D-leucine was not additive. L-leucine inhibited the peak clearance of radiolabelled L-leucine by 78%. BCH also inhibited the peak clearance of L-phenylalanine (66%) and L-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that L-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium. PMID:12101005

  17. Assessment of the metabolic chiral inversion of D-leucine in rat by gas chromatography-mass spectrometry combined with a stable isotope dilution analysis.

    PubMed

    Hasegawa, H; Matsukawa, T; Shinohara, Y; Hashimoto, T

    2000-08-01

    The stereoselective pharmacokinetics of leucine enantiomers in rats has been investigated to evaluate the inversion of D-leucine to L-enantiomer. After a bolus i.v. administration of D- or L-[2H7]leucine to rats, blood samples were obtained over 6 h after administration and analyzed by a stereoselective gas chromatography-mass spectrometry method. Racemic [2H3]leucine was used as an internal standard. The method involved methyl esterification and subsequent chiral derivatization with (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride to form the diastereomeric amide. The derivatization made possible the separation of leucine enantiomers with good gas chromatographic behavior. Plasma concentration of both D- and L-[2H7]leucine declined biexponentially, with elimination half-lives of 60 and 14 min, respectively. In contrast to the L-enantiomer, the D-enantiomer had a lower systemic clearance. When D-[2H7]leucine was administered, the L-enantiomer was found to rapidly appear in plasma. About 30% of an administered dose of the D-isomer was stereospecifically inverted to the L-enantiomer. There was no measurable inversion of the L- to D-enantiomer. This methodology has made it possible to evaluate the pharmacokinetics of each enantiomer of amino acids and estimate of chiral inversion after administration of D-amino acids. PMID:10901701

  18. Structural identification of putative USPs in Catharanthus roseus.

    PubMed

    Bahieldin, Ahmed; Atef, Ahmed; Shokry, Ahmed M; Al-Karim, Saleh; Al Attas, Sanaa G; Gadallah, Nour O; Edris, Sherif; Al-Kordy, Magdy A; Omer, Abdulkader M Shaikh; Sabir, Jamal S M; Ramadan, Ahmed M; Al-Hajar, Abdulrahman S M; Makki, Rania M; Hassan, Sabah M; El-Domyati, Fotouh M

    2015-10-01

    Nucleotide sequences of the C. roseus SRA database were assembled and translated in order to detect putative universal stress proteins (USPs). Based on the known conserved USPA domain, 24 Pfam putative USPA proteins in C. roseus were detected and arranged in six architectures. The USPA-like domain was detected in all architectures, while the protein kinase-like (or PK-like), (tyr)PK-like and/or U-box domains are shown downstream it. Three other domains were also shown to coexist with the USPA domain in C. roseus putative USPA sequences. These domains are tetratricopeptide repeat (or TPR), apolipophorin III (or apoLp-III) and Hsp90 co-chaperone Cdc37. Subsequent analysis divided USPA-like domains based on the ability to bind ATP. The multiple sequence alignment indicated the occurrence of eight C. roseus residues of known features of the bacterial 1MJH secondary structure. The data of the phylogenetic tree indicated several distinct groups of USPA-like domains confirming the presence of high level of sequence conservation between the plant and bacterial USPA-like sequences. PMID:26318047

  19. Leucine-induced activation of translational initiation is partly regulated by the branched-chain {alpha}-keto acid dehydrogenase complex in C2C12 cells

    SciTech Connect

    Nakai, Naoya . E-mail: nakai@hss.osaka-u.ac.jp; Shimomura, Yoshiharu; Tamura, Tomohiro; Tamura, Noriko; Hamada, Koichiro; Kawano, Fuminori; Ohira, Yoshinobu

    2006-05-19

    Branched-chain amino acid leucine has been shown to activate the translational regulators through the mammalian target of rapamycin. However, the leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway. The irreversible and rate-limiting step in the leucine oxidation pathway is catalyzed by the branched-chain {alpha}-keto acid dehydrogenase (BCKDH) complex. The complex contains E1 ({alpha}2{beta}2), E2, and E3 subunits, and its activity is abolished by phosphorylation of the E1{alpha} subunit by BCKDH kinase. The relationship between the activity of BCKDH complex and leucine-mediated activation of the protein translation was investigated using the technique of RNA interference. The activity of BCKDH complex in C2C12 cell was modulated by transfection of small interfering RNA (siRNA) for BCKDH E2 subunit or BCKDH kinase. Transfection of siRNAs decreased the mRNA expression and protein amount of corresponding gene. Suppression of either E2 subunit or kinase produced opposite effects on the cell proliferation and the activation of translational regulators by leucine. Suppression of BCKDH kinase for 48 h resulted in decreasing cell proliferation. In contrast, E2 suppression led to increased amount of total cellular protein. The phosphorylation of p70 S6 kinase by leucine was increased in E2-siRNA transfected C2C12 cells, whereas the leucine's effect was diminished in kinase-siRNA transfected cells. These results suggest that the activation of the translational regulators by leucine was partly regulated by the activity of BCKDH complex.

  20. Structure-activity relations of leucine derivatives reveal critical moieties for cellular uptake and activation of mTORC1-mediated signaling.

    PubMed

    Nagamori, Shushi; Wiriyasermkul, Pattama; Okuda, Suguru; Kojima, Naoto; Hari, Yoshiyuki; Kiyonaka, Shigeki; Mori, Yasuo; Tominaga, Hideyuki; Ohgaki, Ryuichi; Kanai, Yoshikatsu

    2016-04-01

    Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor. PMID:26724922

  1. Protein-leucine ingestion activates a regenerative inflammo-myogenic transcriptome in skeletal muscle following intense endurance exercise.

    PubMed

    Rowlands, David S; Nelson, Andre R; Raymond, Frederic; Metairon, Sylviane; Mansourian, Robert; Clarke, Jim; Stellingwerff, Trent; Phillips, Stuart M

    2016-01-01

    Protein-leucine supplement ingestion following strenuous endurance exercise accentuates skeletal-muscle protein synthesis and adaptive molecular responses, but the underlying transcriptome is uncharacterized. In a randomized single-blind triple-crossover design, 12 trained men completed 100 min of high-intensity cycling then ingested 70/15/180/30 g protein-leucine-carbohydrate-fat (15LEU), 23/5/180/30 g (5LEU), or 0/0/274/30 g (CON) beverages during the first 90 min of a 240 min recovery period. Vastus lateralis muscle samples (30 and 240 min postexercise) underwent transcriptome analysis by microarray followed by bioinformatic analysis. Gene expression was regulated by protein-leucine in a dose-dependent manner affecting the inflammatory response and muscle growth and development. At 30 min, 15LEU and 5LEU vs. CON activated transcriptome networks with gene-set functions involving cell-cycle arrest (Z-score 2.0-2.7, P < 0.01), leukocyte maturation (1.7, P = 0.007), cell viability (2.4, P = 0.005), promyogenic networks encompassing myocyte differentiation and myogenin (MYOD1, MYOG), and a proteinaceous extracellular matrix, adhesion, and development program correlated with plasma lysine, arginine, tyrosine, taurine, glutamic acid, and asparagine concentrations. High protein-leucine dose (15LEU-5LEU) activated an IL-1I-centered proinflammatory network and leukocyte migration, differentiation, and survival functions (2.0-2.6, <0.001). By 240 min, the protein-leucine transcriptome was anti-inflammatory and promyogenic (IL-6, NF- β, SMAD, STAT3 network inhibition), with overrepresented functions including decreased leukocyte migration and connective tissue development (-1.8-2.4, P < 0.01), increased apoptosis of myeloid and muscle cells (2.2-3.0, P < 0.002), and cell metabolism (2.0-2.4, P < 0.01). The analysis suggests protein-leucine ingestion modulates inflammatory-myogenic regenerative processes during skeletal muscle recovery from endurance exercise. Further

  2. Basic facts about Venus

    NASA Technical Reports Server (NTRS)

    Colin, L.

    1983-01-01

    Because of the disturbing influence of the earth's atmosphere on terrestrial and airborne telescopy, radiometry, thermal mapping, spectroscopy, polarimetry and radar astronomy of Venus, major improvements in the body of theory concerning that planet, began with the Mariner 2 planetary exploration program in 1962. The effect of spacecraft exploration culminated with the influx of data yielded by the Pioneer Venus and Venera 11 and 12 missions of 1978. Attention is presently given to the quantitative enhancement of widely accepted, basic facts about Venus that has resulted from the analysis of space probe data, together with an overview of the major features of past and planned planetary missions.

  3. Basic Hitchhiker Payload Requirements

    NASA Technical Reports Server (NTRS)

    Horan, Stephen

    1999-01-01

    This document lists the requirements for the NMSU Hitchhiker experiment payload that were developed as part of the EE 498/499 Capstone Design class during the 1999-2000 academic year. This document is used to describe the system needs as described in the mission document. The requirements listed here are those primarily used to generate the basic electronic and data processing requirements developed in the class design document. The needs of the experiment components are more fully described in the draft NASA hitchhiker customer requirements document. Many of the details for the overall payload are given in full detail in the NASA hitchhiker documentation.

  4. Basic plasma physics II

    NASA Astrophysics Data System (ADS)

    Galeev, A. A.; Sudan, R. N.

    The basic physics of classical ideal plasmas is presented in reviews of recent theoretical and experimental investigations, with an emphasis on nonlinear interactions violating the assumptions of weak turbulence. Topics examined include Kolmogorov spectra, parametric instabilities in magnetoactive plasmas, collapse and self-focusing of Langmuir waves, collective dissipation and transport, spontaneous reconnection of magnetic-field lines in a collisionless plasma, collective-beam/plasma interaction, numerical particle simulations, diagnostic techniques based on the interaction of electromagnetic radiation with a plasma, diagnostics for magnetically confined high-temperature plasmas, and relativistic electron-beam/plasma interaction with self-fields. Diagrams, graphs, spectra, and drawings of experimental apparatus are provided.

  5. Cold, Gas-Phase UV and IR Spectroscopy of Protonated Leucine Enkephalin and its Analogues

    NASA Astrophysics Data System (ADS)

    Burke, Nicole L.; Redwine, James; Dean, Jacob C.; McLuckey, Scott A.; Zwier, Timothy S.

