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Sample records for acid-rich basic leucine

  1. Charged Amino Acid-rich Leucine Zipper-1 (Crlz-1) as a Target of Wnt Signaling Pathway Controls Pre-B Cell Proliferation by Affecting Runx/CBFβ-targeted VpreB and λ5 Genes.

    PubMed

    Choi, Seung-Young; Park, Sung-Kyun; Yoo, Han-Woong; Pi, Joo-Hyun; Kang, Chang-Joong

    2016-07-15

    The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFβ (core binding factor β) into the nucleus to allow Runx (runt-related transcription factor)/CBFβ heterodimerization. Runx/CBFβ then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or β-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/β-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFβ heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively. PMID:27226553

  2. Simulations of temperature and salt concentration effects on bZIP, a basic region leucine zipper.

    PubMed

    Cukier, Robert I

    2012-05-31

    Basic region leucine zipper (bZIP) transcription factors are dimeric proteins that recognize DNA. The monomers consist of a leucine zipper subdomain responsible for dimerization and a highly basic DNA recognition subdomain. Twelve explicit solvent molecular dynamics (MD) trajectories were run on the GCN4 bZIP transcriptional factor in the absence of DNA at three temperatures and two ion concentrations (0 mM with Cl(-) ions to neutralize the bZIP and 200 mM with additional Na(+) and Cl(-) ions) to probe the conformational ensemble that the basic region samples. In most trajectories, the basic region exhibits an alligator-jaw-like opening and closing (only one monomer moves), versus scissor-like motion, by a mainly rigid body, hinge motion centered on three "fork" residues that span the basic region to the coiled coil. In this motion, the α-helical character of the basic region monomers is mostly maintained. A broad range of distances is accessed, consistent with the absence of particular interactions for the basic region monomers. In two of the trajectories, the basic region monomers "collapse" to form a stable state. The coiled coil, leucine zipper subdomain is very stable for all of the trajectories. Ion solvation of the charged residue side chains is transient, on the scale of a few picoseconds. There is no evidence for persistent specific ion salt bridges to charged residues. For 0 mM, only certain basic region positively charged residues are substantially Cl(-) ion salt bridged. For 200 mM, in addition, some basic region negatively (positively) charged residues are salt bridged to Na(+) (Cl(-)) ions. The different ion solvation patterns at the two ion concentrations are not greatly temperature sensitive, and the conformational sampling found in the MD is remarkably unperturbed by ion concentration and/or temperature. PMID:22559083

  3. Basic leucine zipper family in barley: genome-wide characterization of members and expression analysis.

    PubMed

    Pourabed, Ehsan; Ghane Golmohamadi, Farzan; Soleymani Monfared, Peyman; Razavi, Seyed Morteza; Shobbar, Zahra-Sadat

    2015-01-01

    The basic leucine zipper (bZIP) family is one of the largest and most diverse transcription factors in eukaryotes participating in many essential plant processes. We identified 141 bZIP proteins encoded by 89 genes from the Hordeum vulgare genome. HvbZIPs were classified into 11 groups based on their DNA-binding motif. Amino acid sequence alignment of the HvbZIPs basic-hinge regions revealed some highly conserved residues within each group. The leucine zipper heptads were analyzed predicting their dimerization properties. 34 conserved motifs were identified outside the bZIP domain. Phylogenetic analysis indicated that major diversification within the bZIP family predated the monocot/dicot divergence, although intra-species duplication and parallel evolution seems to be occurred afterward. Localization of HvbZIPs on the barley chromosomes revealed that different groups have been distributed on seven chromosomes of barley. Six types of intron pattern were detected within the basic-hinge regions. Most of the detected cis-elements in the promoter and UTR sequences were involved in seed development or abiotic stress response. Microarray data analysis revealed differential expression pattern of HvbZIPs in response to ABA treatment, drought, and cold stresses and during barley grain development and germination. This information would be helpful for functional characterization of bZIP transcription factors in barley.