    2014-06-01

    The conformational preferences of peptide backbones and the resulting hydrogen bonding patterns provide critical biochemical information regarding the structure-function relationship of peptides and proteins. The spectroscopic study of cryogenically-cooled peptide ions in a mass spectrometer probes these H-bonding arrangements and provides information regarding the influence of a charge site. Leucine enkephalin, a biologically active endogenous opiod peptide, has been extensively studied as a model peptide in mass spectrometry. This talk will present a study of the UV and IR spectroscopy of protonated leucine enkephalin [YGGFL+H]+ and two of its analogues: the sodiated [YGGFL+Na]+ and C-terminally methyl esterified [YGGFL-OMe+H]+ forms. All experiments were performed in a recently completed multi-stage mass spectrometer outfitted with a cryocooled ion trap. Ions are generated via nano-electrospray ionization and the analyte of interest is isolated in a linear ion trap. The analyte ions are trapped in a 22-pole ion trap held at 5 K by a closed cycle helium cryostat and interrogated via UV and IR lasers. Photofragments are trapped and isolated in a second LIT and mass analyzed. Double-resonance UV and IR methods were used to assign the conformation of [YGGFL+H]+, using the NH/OH stretch, Amide I, and Amide II regions of the infrared spectrum. The assigned structure contains a single backbone conformation at vibrational/rotational temperatures of 10 K held together with multiple H-bonds that self-solvate the NH3+ site. A "proton wire" between the N and C termini reinforces the H-bonding activity of the COO-H group to the F-L peptide bond, whose cleavage results in formation of the b4 ion, which is a prevalent, low-energy fragmentation pathway for [YGGFL+H]+. The reinforced H-bonding network in conjunction with the mobile proton theory may help explain the prevalence of the b4 pathway. In order to elucidate structural changes caused by modifying this H-bonding activity

  6. Basic space payload fastener

    NASA Technical Reports Server (NTRS)

    Vranish, J. M.; Gorevan, Stephen

    1995-01-01

    A new basic space fastener has been developed and tested by the GSFC. The purposes of this fastener are to permit assembly and servicing in space by astronauts and/or robots and to facilitate qualification of payloads on Earth prior to launch by saving time and money during the systems integration and component testing and qualification processes. The space fastener is a rework of the basic machine screw such that crossthreading is impossible; it is self-locking and will not work its way out during launch (vibration proof); it will not wear out despite repeated use; it occupies a small foot print which is comparable to its machine screw equivalent, and it provides force and exhibits strength comparable to its machine screw equivalent. Construction is ultra-simple and cost effective and the principle is applicable across the full range of screw sizes ranging from a #10 screw to 2.5 cm (1 in) or more. In this paper, the fastener principles of operation will be discussed along with test results and construction details. The new fastener also has considerable potential in the commercial sector. A few promising applications will be presented.

  7. Basic space payload fastener

    NASA Astrophysics Data System (ADS)

    Vranish, J. M.; Gorevan, Stephen

    1995-05-01

    A new basic space fastener has been developed and tested by the GSFC. The purposes of this fastener are to permit assembly and servicing in space by astronauts and/or robots and to facilitate qualification of payloads on Earth prior to launch by saving time and money during the systems integration and component testing and qualification processes. The space fastener is a rework of the basic machine screw such that crossthreading is impossible; it is self-locking and will not work its way out during launch (vibration proof); it will not wear out despite repeated use; it occupies a small foot print which is comparable to its machine screw equivalent, and it provides force and exhibits strength comparable to its machine screw equivalent. Construction is ultra-simple and cost effective and the principle is applicable across the full range of screw sizes ranging from a #10 screw to 2.5 cm (1 in) or more. In this paper, the fastener principles of operation will be discussed along with test results and construction details. The new fastener also has considerable potential in the commercial sector. A few promising applications will be presented.

  8. The Basics of Cheesemaking.

    PubMed

    Kindstedt, Paul S

    2013-10-01

    All cheeses have a common set of principles that involve a complex web of chemical, biochemical, and microbiological changes. These changes first transform milk into fresh or unripened cheese. Although some cheeses are consumed immediately after manufacture, most are subsequently aged or ripened for weeks to years depending on the variety. During aging or ripening, a cheese's sensory characteristics undergo multifaceted and often dramatic changes. The steps performed during the earliest days of the cheesemaking process are especially critical because they establish the chemical characteristics of the cheese at the start of ripening, and these characteristics in turn affect the ripening process. For most cheeses, the key process on the first day of cheesemaking is the fermentation of lactose to lactic acid by bacteria. The rate at which lactic acid is produced profoundly affects the initial chemical characteristics of the cheese, which selectively influence the complex microbial populations that find their way from the milk and surrounding environment into the cheese. This article discusses the basics of cheesemaking by integrating the practical steps that all cheesemakers use with the scientific principles on which those practices are based. The aim is to paint a conceptual picture in which the microbiology of cheese "fits together" with the basic practices of cheesemaking and the scientific principles that underlie them. PMID:26184823

  9. Basic and clinical immunology

    NASA Technical Reports Server (NTRS)

    Chinen, Javier; Shearer, William T.

    2003-01-01

    Progress in immunology continues to grow exponentially every year. New applications of this knowledge are being developed for a broad range of clinical conditions. Conversely, the study of primary and secondary immunodeficiencies is helping to elucidate the intricate mechanisms of the immune system. We have selected a few of the most significant contributions to the fields of basic and clinical immunology published between October 2001 and October 2002. Our choice of topics in basic immunology included the description of T-bet as a determinant factor for T(H)1 differentiation, the role of the activation-induced cytosine deaminase gene in B-cell development, the characterization of CD4(+)CD25(+) regulatory T cells, and the use of dynamic imaging to study MHC class II transport and T-cell and dendritic cell membrane interactions. Articles related to clinical immunology that were selected for review include the description of immunodeficiency caused by caspase 8 deficiency; a case series report on X-linked agammaglobulinemia; the mechanism of action, efficacy, and complications of intravenous immunoglobulin; mechanisms of autoimmunity diseases; and advances in HIV pathogenesis and vaccine development. We also reviewed two articles that explore the possible alterations of the immune system caused by spaceflights, a new field with increasing importance as human space expeditions become a reality in the 21st century.

  10. Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment*

    PubMed Central

    Zhou, Houjiang; Low, Teck Y.; Hennrich, Marco L.; van der Toorn, Henk; Schwend, Thomas; Zou, Hanfa; Mohammed, Shabaz; Heck, Albert J. R.

    2011-01-01

    Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti4+-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO2 enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti4+-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼2000 unique phosphopeptides could be identified by Ti4+-IMAC with HFP and close to 3000 by TiO2. We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti4+-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled

  11. Molecular Dynamics of Rab7::REP1::GGTase-II Ternary Complex and Identification of Their Putative Drug Binding Sites.

    PubMed

    Sindhu, Meenakshi; Saini, Vandana; Piplani, Sakshi; Kumar, A

    2013-01-01

    The structure-function correlation of membrane proteins have been a difficult task, particularly in context to transient protein complexes. The molecular simulation of ternary complex of Rab7::REP1::GGTase-II was carried out to understand the basic structural events occurring during the prenylation event of Rab proteins, using the software YASARA. The study suggested that the C-terminus of Rab7 has to be in completely extended conformation during prenylation to reach the active site of RabGGTase-II. Also, attempt was made to find putative drug binding sites on the ternary complex of Rab7::REP1::GGTase-II using Q-SiteFinder programme. The comprehensive consensus probe generated by the program revealed a total of 10 major pockets as putative drug binding sites on Rab7::REP:: GGTase-II ternary complex. These pockets were found on REP protein and GGTase protein subunits. The Rab7 was found to be devoid of any putative drug binding sites in the ternary complex. The phylogenetic analysis of 60 Rab proteins of human was carried out using PHYLIP and study indicated the close phylogenetic relationship between Rab7 and Rab9 proteins of human and hence with further in silico study, the present observations can be extrapolated to Rab9 proteins. The study paves a good platform for further experimental verifications of the findings and other in silico studies like identifying the potential drug targets by searching the putative drug binding sites, generating pharmacophoric pattern, searching or constructing suitable ligand and docking studies. PMID:23901157

  12. Photodissociation of protonated leucine-enkephalin in the VUV range of 8-40 eV

    SciTech Connect

    Bari, S.; Gonzalez-Magana, O.; Reitsma, G.; Hoekstra, R.; Schlathoelter, T.; Werner, J.; Schippers, S.

    2011-01-14

    Until now, photodissociation studies on free complex protonated peptides were limited to the UV wavelength range accessible by intense lasers. We have studied photodissociation of gas-phase protonated leucine-enkephalin cations for vacuum ultraviolet (VUV) photons energies ranging from 8 to 40 eV. We report time-of-flight mass spectra of the photofragments and various photofragment-yields as a function of photon energy. For sub-ionization energies our results are in line with existing studies on UV photodissociation of leucine-enkephalin. For photon energies exceeding 10 eV we could identify a new dissociation scheme in which photoabsorption leads to a fast loss of the tyrosine side chain. This loss process leads to the formation of a residual peptide that is remarkably cold internally.

  13. Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1

    PubMed Central

    2010-01-01

    Background Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. Results It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. Conclusion The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3. PMID:20509863

  14. Sustained delivery by leucine-modified chitosan spray-dried respirable powders.

    PubMed

    Learoyd, Tristan P; Burrows, Jane L; French, Eddie; Seville, Peter C

    2009-05-01

    The controlled co-delivery of multiple agents to the lung offers potential benefits to patients. This study investigated the preparation and characterisation of highly respirable spray-dried powders displaying the sustained release of two chemically distinct therapeutic agents. Spray-dried powders were produced from 30% (v/v) aqueous ethanol formulations that contained hydrophilic (terbutaline sulphate) and hydrophobic (beclometasone dipropionate) model drugs, chitosan (as a drug release modifier) and leucine (aerosolisation enhancer). The influence of chitosan molecular weight on spray-drying thermal efficiency, aerosol performance and drug release profile was investigated. Resultant powders were physically characterised: with in vitro aerosolisation performance and drug release profile investigated by the Multi-Stage Liquid Impinger and modified USP II dissolution apparatus, respectively. It was found that increased chitosan molecular weight gave increased spray-drying thermal efficiency. The powders generated were of a suitable size for inhalation-with emitted doses over 90% and fine particle fractions up to 72% of the loaded dose. Sustained drug release profiles were observed in dissolution tests for both agents: increased chitosan molecular weight associated with increased duration of drug release. The controlled co-delivery of hydrophilic and hydrophobic entities underlines the capability of spray drying to produce respirable particles with sustained release for delivery to the lung. PMID:19429272

  15. Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma

    PubMed Central

    Satoh, Shunpei; Takatori, Atsushi; Ogura, Atsushi; Kohashi, Kenichi; Souzaki, Ryota; Kinoshita, Yoshiaki; Taguchi, Tomoaki; Hossain, Md. Shamim; Ohira, Miki; Nakamura, Yohko; Nakagawara, Akira

    2016-01-01

    In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1−/− mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB. PMID:27604320