  4. Basic leucine zipper family in barley: genome-wide characterization of members and expression analysis.

    PubMed

    Pourabed, Ehsan; Ghane Golmohamadi, Farzan; Soleymani Monfared, Peyman; Razavi, Seyed Morteza; Shobbar, Zahra-Sadat

    2015-01-01

    The basic leucine zipper (bZIP) family is one of the largest and most diverse transcription factors in eukaryotes participating in many essential plant processes. We identified 141 bZIP proteins encoded by 89 genes from the Hordeum vulgare genome. HvbZIPs were classified into 11 groups based on their DNA-binding motif. Amino acid sequence alignment of the HvbZIPs basic-hinge regions revealed some highly conserved residues within each group. The leucine zipper heptads were analyzed predicting their dimerization properties. 34 conserved motifs were identified outside the bZIP domain. Phylogenetic analysis indicated that major diversification within the bZIP family predated the monocot/dicot divergence, although intra-species duplication and parallel evolution seems to be occurred afterward. Localization of HvbZIPs on the barley chromosomes revealed that different groups have been distributed on seven chromosomes of barley. Six types of intron pattern were detected within the basic-hinge regions. Most of the detected cis-elements in the promoter and UTR sequences were involved in seed development or abiotic stress response. Microarray data analysis revealed differential expression pattern of HvbZIPs in response to ABA treatment, drought, and cold stresses and during barley grain development and germination. This information would be helpful for functional characterization of bZIP transcription factors in barley. PMID:25173685

  5. In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities

    SciTech Connect

    Sera, T.; Schultz, P.G.

    1996-04-02

    A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn{sup {minus}18}, Ala{sup {minus}15}, Val{sup {minus}14}, Ser{sup {minus}11}, And Arg{sup {minus}10}. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5{prime}-A{sup 5}T{sup 4}T{sup 3}G{sup 2}C{sup 1}G{sup 1{prime}}C{sup 2{prime}}A{sup 3}A{sup 4{prime}}T{sup 5{prime}}-3{prime}. Mutants containing the sequence Leu{sup {minus}18}Tyr{sup {minus}15}Xaa{sup {minus}14}-Tyr{sup {minus}11}Arg{sup {minus}10}, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5{prime}-ACTCYCGY{prime}GAT-3{prime} (Xaa=asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5{prime}-ATTACGTAAT-3{prime}, mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed. 28 refs., 1 fig., 4 tabs.

  6. Action of a Basic Copolymer of Ornithine and Leucine on Cells of Staphylococcus aureus

    PubMed Central

    Shenfeld, Avner; Flowers, Harold M.; Katchalski, Ephraim

    1974-01-01

    A basic, random copolymer of l-ornithine and l-leucine (OL; molar ratios 1:1) was bactericidal to a sensitive (S) strain of Staphylococcus aureus at low concentration. Resistant cells (R) were selected from the culture medium and, after serial transfers to solutions containing increasing amounts of the polymer, grew well in the presence of very high concentrations of it (1,000 μg/ml). S cells bound much more OL than did R cells, but no difference in binding was shown between separated cell walls or cell membranes of S and R. The binding of OL and sensitivity to it were not dependent on the teichoic acid-content of the cells. Bound OL was only partially removed from the cells by a variety of reagents, such as sodium dodecyl sulfate, Triton X-100, dilute trichloroacetic acid, and Ba(OH)2, and the extent of removal was similar for R and S cells. Images PMID:4840430

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

    PubMed

    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

  8. 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

  9. Transcriptional Activation by ETS and Leucine Zipper-Containing Basic Helix-Loop-Helix Proteins

    PubMed Central

    Tian, Gang; Erman, Batu; Ishii, Haruhiko; Gangopadhyay, Samudra S.; Sen, Ranjan

    1999-01-01

    The immunoglobulin μ heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the μ enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families. PMID:10082562

  10. 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.

  11. 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

  12. The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development.

    PubMed Central

    Hallsson, Jón H; Haflidadóttir, Benedikta S; Stivers, Chad; Odenwald, Ward; Arnheiter, Heinz; Pignoni, Francesca; Steingrímsson, Eiríkur

    2004-01-01

    The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes. PMID:15166150

  13. Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

    PubMed

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

    2011-02-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

  14. Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome▿ †

    PubMed Central

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M.; Young, Patrick

    2011-01-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n’ collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis. PMID:21149573

  15. 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

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

    2012-11-01

    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 nonreducing conditions. Our data indicate that removal of this region results in a loss of α-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.

  16. 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

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

    2012-11-01

    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 nonreducing conditions. Our data indicate that removal of this region results in a loss of α-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

  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. Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

    PubMed

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

    2015-03-27

    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.