  16. Diatom Mimics: Directing the Formation of Biosilica Nanoparticles by Controlled Folding of Lysine-Leucine Peptides

    PubMed Central

    Baio, Joe E.; Zane, Ariel; Jaeger, Vance; Roehrich, Adrienne M.; Lutz, Helmut; Pfaendtner, Jim; Drobny, Gary P.; Weidner, Tobias

    2015-01-01

    Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom's biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 310 helix, the morphology of the nanoparticles changes to elongated rod-like structures. β-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids. PMID:25285787

  17. Diatom mimics: directing the formation of biosilica nanoparticles by controlled folding of lysine-leucine peptides.

    PubMed

    Baio, Joe E; Zane, Ariel; Jaeger, Vance; Roehrich, Adrienne M; Lutz, Helmut; Pfaendtner, Jim; Drobny, Gary P; Weidner, Tobias

    2014-10-29

    Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom's biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 310 helix, the morphology of the nanoparticles changes to elongated rod-like structures. β-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids. PMID:25285787

  18. Activity of leucine aminopeptidase of Telchin licus licus: an important insect pest of sugarcane.

    PubMed

    Valencia, Jorge W Arboleda; de Sá, Maria Fátima Grossi; Jiménez, Arnubio Valencia

    2014-06-01

    The enzymatic activity of leucine aminopeptidase (EC 3.4.11.1) from the intestinal tract of sugarcane giant borer (Telchin licus licus) was assayed by using a simple and sensitive spectrophotometric assay that uses L-leucyl-2- naphthylamide as substrate. In this assay, L-leucyl-2-naphthylamide is hydrolyzed to produce 2-naphthylamine and Lleucine. The product 2-naphthylamine reacts with Fast Black K and can be monitored using a continuous spectrophotometric measurement at 590 nm. The data on the kinetic parameters indicates that the Km for the L-leucyl-2- naphthylamide at pH 7.0 was found to be lower than those found for other LAP substrates. The Km and Vmax for the LAP were determined to be 84.03 µM and 357.14 enzymatic units mg(-1), respectively. A noticeable difference of LAP activity between the two insect orders tested was observed. This method could be used to screen for natural LAP inhibitors. PMID:24410745

  19. Preferentially Expressed Antigen in Melanoma (PRAME) and the PRAME Family of Leucine-Rich Repeat Proteins.

    PubMed

    Hermes, Nora; Kewitz, Stefanie; Staege, Martin S

    2016-01-01

    Preferentially expressed antigen in melanoma (PRAME) is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia cells. Expression of PRAME and other members of the PRAME family is regulated epigenetically. PRAME interacts with varying pathways that might be directly involved in the malignant phenotype of cancer cells. For instance, PRAME is able to dominantly repress retinoic acid signaling in these cells. On the other hand, PRAME-derived peptides can be recognized as epitopes by cytotoxic T cells and PRAME represents an attractive target for immunological treatment strategies. PMID:26694250

  20. Function and dysfunction of leucine-rich repeat kinase 2 (LRRK2): Parkinson's disease and beyond.

    PubMed

    Bae, Jae Ryul; Lee, Byoung Dae

    2015-05-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). As such, functions and dysfunctions of LRRK2 in PD have been the subject of extensive investigation. In addition to PD, increasing evidence is suggesting that LRRK2 is associated with a wide range of diseases. Genome-wide association studies have implicated LRRK2 in Crohn's disease (CD) and leprosy, and the carriers with pathogenic mutations of LRRK2 show increased risk to develop particular types of cancer. LRRK2 mutations are rarely found in Alzheimer's disease (AD), but LRRK2 might play a part in tauopathies. The association of LRRK2 with the pathogenesis of apparently unrelated diseases remains enigmatic, but it might be related to the yet unknown diverse functions of LRRK2. Here, we reviewed current knowledge on the link between LRRK2 and several diseases, including PD, AD, CD, leprosy, and cancer, and discussed the possibility of targeting LRRK2 in such diseases. PMID:25703537

  1. [B17-D-leucine]insulin and [B17-norleucine]insulin: synthesis and biological properties.

    PubMed

    Knorr, R; Danho, W; Büllesbach, E E; Gattner, H G; Zahn, H; King, G L; Kahn, C R

    1983-11-01

    The chemical synthesis of two porcine insulin analogues is described. Leucine in position B17 of the native molecule was substituted by its D-enantiomer and by L-norleucine, respectively. Both B-chain derivatives were synthesized by fragment condensation and purified as di-S-sulphonates by gel filtration followed by ion exchange chromatography on SP-Sephadex at pH3. Combination with native sulphhydryl A-chain yielded [DLeuB17]insulin and [NleB17]insulin. Both insulin analogues were isolated by gel filtration followed by ion exchange chromatography on CM-cellulose at pH 4.0. Biological activities of the analogues were determined relative to native pork insulin: 1) glucose oxidation in rat epididymal adipocytes was 6% for [DLeuB17]insulin and 16% for [NleB17]insulin, 2) receptor-binding affinity tested with cultured human fibroblasts and with rat adipocytes was 3% for [DLeuB17]insulin and 26% for [NleB17]insulin, and 3) thymidine incorporation into DNA of human fibroblasts was 35% for [DLeuB17]insulin and 100% for [NleB17]insulin. PMID:6363268

  2. Gas-phase synthesis of solid state DNA nanoparticles stabilized by l-leucine.

    PubMed

    Raula, Janne; Hanzlíková, Martina; Rahikkala, Antti; Hautala, Juho; Kauppinen, Esko I; Urtti, Arto; Yliperttula, Marjo

    2013-02-28

    Aerosol flow reactor is used to generate solid-state nanoparticles in a one-step process that is based on drying of aerosol droplets in continuous flow. We investigated the applicability of aerosol flow reactor method to prepare solid state DNA nanoparticles. Precursor solutions of plasmid DNA with or without complexing agent (polyethylenimine), coating material (l-leucine) and mannitol (bulking material) were dispersed to nanosized droplets and instantly dried in laminar heat flow. Particle morphology, integrity and stability were studied by scanning electron microscopy. The stability of DNA was studied by gel electrophoresis. Plasmid DNA as such degraded in the aerosol flow process. Complexing agent protected DNA from degradation and coating material enabled production of dispersed, non-aggregated, nanoparticles. The resulting nanoparticles were spherical and their mean diameter ranged from 65 to 125nm. The nanoparticles were structurally stable at room temperature and their DNA content was about 10%. We present herein the proof of principle for the production of dispersed solid state nanoparticles with relevant size and intact plasmid DNA. PMID:23352859

  3. Molecular dynamics simulations of Na{sup +} and leucine transport by LeuT

    SciTech Connect

    Chen, Rong Chung, Shin-Ho

    2015-08-14

    Molecular dynamics simulations are used to gain insight into the binding of Na{sup +} and leucine substrate to the bacterial amino acid transporter LeuT, focusing on the crystal structures of LeuT in the outward-open and inward-open states. For both conformations of LeuT, a third Na{sup +} binding site involving Glu290 in addition to the two sites identified from the crystal structures is observed. Once the negative charge from Glu290 in the inward-open LeuT is removed, the ion bound to the third site is ejected from LeuT rapidly, suggesting that the protonation state of Glu290 regulates Na{sup +} binding and release. In Cl{sup −}-dependent transporters where Glu290 is replaced by a neutral serine, a Cl{sup −} ion would be required to replace the role of Glu290. Thus, the simulations provide insights into understanding Na{sup +} and substrate transport as well as Cl{sup −}-independence of LeuT. - Highlights: • Ion binding site involving Glu290 is identified in the outward- and inward-open LeuT. • Sodium is released from inward-open LeuT once the side chain of Glu290 is protonated. • Protonation state of Glu290 regulates sodium binding and transport in LeuT.

  4. The role of leucine and its metabolites in protein and energy metabolism.

    PubMed

    Duan, Yehui; Li, Fengna; Li, Yinghui; Tang, Yulong; Kong, Xiangfeng; Feng, Zemeng; Anthony, Tracy G; Watford, Malcolm; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2016-01-01

    Leucine (Leu) is a nutritionally essential branched-chain amino acid (BCAA) in animal nutrition. It is usually one of the most abundant amino acids in high-quality protein foods. Leu increases protein synthesis through activation of the mammalian target of rapamycin (mTOR) signaling pathway in skeletal muscle, adipose tissue and placental cells. Leu promotes energy metabolism (glucose uptake, mitochondrial biogenesis, and fatty acid oxidation) to provide energy for protein synthesis, while inhibiting protein degradation. Approximately 80 % of Leu is normally used for protein synthesis, while the remainder is converted to α-ketoisocaproate (α-KIC) and β-hydroxy-β-methylbutyrate (HMB) in skeletal muscle. Therefore, it has been hypothesized that some of the functions of Leu are modulated by its metabolites. Both α-KIC and HMB have recently received considerable attention as nutritional supplements used to increase protein synthesis, inhibit protein degradation, and regulate energy homeostasis in a variety of in vitro and in vivo models. Leu and its metabolites hold great promise to enhance the growth and health of animals (including humans, birds and fish). PMID:26255285

  5. Controlled Localization of Functionally Active Proteins to Inclusion Bodies Using Leucine Zippers

    PubMed Central

    Choi, Su-Lim; Lee, Sang Jun; Yeom, Soo-Jin; Kim, Hyun Ju; Rhee, Young Ha; Jung, Heung-Chae; Lee, Seung-Goo

    2014-01-01

    Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8–1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli. PMID:24897378

  6. Leucine-rich pentatricopeptide-repeat containing protein regulates mitochondrial transcription.

    PubMed

    Sondheimer, Neal; Fang, Ji-Kang; Polyak, Erzsebet; Falk, Marni J; Avadhani, Narayan G

    2010-09-01

    Mitochondrial function depends upon the coordinated expression of the mitochondrial and nuclear genomes. Although the basal factors that carry out the process of mitochondrial transcription are known, the regulation of this process is incompletely understood. To further our understanding of mitochondrial gene regulation, we identified proteins that bound to the previously described point of termination for the major mRNA-coding transcript H2. One was the leucine-rich pentatricopeptide-repeat containing protein (LRPPRC), which has been linked to the French-Canadian variant of Leigh syndrome. Cells with reduced expression of LRPPRC had a reduction in oxygen consumption. The expression of mitochondrial mRNA and tRNA was dependent upon LRPPRC levels, but reductions in LRPPRC did not affect the expression of mitochondrial rRNA. Reduction of LRPPRC levels interfered with mitochondrial transcription in vitro but did not affect the stability of mitochondrial mRNAs or alter the expression of nuclear genes responsible for mitochondrial transcription in vivo. These findings demonstrate the control of mitochondrial mRNA synthesis by a protein that has an established role in regulating nuclear transcription and a link to mitochondrial disease. PMID:20677761