  20. Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

    PubMed

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

    2015-03-27

    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

  1. Redox-mediated Mechanisms Regulate DNA Binding Activity of the G-group of Basic Region Leucine Zipper (bZIP) Transcription Factors in Arabidopsis*

    PubMed Central

    Shaikhali, Jehad; Norén, Louise; de Dios Barajas-López, Juan; Srivastava, Vaibhav; König, Janine; Sauer, Uwe H.; Wingsle, Gunnar; Dietz, Karl-Josef; Strand, Åsa

    2012-01-01

    Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys330 in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors. PMID:22718771

  2. Seasonal abscisic acid signal and a basic leucine zipper transcription factor, DkbZIP5, regulate proanthocyanidin biosynthesis in persimmon fruit.

    PubMed

    Akagi, Takashi; Katayama-Ikegami, Ayako; Kobayashi, Shozo; Sato, Akihiko; Kono, Atsushi; Yonemori, Keizo

    2012-02-01

    Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5.

  3. 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

  4. The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein

    PubMed Central

    Lipps, Georg; Ibanez, Pablo; Stroessenreuther, Thomas; Hekimian, Katya; Krauss, Gerhard

    2001-01-01

    The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by ∼60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins. PMID:11812827

  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. Specific Association of Transcripts of tbzF and tbz17, Tobacco Genes Encoding Basic Region Leucine Zipper-Type Transcriptional Activators, with Guard Cells of Senescing Leaves and/or Flowers1

    PubMed Central

    Yang, Seung Hwan; Berberich, Thomas; Sano, Hiroshi; Kusano, Tomonobu

    2001-01-01

    Induction by low temperature is a common feature of the lip19 subfamily members of the basic region leucine zipper gene family in plants. Here, we characterize two tobacco (Nicotiana tabacum) genes, tbzF and tbz17, belonging to the lip19 subfamily, whose gene products, TBZF and TBZ17, show 73% identity and are located in nuclei. They preferentially bind to DNA fragments spanning A-box/G-box and C-box/G-box hybrid motifs and show transactivation activity in cobombarded tobacco BY-2 cells, indicating they function as transcriptional activators. Transcripts of tbzF were detected at a high level in senescing leaves and flowers. In contrast, tbz17 transcripts could be shown to accumulate in aged leaves but not in flowers. In situ hybridization analysis revealed transcripts of tbzF and tbz17 to be predominantly located in guard cells and vascular tissues of senescing leaves. These results suggest that TBZF and TBZ17 are both involved in controlling gene transcription related to functions of guard cells in senescing leaves and that TBZF bifunctionally acts in floral development. PMID:11553731

  7. 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

  8. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    PubMed

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  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 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

  12. Preparation method and stability of ellagic acid-rich pomegranate fruit peel extract.

    PubMed

    Panichayupakaranant, Pharkphoom; Itsuriya, Atcharaporn; Sirikatitham, Anusak

    2010-02-01

    A simple one-step purification using liquid-liquid extraction for preparing pomegranate peel extract rich in ellagic acid has been demonstrated. The method involved partitioning of the 10% v/v water in methanol extract of pomegranate peel between ethyl acetate and 2% aqueous acetic acid. This method was capable of increasing the ellagic acid content of the extract from 7.06% to 13.63% w/w. Moreover, the antioxidant activity of the extract evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was also increased (ED(50) from 38.21 to 14.91 micro/mL). Stability evaluations of the ellagic acid-rich pomegranate peel extract in several conditions through a period of four months found that the extracts were stable either kept under light or protected from light. The extracts were also stable under 4 degrees +/- 2 degrees C, 30 degrees +/- 2 degrees C and accelerated conditions at 45 degrees C with 75% relative humidity. However, study on the effect of pH on stability of the extract in the form of solution revealed that the extract was not stable in all tested pH (5.5, 7 and 8). These results indicated that the ellagic acid-rich pomegranate peel extract was stable when it was kept as dried powder, but it was not stable in any aqueous solution. PMID:20645841

  13. 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.

  14. 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.

  15. Binding of Ca2+ to Glutamic Acid-Rich Polypeptides from the Rod Outer Segment

    PubMed Central

    Haber-Pohlmeier, S.; Abarca-Heidemann, K.; Körschen, H. G.; Dhiman, H. Kaur; Heberle, J.; Schwalbe, H.; Klein-Seetharaman, J.; Kaupp, U. B.; Pohlmeier, A.

    2007-01-01

    Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca2+ from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca2+ to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca2+ binding was investigated by means of a Ca2+-sensitive electrode. In all cases, Ca2+ binds with low affinity; the half-maximum binding constant K1/2 ranges from 6 to 16 mM. The binding stoichiometry between Ca2+ ions and carboxylic groups is ∼1:1; an exception is GARP2, where a binding stoichiometry of ∼1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca2+ binding. PMID:17218469

  16. 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…

  17. 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.