  7. Role of glucocorticoid-induced leucine zipper (GILZ) in bone acquisition.

    PubMed

    Pan, Guodong; Cao, Jay; Yang, Nianlan; Ding, Kehong; Fan, Cheng; Xiong, Wen-Cheng; Hamrick, Mark; Isales, Carlos M; Shi, Xing-Ming

    2014-07-11

    Glucocorticoids (GCs) have both anabolic and catabolic effects on bone. However, no GC anabolic effect mediator has been identified to date. Here we show that targeted expression of glucocorticoid-induced leucine zipper (GILZ), a GC anti-inflammatory effect mediator, enhances bone acquisition in mice. Transgenic mice, in which the expression of GILZ is under the control of a 3.6-kb rat type I collagen promoter, exhibited a high bone mass phenotype with significantly increased bone formation rate and osteoblast numbers. The increased osteoblast activity correlates with enhanced osteogenic differentiation and decreased adipogenic differentiation of bone marrow stromal cell cultures in vitro. In line with these changes, the mRNA levels of key osteogenic regulators (Runx2 and Osx) increased, and the level of adipogenic regulator peroxisome proliferator-activated receptor (PPAR) γ2 decreased significantly. We also found that GILZ physically interacts with C/EBPs and disrupts C/EBP-mediated PPARγ gene transcription. In conclusion, our results showed that GILZ is capable of increasing bone acquisition in vivo, and this action is mediated via a mechanism involving the inhibition of PPARγ gene transcription and shifting of bone marrow MSC/progenitor cell lineage commitment in favor of the osteoblast pathway. PMID:24860090

  8. Dual leucine zipper kinase is required for excitotoxicity-induced neuronal degeneration

    PubMed Central

    Pozniak, Christine D.; Sengupta Ghosh, Arundhati; Gogineni, Alvin; Hanson, Jesse E.; Lee, Seung-Hye; Larson, Jessica L.; Solanoy, Hilda; Bustos, Daisy; Li, Hong; Ngu, Hai; Jubb, Adrian M.; Ayalon, Gai; Wu, Jiansheng; Scearce-Levie, Kimberly; Zhou, Qiang; Weimer, Robby M.; Kirkpatrick, Donald S.

    2013-01-01

    Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid–induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss. PMID:24166713

  9. A framework for interpreting the leucine-rich repeats of the Listeria internalins

    PubMed Central

    Marino, Michael; Braun, Laurence; Cossart, Pascale; Ghosh, Partho

    2000-01-01

    The surface protein InlB of the bacterial pathogen Listeria monocytogenes is required for inducing phagocytosis in various nonphagocytic mammalian cell types in vitro. InlB causes tyrosine phosphorylation of host cell adaptor proteins, activation of phosphoinositide 3-kinase, and rearrangements of the actin cytoskeleton. These events lead to phagocytic uptake of the bacterium by the host cell. InlB belongs to the internalin family of Listeria proteins, which also includes InlA, another surface protein involved in host cell invasion. The internalins are the largest class of bacterial proteins containing leucine-rich repeats (LRR), a motif associated with protein–protein interactions. The LRR motif is found in a functionally diverse array of proteins, including those involved in the plant immune system and in the mammalian innate immune response. Structural and functional interpretations of the sequences of internalin family members are presented in light of the recently determined x-ray crystal structure of the InlB LRR domain. PMID:10922035

  10. Capping motifs stabilize the leucine-rich repeat protein PP32 and rigidify adjacent repeats.

    PubMed

    Dao, Thuy P; Majumdar, Ananya; Barrick, Doug

    2014-06-01

    Capping motifs are found to flank most β-strand-containing repeat proteins. To better understand the roles of these capping motifs in organizing structure and stability, we carried out folding and solution NMR studies on the leucine-rich repeat (LRR) domain of PP32, which is composed of five tandem LRR, capped by α-helical and β-hairpin motifs on the N- and C-termini. We were able to purify PP32 constructs lacking either cap and containing destabilizing substitutions. Removing the C-cap results in complete unfolding of PP32. Removing the N-cap has a much less severe effect, decreasing stability but retaining much of its secondary structure. In contrast, the dynamics and tertiary structure of the first two repeats are significantly perturbed, based on (1)H-(15)N relaxation studies, chemical shift perturbations, and residual dipolar couplings. However, more distal repeats (3 to C-cap) retain their native tertiary structure. In this regard, the N-cap drives the folding of adjacent repeats from what appears to be a molten-globule-like state. This interpretation is supported by extensive analysis using core packing substitutions in the full-length and N-cap-truncated PP32. This work highlights the importance of caps to the stability and structural integrity of β-strand-containing LRR proteins, and emphasizes the different contributions of the N- and C-terminal caps. PMID:24659532

  11. Maternal embryonic leucine zipper kinase: key kinase for stem cell phenotype in glioma and other cancers.

    PubMed

    Ganguly, Ranjit; Hong, Christopher S; Smith, Luke G F; Kornblum, Harley I; Nakano, Ichiro

    2014-06-01

    Maternal embryonic leucine zipper kinase (MELK) is a member of the snf1/AMPK family of protein serine/threonine kinases that has recently gained significant attention in the stem cell and cancer biology field. Recent studies suggest that activation of this kinase is tightly associated with extended survival and accelerated proliferation of cancer stem cells (CSC) in various organs. Overexpression of MELK has been noted in various cancers, including colon, breast, ovaries, pancreas, prostate, and brain, making the inhibition of MELK an attractive therapeutic strategy for a variety of cancers. In the experimental cancer models, depletion of MELK by RNA interference or small molecule inhibitors induces apoptotic cell death of CSCs derived from glioblastoma multiforme and breast cancer, both in vitro and in vivo. Mechanism of action of MELK includes, yet may not be restricted to, direct binding and activation of the oncogenic transcription factors c-JUN and FOXM1 in cancer cells but not in the normal counterparts. Following these preclinical studies, the phase I clinical trial for advanced cancers with OTSSP167 started in 2013, as the first-in-class MELK inhibitor. This review summarizes the current molecular understanding of MELK and the recent preclinical studies about MELK as a cancer therapeutic target. PMID:24795222

  12. Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia.

    PubMed

    Alachkar, Houda; Mutonga, Martin B G; Metzeler, Klaus H; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

    2014-12-15

    Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients. PMID:25365263

  13. Maternal embryonic leucine zipper kinase regulates pancreatic ductal, but not β-cell, regeneration.

    PubMed

    Chung, Cheng-Ho; Miller, Amber; Panopoulos, Andreas; Hao, Ergeng; Margolis, Robert; Terskikh, Alexey; Levine, Fred

    2014-09-01

    The maternal embryonic leucine zipper kinase (MELK) is expressed in stem/progenitor cells in some adult tissues, where it has been implicated in diverse biological processes, including the control of cell proliferation. Here, we described studies on its role in adult pancreatic regeneration in response to injury induced by duct ligation and β-cell ablation. MELK expression was studied using transgenic mice expressing GFP under the control of the MELK promoter, and the role of MELK was studied using transgenic mice deleted in the MELK kinase domain. Pancreatic damage was initiated using duct ligation and chemical beta-cell ablation. By tracing MELK expression using a MELK promoter-GFP transgene, we determined that expression was extremely low in the normal pancreas. However, following duct ligation and β-cell ablation, it became highly expressed in pancreatic ductal cells while remaining weakly expressed in α-cells and β- cells. In a mutant mouse in which the MELK kinase domain was deleted, there was no effect on pancreatic development. There was no apparent effect on islet regeneration, either. However, following duct ligation there was a dramatic increase in the number of small ducts, but no change in the total number of duct cells or duct cell proliferation. In vitro studies indicated that this was likely due to a defect in cell migration. These results implicate MELK in the control of the response of the pancreas to injury, specifically controlling cell migration in normal and transformed pancreatic duct cells. PMID:25194022

  14. Nurit, a novel leucine-zipper protein, expressed uniquely in the spermatid flower-like structure.

    PubMed

    Feige, Erez; Chen, Aviva; Motro, Benny

    2002-09-01

    Spermatozoa formation involves drastic morphological and cellular reconstructions. However, the molecular mechanisms driving this process remain elusive. We describe the cloning of a novel murine spermatid-specific gene, designated nurit, identified in a two-hybrid screen for proteins that binds the Nek1 kinase. Nurit protein harbors a leucine-zipper motif, and two additional coiled-coil regions. The C-terminal coiled-coil domain mediates homodimerization of the protein. Nurit homologues are found in primates, pig and rodents. nurit is transcribed through the elongation stage of the spermatids, but is absent from mature spermatozoa. Interestingly, immunogold electron microscopy revealed that the protein is restricted, from its first detectable appearance, to a unique spermatid organelle called the 'flower-like structure'. The function of this structure is unknown, though it may be involved in transporting proteins designated to be discarded via the residual bodies. Nurit is the first marker of the flower-like structure, and its study may provide an excellent opportunity to dissect the function of this organelle. PMID:12204287

  15. Key roles for the small leucine-rich proteoglycans in renal and pulmonary pathophysiology

    PubMed Central

    Nastase, Madalina V.; Iozzo, Renato V.; Schaefer, Liliana

    2014-01-01

    Background Small leucine-rich proteoglycans (SLRPs) are molecules that have signaling roles in a multitude of biological processes. In this respect, SLRPs play key roles in the evolution of a variety of diseases throughout the human body. Scope of Review We will critically review current developments in the roles of SLRPs in several types of disease of the kidney and lungs. Particular emphasis will be given to the roles of decorin and biglycan, the best characterized members of the SLRP gene family. Major Conclusions In both renal and pulmonary disorders, SLRPs are essential elements that regulate several pathophysiological processes including fibrosis, inflammation and tumor progression. Decorin has remarkable antifibrotic and antitumorigenic properties and is considered a valuable potential treatment of these diseases. Biglycan can modulate inflammatory processes in lung and renal inflammation and is a potential target in the treatment of inflammatory conditions. General significance SLRPs can serve as either treatment targets or as potential treatment in renal or lung disease. PMID:24508120

  16. Escherichia coli d-Malate Dehydrogenase, a Generalist Enzyme Active in the Leucine Biosynthesis Pathway*

    PubMed Central

    Vorobieva, Anastassia A.; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-01-01

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB− strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

  17. Anti-leucine-rich glioma-inactivated 1 limbic encephalitis: A case report and literature review

    PubMed Central

    LIU, JINGYAO; LI, MIN; LI, GUIBO; ZHOU, CHUNKUI; ZHANG, RENSHENG

    2016-01-01

    This study describes the case of a 41-year-old woman admitted for anterograde memory loss, right facial grimacing and right arm posturing that had begun 1 month previously. Cranial magnetic resonance-diffusion weighted imaging and -fluid-attenuated inversion recovery imaging revealed a hyperintense signal in the left hippocampus and right basal ganglia, but no contrast enhancement. An electroencephalogram revealed rhythmic sharp and slow waves and rhythmic θ build-ups in the left temporal area. Single-photon emission computed tomography showed increased regional blood flow perfusion in the left cerebral frontal lobe and the right basal ganglia. The cerebrospinal fluid was normal, with the exception of the presence of leucine-rich glioma-inactivated 1 (LGI1) antibodies, and LGI1 antibodies were also found in the blood serum. The presence of the antibodies, the faciobrachial dystonic seizures (FBDSs) and the memory loss indicated limbic encephalitis. After 3 months of immunotherapy, the patient was free from epileptic seizures and had undergone a partial memory restoration. FBDSs alone justify the immediate initiation of immunotherapy, even prior to laboratory confirmation of the disease, as early treatment limits the duration of the illness. PMID:26889260

  18. Spectroscopic evidence of β-turn in N-glycated peptidomimetics related to leucine-enkephalin

    NASA Astrophysics Data System (ADS)

    Vass, E.; Hollósi, M.; Kveder, M.; Kojić-Prodić, B.; Čudić, M.; Horvat, Š.

    2000-11-01

    The conformational differences caused by N-glycation of the amide bond in endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) have been explored in solution using FTIR spectroscopy, NMR and molecular modelling. The compounds studied include protected and unprotected enkephalin analogues N-alkylated at the second (Gly 2) amino acid residue with a 6-deoxy- D-galactose moiety ( 1- 3). Comparison of the amide I component bands in the FTIR spectra, measured in trifluoroethanol (TFE), CHCl 3 and DMSO, revealed significant differences in the intensity as well as shifts in component band frequencies for glycopeptides 1- 3. We found that only the FTIR spectrum of the fully protected compound 1 indicated the presence of a higher population of β-turns, while the spectra of the partially protected and unprotected glycopeptides 2 and 3 reflected the dominance of unordered or open structures, with some low population of turns. The observed NOE connectivities in CDCl 3 for both isomers of the fully protected compound 1, the all-trans one and another with Tyr 1-Gly 2 peptide bond in cis conformation, indicate the presence of a β-like turn conformation. Molecular dynamics simulations of the glycopeptide 1 obtained by unconstrained energy minimization of trans- and cis- 1 shows that one of trans form conformations is consistent with β-turn whereas cis isomer has revealed less-compact turn.

  19. Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms.

    PubMed

    Shanmugam, V

    2005-01-01

    Plant-pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG-PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management. PMID:15782942

  20. Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

    PubMed Central

    Lee, Yu-Ran; Na, Byoung-Kuk; Moon, Eun-Kyung; Song, Su-Min; Joo, So-Young; Kong, Hyun-Hee; Goo, Youn-Kyoung; Chung, Dong-Il; Hong, Yeonchul

    2015-01-01

    Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba. PMID:26075721

  1. The estrous cycle modulates small leucine-rich proteoglycans expression in mouse uterine tissues.

    PubMed

    Salgado, Renato M; Favaro, Rodolfo R; Martin, Sebastian San; Zorn, Telma M T

    2009-01-01

    In the pregnant mouse uterus, small leucine-rich proteoglycans (SLRPs) are drastically remodeled within a few hours after fertilization, suggesting that ovarian hormone levels modulate their synthesis and degradation. In this study, we followed by immunoperoxidase approach, the presence of four members of the SLRP family (decorin, lumican, biglycan, and fibromodulin) in the uterine tissues along the estrous cycle of the mouse. All molecules except fibromodulin, which predominates in the myometrium, showed a striking modulation in their distribution in the endometrial stroma, following the rise in the level of estrogen. Moreover, notable differences in the distribution of SLRPs were observed between superficial and deep stroma, as well as between the internal and external layers of the myometrium. Only biglycan and fibromodulin were expressed in the luminal and glandular epithelia. All four SLRPs were found in cytoplasmic granules of mononucleated cells. The pattern of distribution of the immunoreaction for these molecules in the uterine tissues was found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation. In addition, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of the decidua. Anat Rec, 2008. (c) 2008 Wiley-Liss, Inc. PMID:18951514

  2. Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma.

    PubMed

    Satoh, Shunpei; Takatori, Atsushi; Ogura, Atsushi; Kohashi, Kenichi; Souzaki, Ryota; Kinoshita, Yoshiaki; Taguchi, Tomoaki; Hossain, Md Shamim; Ohira, Miki; Nakamura, Yohko; Nakagawara, Akira

    2016-01-01

    In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1-/- mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB. PMID:27604320

  3. Regulation of taste-active components of meat by dietary leucine.

    PubMed

    Imanari, M; Kadowaki, M; Fujimura, S

    2007-04-01

    1. Regulation of meat taste is one effective method for improvement of meat quality. In this study, effects of dietary leucine (Leu) content on taste-active components, especially free glutamate (Glu), in meat were investigated. 2. Broiler chickens (28 d old) were fed on diets with graded dietary Leu content (100, 130 or 150% of Leu requirement in NRC, 1994) for 10 d before marketing. Taste-active components of meat (free amino acids and ATP metabolites) and sensory score of meat soup were estimated. 3. Free Glu content, the main taste-active component of meat, was significantly increased by dietary Leu. Compared with the Leu 130% group, free Glu was increased by 17% in the Leu 100% group. Free Glu of meat tended to decrease in the Leu 150% group. In contrast, inosine monophosphate content in meat did not change among all groups. 4. Sensory evaluation of meat soup from the Leu 100 and 150% groups showed that they had different meat tastes. Sensory scores of overall preference, umami taste and chicken-like taste were significantly higher in the Leu 100% group. 5. These results suggest that dietary Leu content is a regulating factor of free Glu in meat. Decreasing dietary Leu induces an increase in the free Glu content of meat and improves meat taste. PMID:17453808

  4. The role and effects of glucocorticoid-induced leucine zipper in the context of inflammation resolution.

    PubMed

    Vago, Juliana P; Tavares, Luciana P; Garcia, Cristiana C; Lima, Kátia M; Perucci, Luiza O; Vieira, Érica L; Nogueira, Camila R C; Soriani, Frederico M; Martins, Joilson O; Silva, Patrícia M R; Gomes, Karina B; Pinho, Vanessa; Bruscoli, Stefano; Riccardi, Carlo; Beaulieu, Elaine; Morand, Eric F; Teixeira, Mauro M; Sousa, Lirlândia P

    2015-05-15

    Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate or mimic several actions of GC. This study assessed the role of GILZ in self-resolving and GC-induced resolution of neutrophilic inflammation induced by LPS in mice. GILZ expression was increased during the resolution phase of LPS-induced pleurisy, especially in macrophages with resolving phenotypes. Pretreating LPS-injected mice with trans-activator of transcription peptide (TAT)-GILZ, a cell-permeable GILZ fusion protein, shortened resolution intervals and improved resolution indices. Therapeutic administration of TAT-GILZ induced inflammation resolution, decreased cytokine levels, and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the activation of the survival-controlling proteins ERK1/2, NF-κB and Mcl-1. GILZ deficiency was associated with an early increase of annexin A1 (AnxA1) and did not modify the course of neutrophil influx induced by LPS. Dexamethasone treatment resolved inflammation and induced GILZ expression that was dependent on AnxA1. Dexamethasone-induced resolution was not altered in GILZ(-/-) mice due to compensatory expression and action of AnxA1. Our results show that therapeutic administration of GILZ efficiently induces a proapoptotic program that promotes resolution of neutrophilic inflammation induced by LPS. Alternatively, a lack of endogenous GILZ during the resolution of inflammation is compensated by AnxA1 overexpression. PMID:25876761

  5. Effects of Leucin-Enkephalins on Surface Characteristics and Morphology of Model Membranes Composed of Raft-Forming Lipids.

    PubMed

    Tsanova, Asya; Jordanova, A; Lalchev, Z

    2016-06-01

    During the last decades opioid peptides, like enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are subject to extensive studies due to their antinociceptive action in organism. According to the membrane catalysis theory, in order to adopt a proper conformation for binding to their receptors, opioid peptides interact with the lipid phase of the membrane receptor surrounding. With this regard, the aim of the present work was to study the effects of synthetic leucine-enkephalin and leucine-enkephalinamide on surface characteristics and morphology of lipid monolayers, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol alone and with their mixtures. The lipids were chosen to represent a model of a membrane raft, since it is known that G-protein-coupled receptors, including opioid receptors, are located preferably in membrane rafts. By using Langmuir's monolayer method, the change in surface pressure of the model membranes before and after the addition of the synthetic enkephalins was studied, and the compressional moduli of the lipids and lipid-peptides monolayers were determined. In addition, by Brewster angle microscopy, the surface morphology of the lipid monolayers alone and after the injection of both enkephalins was monitored. Our results showed that both leucine-enkephalins affected the lipid monolayers surface characteristics, and led to an increase in surface density of the mixed surface lipids/enkephalins films at loose lipid packing. This effect was more pronounced for the enkephalinamide, suggesting a different mechanism of interaction for the amidated enkephalin with the lipid phase, as compared to leucine-enkephalin. PMID:26661722

  6. Hypoxia and Leucine Deprivation Induce Human Insulin-Like Growth Factor Binding Protein-1 Hyperphosphorylation and Increase Its Biological Activity

    PubMed Central

    Seferovic, Maxim D.; Ali, Rashad; Kamei, Hiroyasu; Liu, Suya; Khosravi, Javad M.; Nazarian, Steven; Han, Victor K. M.; Duan, Cunming; Gupta, Madhulika B.

    2009-01-01

    Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)−10 m and 6.40E−09 m] relative to the IGFBP-1 from the controls (dissociation constant ∼1.54E−07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction. PMID:18772238

  7. Leucine-enriched essential amino acids attenuate inflammation in rat muscle and enhance muscle repair after eccentric contraction.

    PubMed

    Kato, Hiroyuki; Miura, Kyoko; Nakano, Sayako; Suzuki, Katsuya; Bannai, Makoto; Inoue, Yoshiko

    2016-09-01

    Eccentric exercise results in prolonged muscle damage that may lead to muscle dysfunction. Although inflammation is essential to recover from muscle damage, excessive inflammation may also induce secondary damage, and should thus be suppressed. In this study, we investigated the effect of leucine-enriched essential amino acids on muscle inflammation and recovery after eccentric contraction. These amino acids are known to stimulate muscle protein synthesis via mammalian target of rapamycin (mTOR), which, is also considered to alleviate inflammation. Five sets of 10 eccentric contractions were induced by electrical stimulation in the tibialis anterior muscle of male SpragueDawley rats (8-9 weeks old) under anesthesia. Animals received a 1 g/kg dose of a mixture containing 40 % leucine and 60 % other essential amino acids or distilled water once a day throughout the experiment. Muscle dysfunction was assessed based on isometric dorsiflexion torque, while inflammation was evaluated by histochemistry. Gene expression of inflammatory cytokines and myogenic regulatory factors was also measured. We found that leucine-enriched essential amino acids restored full muscle function within 14 days, at which point rats treated with distilled water had not fully recovered. Indeed, muscle function was stronger 3 days after eccentric contraction in rats treated with amino acids than in those treated with distilled water. The amino acid mix also alleviated expression of interleukin-6 and impeded infiltration of inflammatory cells into muscle, but did not suppress expression of myogenic regulatory factors. These results suggest that leucine-enriched amino acids accelerate recovery from muscle damage by preventing excessive inflammation. PMID:27168073

  8. D-Amino acids influence ultrasonic calling in mice pups: effects of D-phenylalanine and D-leucine.

    PubMed

    Albonetti, M E; D'Udine, B; Oliverio, A

    1985-06-24

    6-day-old mice pups were injected with D-amino acids (D-phenyl-alanine + D-leucine), and their ultrasonic distress vocalizations were measured. D-Amino acids, which exert opioid-like effects, reduce the number of ultrasonic calls without affecting the activity of the pups. This effect is reversed by naloxone, an opioid antagonist. The role of endogenous opioids in modulating early attachment is discussed. PMID:4041020

  9. BasicODT

    Energy Science and Technology Software Center (ESTSC)

    2007-09-25

    BasicODT is a Monte Carlo simulation that numerically implements One-Dimensional Turbulence (ODT), a stochastic model of turbulent flow that was developed by the author of the code. This code is set up to simulate channel flow, which is the flow between two parallel flat walls driven by a fixed pressure gradient, with no-slip conditions at the walls. The code writes output files containing flow statistics gathered during the simulation. The code is accompanied by documentationmore » that explains how ODT modeling principles are numerically implemented within the code. The code and documentation are intended as an introduction to ODT for use as a learning tool for people who are unfamiliar with the model and its numerical implementation. ODT is fully described in published literature.« less

  10. Basic trauma life support.

    PubMed

    Werman, H A; Nelson, R N; Campbell, J E; Fowler, R L; Gandy, P

    1987-11-01

    The impact of traumatic injuries on modern society in terms of morbidity, mortality, and economic cost is enormous. Studies have shown that both advanced life support skills and rapid stabilization and transport of the trauma victim have a beneficial effect on the patient's ultimate outcome. The Basic Trauma Life Support (BTLS) course was designed to provide pre-hospital care providers with the skills necessary to provide a thorough assessment, initial resuscitation, and rapid transportation of the trauma victim. Early studies suggest that the material is easily learned by prehospital care providers and that the on-scene time for trauma cases is reduced following training in BTLS. More widespread training in BTLS may have a significant effect on the mortality and morbidity associated with traumatic injuries. PMID:3662184

  11. Basic properties and variability

    NASA Technical Reports Server (NTRS)

    Querci, Francois R.

    1987-01-01

    Giant and supergiant M, S, and C stars are discussed in this survey of research. Basic properties as determined by spectra, chemical composition, photometry, or variability type are discussed. Space motions and space distributions of cool giants are described. Distribution of these stars in our galaxy and those nearby is discussed. Mira variables in particular are surveyed with emphasis on the following topics: (1) phase lag phenomenon; (2) Mira light curves; (3) variations in color indices; (4) determination of multiple periods; (5) correlations between quantities such as period length, light-curve shape, infrared (IR) excess, and visible and IR color diagram; (6) semiregular (SR) variables and different time scales in SR light variations; (7) irregular variable Lb and Lc stars; (8) different time-scale light variations; (9) hydrogen-deficient carbon (HdC) stars, in particular RCB stars; and (10) irreversible changes and rapid evolution in red variable stars.

  12. Basic memory module

    NASA Technical Reports Server (NTRS)

    Tietze, F. C.

    1974-01-01

    Construction and electrical characterization of the 4096 x 2-bit Basic Memory Module (BMM) are reported for the Space Ultrareliable Modular Computer (SUMC) program. The module uses four 2K x 1-bit N-channel FET, random access memory chips, called array chips, and two sense amplifier chips, mounted and interconnected on a ceramic substrate. Four 5% tolerance power supplies are required. At the Module, the address, chip select, and array select lines require a 0-8.5 V MOS signal level. The data output, read-strobe, and write-enable lines operate at TTl levels. Although the module is organized as 4096 x 2 bits, it can be used in a 8196 x 1-bit application with appropriate external connections. A 4096 x 1-bit organization can be obtained by depopulating chips.

  13. L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q- syndrome in a TP53-independent way.

    PubMed

    Narla, Anupama; Payne, Elspeth M; Abayasekara, Nirmalee; Hurst, Slater N; Raiser, David M; Look, A Thomas; Berliner, Nancy; Ebert, Benjamin L; Khanna-Gupta, Arati

    2014-11-01

    Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner. PMID:25098371

  14. Number and phylogenetic affiliation of bacteria assimilating dimethylsulfoniopropionate and leucine in the ice-covered coastal Arctic Ocean

    NASA Astrophysics Data System (ADS)

    Vila-Costa, Maria; Simó, Rafel; Alonso-Sáez, Laura; Pedrós-Alió, Carlos

    2008-12-01

    The ability of bacteria to assimilate sulfur from dimethylsulfoniopropionate (DMSP) was examined in the western Arctic Ocean by combining microautoradiography and fluorescence in situ hybridization (FISH). Assimilation of leucine was also measured for comparative purposes since leucine is considered a universal substrate for bacteria, which use it for protein synthesis. Samples were collected at 3 m depth, through a hole in the ice, in the CASES (Canadian Arctic Shelf Ecosystem Study) overwintering station in Franklin Bay (eastern Beaufort Sea) in March and May 2004 to compare two contrasting situations: winter and early spring. FISH counts indicated that the bacterial assemblage consisted of α- (up to 60% of the EUB positive cells), β- (up to 10%) and γ-proteobacteria (around 20%), and Bacteroidetes (up to 60%). The β-proteobacteria were not active with any of the two substrates tested. The remaining groups were much less efficient at assimilating DMSP-sulfur (5% of the cells) than leucine (20-35%) both in winter and in spring. Only the Roseobacter group of α-proteobacteria showed a similar assimilation of both substrates.

  15. Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo.

    PubMed Central

    Kirkpatrick, C R; Schimmel, P

    1995-01-01

    The product of the Saccharomyces cerevisiae LEU3 gene, Leu3p, is a transcriptional activator which regulates leucine biosynthesis in response to intracellular levels of leucine through the biosynthetic intermediate alpha-isopropylmalate. We devised a novel assay to examine the DNA site occupancy of Leu3p under different growth conditions, using a reporter gene with internal Leu3p-binding sites. Expression of the reporter is inhibited by binding of nuclear Leu3p to these sites; inhibition is dependent on the presence of the sites in the reporter, on the integrity of the Leu3p DNA-binding domain, and, surprisingly, on the presence of a transcriptional activation domain in the inhibiting protein. By this assay, Leu3p was found to occupy its binding site under all conditions tested, including high and low levels of leucine and in the presence and absence of alpha-isopropylmalate. The localization of Leu3p to the nucleus was confirmed by immunofluorescence staining of cells expressing epitope-tagged Leu3p derivatives. We conclude that Leu3p regulates transcription in vivo without changing its intracellular localization and DNA site occupancy. PMID:7623798

  16. A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase

    PubMed Central

    Sträter, Norbert; Sun, Lee; Kantrowitz, E. R.; Lipscomb, William N.

    1999-01-01

    The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when l-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces kcat/Km about 10,000-fold. PMID:10500145

  17. Distinct patterns of the histone marks associated with recruitment of the methionine chain-elongation pathway from leucine biosynthesis.

    PubMed

    Xue, Ming; Long, Jingcheng; Jiang, Qinlong; Wang, Minghui; Chen, Sixue; Pang, Qiuying; He, Yan

    2015-02-01

    Aliphatic glucosinolates (GLSs) are derived from chain-elongated methionine produced by an iterative three-step process, known to be evolutionarily recruited from leucine biosynthesis. The divergence of homologous genes between two pathways is mainly linked to the alterations in biochemical features. In this study, it was discovered that a distinct pattern of histone modifications is associated with and/or contributes to the divergence of the two pathways. In general, genes involved in leucine biosynthesis were robustly associated with H3k4me2 and H3K4me3. In contrast, despite the considerable abundances of H3K4me2 observed in some of genes involved in methionine chain elongation, H3K4me3 was completely missing. This H3K4m3-depleted pattern had no effect on gene transcription, whereas it seemingly co-evolved with the entire pathway of aliphatic GLS biosynthesis. The results reveal a novel association of the epigenetic marks with plant secondary metabolism, and may help to understand the recruitment of the methionine chain-elongation pathway from leucine biosynthesis. PMID:25428994

  18. Involvement of the Neutral Amino Acid Transporter SLC6A15 and Leucine in Obesity-Related Phenotypes

    PubMed Central

    Drgonova, Jana; Jacobsson, Josefin A.; Han, Joan C.; Yanovski, Jack A.; Fredriksson, Robert; Marcus, Claude; Schiöth, Helgi B.; Uhl, George R.

    2013-01-01

    Brain pathways, including those in hypothalamus and nucleus of the solitary tract, influence food intake, nutrient preferences, metabolism and development of obesity in ways that often differ between males and females. Branched chain amino acids, including leucine, can suppress food intake, alter metabolism and change vulnerability to obesity. The SLC6A15 (v7-3) gene encodes a sodium-dependent transporter of leucine and other branched chain amino acids that is expressed by neurons in hypothalamus and nucleus of the solitary tract. We now report that SLC6A15 knockout attenuates leucine's abilities to reduce both: a) intake of normal chow and b) weight gain produced by access to a high fat diet in gender-selective fashions. We identify SNPs in the human SLC6A15 that are associated with body mass index and insulin resistance in males. These observations in mice and humans support a novel, gender-selective role for brain amino acid compartmentalization mediated by SLC6A15 in diet and obesity-associated phenotypes. PMID:24023709

  19. Asymmetrical transfer of alpha-aminoisobutyric acid (AIB), leucine and lysine across the in vitro perfused human placenta.

    PubMed

    Schneider, H; Proegler, M; Sodha, R; Dancis, J

    1987-01-01

    The mechanism for establishing transplacental gradients for leucine, lysine and alpha-aminoisobutyric acid (AIB) has been investigated in the perfused human placenta. Experiments were done with either the maternal or the fetal circulation closed and the donor circulation open. Transfer of the amino acids towards the fetal side was more rapid than it was in the reverse direction. When the maternal perfusate was recirculated, the amino acid concentrations were maintained at a considerably lower level in the maternal circulation than in the open fetal circuit. When the fetal circuit was closed, the concentrations approached or slightly exceeded those in the maternal perfusate over a period of three hours. Within the placenta, higher concentrations were established during the experiments with transfer towards the fetal side than in the reverse direction. Of the three amino acids, leucine was transferred most rapidly across the placenta while AIB reached the highest concentrations in the placental tissue. The asymmetry of the transplacental amino acid flux is favoured by rapid uptake from the maternal circulation and transfer towards the fetus. Both rates exceed those observed in the reverse direction. The transfer rate of D-leucine was 1.7 times that of L-glucose. For in vitro studies of the transfer rate of physiological compounds a correction for diffusion is required. The results may differ considerably depending on which marker is used as the basis. PMID:3112761

  20. GCN2 contributes to mTORC1 inhibition by leucine deprivation through an ATF4 independent mechanism.

    PubMed

    Averous, Julien; Lambert-Langlais, Sarah; Mesclon, Florent; Carraro, Valérie; Parry, Laurent; Jousse, Céline; Bruhat, Alain; Maurin, Anne-Catherine; Pierre, Philippe; Proud, Christopher G; Fafournoux, Pierre

    2016-01-01

    It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids. PMID:27297692

  1. Improved muscle function and quality after diet intervention with leucine-enriched whey and antioxidants in antioxidant deficient aged mice.

    PubMed

    van Dijk, Miriam; Dijk, Francina J; Bunschoten, Annelies; van Dartel, Dorien A M; van Norren, Klaske; Walrand, Stephane; Jourdan, Marion; Verlaan, Sjors; Luiking, Yvette

    2016-04-01

    Antioxidant (AOX) deficiencies are commonly observed in older adults and oxidative stress has been suggested to contribute to sarcopenia. Here we investigate if 1) low levels of dietary antioxidants had a negative impact on parameters of muscle mass, function and quality, and 2) to study if nutritional interventions with AOX and/or leucine-enriched whey protein could improve these muscle parameters in aged mice. 18-months-old mice were fed a casein-based antioxidant-deficient (lowox) diet or a casein-based control-diet (CTRL) for 7 months. During the last 3 months, lowox-mice were subjected to either: a) continued lowox, b) supplementation with vitamin A/E, Selenium and Zinc (AOX), c) substitution of casein with leucine-enriched whey protein (PROT) or d) a combination of both AOX and PROT (TOTAL). After 7 months lowox-mice displayed lower muscle strength and more muscle fatigue compared to CTRL. Compared to lowox-mice, PROT-mice showed improved muscle power, grip strength and less muscle fatigue. AOX-mice showed improved oxidative status, less muscle fatigue, improved grip strength and mitochondrial dynamics compared to lowox-mice. The TOTAL-mice showed the combined effects of both interventions compared to lowox-mice. In conclusion, nutritional intervention with AOX and/or leucine-enriched whey protein can play a role in improving muscle health in a AOX-deficient mouse model. PMID:26943770

  2. Point mutation in a leucine-rich repeat of platelet glycoprotein Ib alpha resulting in the Bernard-Soulier syndrome.

    PubMed Central

    Ware, J; Russell, S R; Marchese, P; Murata, M; Mazzucato, M; De Marco, L; Ruggeri, Z M

    1993-01-01

    Leucine-rich repeats are a conserved structural motif, of yet undefined significance, found in a group of proteins from different species. Among these are the four components of the human platelet glycoprotein Ib-IX-V complex, a membrane receptor that performs an essential role in the thrombogenic function of platelets by interacting with the adhesive protein, von Willebrand factor. We have found that a single amino acid substitution (Ala156-->Val) within one of the six leucine-rich repeats in the alpha-subunit of glycoprotein Ib results in a variant form of the congenital bleeding disorder, Bernard-Soulier syndrome, characterized by giant dysfunctional platelets. Genetic studies of the propositus and his family members were complemented by immunological and functional analysis of expressed recombinant GP Ib alpha fragments to demonstrate that the observed mutation is the cause of defective von Willebrand factor binding. These studies define the molecular basis of the Bernard-Soulier syndrome within this family and demonstrate that structural integrity of a leucine-rich repeat is necessary for normal function of the glycoprotein Ib-IX-V receptor complex and, possibly, for normal platelet morphology. Images PMID:7690774

  3. Improved muscle function and quality after diet intervention with leucine-enriched whey and antioxidants in antioxidant deficient aged mice

    PubMed Central

    van Dijk, Miriam; Dijk, Francina J.; Bunschoten, Annelies; van Dartel, Dorien A.M.; van Norren, Klaske; Walrand, Stephane; Jourdan, Marion; Verlaan, Sjors; Luiking, Yvette

    2016-01-01

    Antioxidant (AOX) deficiencies are commonly observed in older adults and oxidative stress has been suggested to contribute to sarcopenia. Here we investigate if 1) low levels of dietary antioxidants had a negative impact on parameters of muscle mass, function and quality, and 2) to study if nutritional interventions with AOX and/or leucine-enriched whey protein could improve these muscle parameters in aged mice. 18-months-old mice were fed a casein-based antioxidant-deficient (lowox) diet or a casein-based control-diet (CTRL) for 7 months. During the last 3 months, lowox-mice were subjected to either: a) continued lowox, b) supplementation with vitamin A/E, Selenium and Zinc (AOX), c) substitution of casein with leucine-enriched whey protein (PROT) or d) a combination of both AOX and PROT (TOTAL). After 7 months lowox-mice displayed lower muscle strength and more muscle fatigue compared to CTRL. Compared to lowox-mice, PROT-mice showed improved muscle power, grip strength and less muscle fatigue. AOX-mice showed improved oxidative status, less muscle fatigue, improved grip strength and mitochondrial dynamics compared to lowox-mice. The TOTAL-mice showed the combined effects of both interventions compared to lowox-mice. In conclusion, nutritional intervention with AOX and/or leucine-enriched whey protein can play a role in improving muscle health in a AOX-deficient mouse model. PMID:26943770

  4. GCN2 contributes to mTORC1 inhibition by leucine deprivation through an ATF4 independent mechanism

    PubMed Central

    Averous, Julien; Lambert-Langlais, Sarah; Mesclon, Florent; Carraro, Valérie; Parry, Laurent; Jousse, Céline; Bruhat, Alain; Maurin, Anne-Catherine; Pierre, Philippe; Proud, Christopher G.; Fafournoux, Pierre

    2016-01-01

    It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids. PMID:27297692

  5. Basic Blood Tests (For Parents)

    MedlinePlus

    ... What to Say Vaccines: Which Ones & When? Smart School Lunches Emmy-Nominated Video "Cerebral Palsy: Shannon's Story" 5 Things to Know About Zika & Pregnancy Basic Blood Chemistry Tests KidsHealth > For Parents > Basic Blood Chemistry Tests ...

  6. The Basic Leucine Zipper Domain Transcription Factor Atf1 Directly Controls Cdc13 Expression and Regulates Mitotic Entry Independently of Wee1 and Cdc25 in Schizosaccharomyces pombe

    PubMed Central

    Bandyopadhyay, Sushobhana; Dey, Isha; Suresh, Megalakshmi

    2014-01-01

    Progression into mitosis is a major point of regulation in the Schizosaccharomyces pombe cell cycle, and its proper control is essential for maintenance of genomic stability. Investigation of the G2/M progression event in S. pombe has revealed the existence of a complex regulatory process that is responsible for making the decision to enter mitosis. Newer aspects of this regulation are still being revealed. In this paper, we report the discovery of a novel mode of regulation of G2/M progression in S. pombe. We show that the mitogen-activated protein kinase (MAPK)-regulated transcription factor Atf1 is a regulator of Cdc13 (mitotic cyclin) transcription and is therefore a prominent player in the regulation of mitosis in S. pombe. We have used genetic approaches to study the effect of overexpression or deletion of Atf1 on the cell length and G2/M progression of S. pombe cells. Our results clearly show that Atf1 overexpression accelerates mitosis, leading to an accumulation of cells with shorter lengths. The previously known major regulators of entry into mitosis are the Cdc25 phosphatase and the Wee1 kinase, which modulate cyclin-dependent kinase (CDK) activity. The significantly striking aspect of our discovery is that Atf1-mediated G2/M progression is independent of both Cdc25 and Wee1. We have shown that Atf1 binds to the Cdc13 promoter, leading to activation of Cdc13 expression. This leads to enhanced nuclear localization of CDK Cdc2, thereby promoting the G2/M transition. PMID:24728197

  7. BULGARIAN, BASIC COURSE, VOLUME 1.

    ERIC Educational Resources Information Center

    HODGE, CARLETON T.; AND OTHERS

    A BASIC COURSE IN BULGARIAN HAS BEEN PREPARED IN TWO VOLUMES. THIS VOLUME, VOLUME 1, IS DIVIDED INTO THREE PARTS--BASIC SENTENCES, NOTES, AND DRILLS. AN ADDITIONAL PART INCLUDES READING PASSAGES. THE BASIC SENTENCES ARE NORMAL DIALOG MATERIAL, MEANT TO BE MEMORIZED. THE NOTES EXPLAIN THE GRAMMATICAL STRUCTURE OF THE LANGUAGE AND ARE DIVIDED INTO…

  8. Korean Basic Course. Volume Two.

    ERIC Educational Resources Information Center

    Park, B. Nam

    Volume Two of the Korean Basic Course contains Units 29 through 47. Most units consist of (1) a basic dialog, (2) notes on the basic dialog, (3) additional vocabulary and phrases, (4) grammar notes, (5) drills, (6) a supplementary dialog for comprehension, (7) a narrative for comprehension and reading, and (8) exercises. Two of the last units…

  9. "New Voices": Enduring Basic Writing

    ERIC Educational Resources Information Center

    Bourgeois, Pamela

    2008-01-01

    This article addresses the issue of basic writing, demonstrating how one university's basic writing program acts as a steward of writing. The assumption that basic writers only consume resources rather than contribute to academic excellence is rejected. What links the author responses to this issue is a publication of student writing entitled…

  10. The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2.

    PubMed

    Keestra, A Marijke; de Zoete, Marcel R; van Aubel, Rémon A M H; van Putten, Jos P M

    2007-06-01

    The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-kappaB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)(3)-Lys (Pam(3)CSK(4)), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6-16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2. PMID:17513760

  11. Hundreds of putatively functional small open reading frames in Drosophila

    PubMed Central

    2011-01-01

    Background The relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization. Results Using bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of Drosophila melanogaster, and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between D. melanogaster and Drosophila pseudoobscura, their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data. Conclusions The combination of these analyses led us to postulate the existence of at least 401 functional smORFs in Drosophila, with the possibility that as many as 4,561 such functional smORFs may exist. PMID:22118156

  12. Categorization of Putative Factors Against Rhopalosiphum padi (L.) (Heteroptera: Aphididae).

    PubMed

    Zeb, Qamar; Rondon, Silvia I; Naeem, Mohammad; Khan, Shah Alam; Goyer, Aymeric; Vleet, Steve Van; Corp, Mary K

    2016-02-01

    The bird cherry-oat aphid, Rhopalosiphum padi (L.) (Heteroptera: Aphididae), causes heavy losses to wheat crops worldwide by direct damage and virus transmission. This study was conducted to identify putative resistance mechanisms in four wheat varieties (Bobtail, Ladd, Stephens, and Skiles) and one advanced line (YS434)where R. padi was subjected to choice and no-choice tests. Antixenosis, antibiosis and tolerance studies were conducted in controlled environmental conditions at temperature of 20±5°C, 50–65% RH, and a photoperiod of 14:10 (L:D) h. Based on the antixenosis test, the variety Skiles was found susceptible to R. padi, while the line YS434 showed a significant level of resistance; the varieties Bobtail, Ladd, and Stephens showed intermediate response. In the antibiosis experiment, R. padi produced less progeny on the variety Skiles as compared with other varieties, but the developmental time for nymphs was also significantly shorter on Skiles and recorded higher intrinsic rate of natural increase (r(m)) values as compared with the varieties YS434, Bobtail, and Ladd. In the tolerance tests, the variety Ladd showed significantly lower tolerance index value than YS434, followed by Skiles, Bobtail, and Stephens. The plant resistance index value was greater for the variety Ladd, followed by Stephens, YS434, and Bobtail. In conclusion, this study provides baseline information that will contribute to the identification of putative resistance factors for a future breeding program against this aphid. PMID:26568060

  13. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    SciTech Connect

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  14. Evidence for a putative biomarker for substance dependence.

    PubMed

    Taylor, Jeanette; James, Lisa M

    2009-09-01

    Electrodermal response modulation (ERM) reflects the reduction in skin conductance response to an aversive stimulus that is temporally predictable relative to when it is unpredictable. Poor ERM is associated with substance dependence (SD). It was hypothesized that ERM is a putative biomarker for SD rather than for externalizing disorders generally. Participants included 83 controls (no SD, antisocial personality disorder [PD] or borderline PD), 52 participants with SD only (SD and no PD), 12 with PD only (antisocial and/or borderline PD and no SD), and 35 comorbid (having SD and PD). Diagnoses at definite and probable certainty levels were used and were determined by semistructured clinical interviews. ERM was calculated from skin conductance responses to predictable and unpredictable 2-s 110-dB white noise blasts. As expected, the SD-only and comorbid groups had significantly lower ERM scores than the control group, which did not differ significantly from the PD-only group. Results provide preliminary evidence that ERM is a putative biomarker for SD. Future research should examine cognitive correlates of ERM in an effort to understand why it relates to SD. PMID:19769433

  15. Tuberculosis and nature's pharmacy of putative anti-tuberculosis agents.

    PubMed

    Chinsembu, Kazhila C

    2016-01-01

    Due to the growing problem of drug resistant Mycobacterium tuberculosis strains, coupled with the twinning of tuberculosis (TB) to human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), the burden of TB is now difficult to manage. Therefore, new antimycobacterial agents are being sought from natural sources. This review focuses on natural antimycobacterial agents from endophytes and medicinal plants of Africa, Europe, Asia, South America and Canada. In the countries mentioned in this review, numerous plant species display putative anti-TB activity. Several antimycobacterial chemical compounds have also been isolated, including: ellagitannin punicalagin, allicin, anthraquinone glycosides, iridoids, phenylpropanoids, beta-sitosterol, galanthimine, crinine, friedelin, gallic acid, ellagic acids, anthocyanidin, taraxerol, termilignan B, arjunic acid, glucopyranosides, 1-epicatechol, leucopelargonidol, hydroxybenzoic acids, benzophenanthridine alkaloids, neolignans, and decarine. These compounds may provide leads to novel and more efficacious drugs to lessen the global burden of TB and drug-resistant M. tuberculosis strains. If there is a long-term remedy for TB, it must lie in nature's pharmacy of putative antimycobacterial agents. PMID:26464047

  16. Brain response to putative pheromones in homosexual men.

    PubMed

    Savic, Ivanka; Berglund, Hans; Lindström, Per

    2005-05-17

    The testosterone derivative 4,16-androstadien-3-one (AND) and the estrogen-like steroid estra-1,3,5(10),16-tetraen-3-ol (EST) are candidate compounds for human pheromones. AND is detected primarily in male sweat, whereas EST has been found in female urine. In a previous positron emission tomography study, we found that smelling AND and EST activated regions covering sexually dimorphic nuclei of the anterior hypothalamus, and that this activation was differentiated with respect to sex and compound. In the present study, the pattern of activation induced by AND and EST was compared among homosexual men, heterosexual men, and heterosexual women. In contrast to heterosexual men, and in congruence with heterosexual women, homosexual men displayed hypothalamic activation in response to AND. Maximal activation was observed in the medial preoptic area/anterior hypothalamus, which, according to animal studies, is highly involved in sexual behavior. As opposed to putative pheromones, common odors were processed similarly in all three groups of subjects and engaged only the olfactory brain (amygdala, piriform, orbitofrontal, and insular cortex). These findings show that our brain reacts differently to the two putative pheromones compared with common odors, and suggest a link between sexual orientation and hypothalamic neuronal processes. PMID:15883379

  17. Chloroplast diversity in a putative hybrid swarm of Ponderosae (Pinaceae).

    PubMed

    Epperson, Bryan K; Telewski, Frank W; Willyard, Ann

    2009-03-01

    The Ponderosae subsection of the genus Pinus contains numerous taxa in disjunct mountain ranges of southern Arizona and New Mexico, differing for several leaf and cone traits, key among which is the number of leaf needles per fascicle. Trees with three needles are often found together with trees having five needles and mixed numbers. One taxonomic hypothesis is that there are swarms of hybrids between P. ponderosa and P. arizonica. A second hypothesis is that there are spatial mixtures of two separate taxa, five-needle P. arizonica and a "taxon X" containing three needle and mixed needle trees. We genotyped chloroplasts in one putative hybrid swarm on Mt. Lemmon using microsatellite markers and show that cpDNA is almost completely differentiated between two separate morphotypes corresponding to P. arizonica and "taxon X." Little if any introgression has occurred on Mt. Lemmon, and the simplest explanation is that little or no effective hybridization has occurred. Further results indicate that not only is taxon X not of hybrid origin, it is more closely related to nonregional Ponderosae other than P. ponderosa and P. arizonica. The results further suggest that other putative hybrid swarms in the region are also spatial mixtures of distinct taxa. PMID:21628225

  18. Basic science of osteoarthritis.

    PubMed

    Cucchiarini, Magali; de Girolamo, Laura; Filardo, Giuseppe; Oliveira, J Miguel; Orth, Patrick; Pape, Dietrich; Reboul, Pascal

    2016-12-01

    Osteoarthritis (OA) is a prevalent, disabling disorder of the joints that affects a large population worldwide and for which there is no definitive cure. This review provides critical insights into the basic knowledge on OA that may lead to innovative end efficient new therapeutic regimens. While degradation of the articular cartilage is the hallmark of OA, with altered interactions between chondrocytes and compounds of the extracellular matrix, the subchondral bone has been also described as a key component of the disease, involving specific pathomechanisms controlling its initiation and progression. The identification of such events (and thus of possible targets for therapy) has been made possible by the availability of a number of animal models that aim at reproducing the human pathology, in particular large models of high tibial osteotomy (HTO). From a therapeutic point of view, mesenchymal stem cells (MSCs) represent a promising option for the treatment of OA and may be used concomitantly with functional substitutes integrating scaffolds and drugs/growth factors in tissue engineering setups. Altogether, these advances in the fundamental and experimental knowledge on OA may allow for the generation of improved, adapted therapeutic regimens to treat human OA. PMID:27624438

  19. [Basic research in pulmonology].

    PubMed

    Gea, Joaquim

    2008-11-01

    This is a review of the articles dealing with basic science published in recent issues of Archivos de Bronconeumología. Of particular interest with regard to chronic obstructive pulmonary disease were an article on extrapulmonary inflammation and oxidative stress and another on bronchial remodeling. The articles relating to asthma included a review on the use of drugs that block free immunoglobulin-E and an article about the contribution of experimental models to our knowledge of this disease. Two of the most interesting articles on the topic of lung cancer dealt with gene therapy and resistance to chemotherapy. Also notable were 2 studies that investigated ischemia-reperfusion injury. One evaluated tissue resistance to injury while the other analyzed the role played by interleukin-8 in this process. On the topic of pulmonary fibrosis, an article focused on potential biomarkers of progression and prognosis; others dealt with the contribution of experimental models to our understanding of this disorder and the fibrogenic role of transforming growth factor b. In the context of both sleep apnea syndrome and pulmonary infection, studies investigating the role of oxidative stress were published. Finally, 2 studies analyzed the diagnosis and treatment of tuberculosis and other pulmonary infections. PMID:19007569

  20. Basics of cytology

    PubMed Central

    Al-Abbadi, Mousa A.

    2011-01-01

    This overview is intended to give a general outline about the basics of Cytopathology. This is a field that is gaining tremendous momentum all over the world due to its speed, accuracy and cost effectiveness. This review will include a brief description about the history of cytology from its inception followed by recent developments. Discussion about the different types of specimens, whether exfoliative or aspiration will be presented with explanation of its rule as a screening and diagnostic test. A brief description of the indications, utilization, sensitivity, specificity, cost effectiveness, speed and accuracy will be carried out. The role that cytopathology plays in early detection of cancer will be emphasized. The ability to provide all types of ancillary studies necessary to make specific diagnosis that will dictate treatment protocols will be demonstrated. A brief description of the general rules of cytomorphology differentiating benign from malignant will be presented. Emphasis on communication between clinicians and pathologist will be underscored. The limitations and potential problems in the form of false positive and false negative will be briefly discussed. Few representative examples will be shown. A brief description of the different techniques in performing fine needle aspirations will be presented. General recommendation for the safest methods and hints to enhance the sensitivity of different sample procurement will be given. It is hoped that this review will benefit all practicing clinicians that may face certain diagnostic challenges requiring the use of cytological material. PMID:23210005