  18. Toxicity of leucine-containing peptides in Escherichia coli caused by circumvention of leucine transport regulation.

    PubMed Central

    Tavori, H; Kimmel, Y; Barak, Z

    1981-01-01

    A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu, Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. When peptide transport was impaired by mutations, strains became resistant to the respective LCP. Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable. Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium. The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures. We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormality high concentrations of leucine inside the cells. This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria. Images PMID:7012134

  19. 21 CFR 582.5406 - Leucine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 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...

  20. 21 CFR 582.5406 - Leucine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 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...

  1. 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...

  2. 21 CFR 582.5406 - Leucine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 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...

  3. 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...

  4. Role of leucine in hepatic ketogenesis

    SciTech Connect

    Kulaylat, M.N.; Frexes-Steed, M.; Geer, R.; Williams, P.E.; Abumrad, N.N.

    1988-03-01

    Isolated hepatocyte studies demonstrated that leucine can be a precursor of ketone bodies. In this study we examine the relative contribution of leucine to hepatic ketogenesis in vivo. Three groups of conscious dogs with long-term indwelling catheters in the femoral artery, hepatic vein, and portal vein were studied. Group I (n = 3) animals were fasted overnight for 24 hours, and those in groups II and III (n = 4, each) were fasted for 62 to 68 hours (designated 3-day fast). Groups I and III received intravenous saline solution (0.9%) and served as controls. In group II selective acute insulin deficiency (SAID) was induced by a peripheral intravenous somatostatin (SRIF) infusion and intraportal glucagon (0.55 ng/body weight/min). Net hepatic production (NHP) of ketone bodies (kb) and leucine (leu) was measured by the arteriovenous difference technique. Hepatic conversion of leucine to ketone bodies was measured by continuous infusion of L-U-(/sup 14/C)-leucine and by determination of the appearance of (/sup 14/C)-ketone bodies across the liver. In the group fasted overnight NHPleu was 0.02 +/- 0.01 mumol/kg/min, a value not different from zero. NHPkb was 3.1 +/- 0.1 mumol/kg/min and hepatic conversion of leucine to ketone bodies accounted for 3.5% of NHPkb. Insulin deficiency after 3 day's fasting resulted in a near 70% increase in NHPleu (from basal values of 0.31 +/- 0.1 mumol/kg/min to 0.52 +/- 0.06 mumol/kg/min during SAID, p less than 0.01). NHPkb increased from 11.0 +/- 1.0 to 15.5 mumol/kg/min (p less than 0.05). The rate of leucine conversion to ketone bodies (L-C) increased from 1.1 +/- 0.25 to 2.4 +/- 0.3 mumol/kg/min (p less than 0.01) with SAID.

  5. Bioprospecting the Curculigoside-Cinnamic Acid-Rich Fraction from Molineria latifolia Rhizome as a Potential Antioxidant Therapeutic Agent.

    PubMed

    Ooi, Der Jiun; Chan, Kim Wei; Sarega, Nadarajan; Alitheen, Noorjahan Banu; Ithnin, Hairuszah; Ismail, Maznah

    2016-06-17

    Increasing evidence from both experimental and clinical studies depicts the involvement of oxidative stress in the pathogenesis of various diseases. Specifically, disruption of homeostatic redox balance in accumulated body fat mass leads to obesity-associated metabolic syndrome. Strategies for the restoration of redox balance, potentially by exploring potent plant bioactives, have thus become the focus of therapeutic intervention. The present study aimed to bioprospect the potential use of the curculigoside-cinnamic acid-rich fraction from Molineria latifolia rhizome as an antioxidant therapeutic agent. The ethyl acetate fraction (EAF) isolated from M. latifolia rhizome methanolic extract (RME) contained the highest amount of phenolic compounds, particularly curculigoside and cinnamic acid. EAF demonstrated glycation inhibitory activities in both glucose- and fructose-mediated glycation models. In addition, in vitro chemical-based and cellular-based antioxidant assays showed that EAF exhibited high antioxidant activities and a protective effect against oxidative damage in 3T3-L1 preadipocytes. Although the efficacies of individual phenolics differed depending on the structure and concentration, a correlational study revealed strong correlations between total phenolic contents and antioxidant capacities. The results concluded that enriched phenolic contents in EAF (curculigoside-cinnamic acid-rich fraction) contributed to the overall better reactivity. Our data suggest that this bioactive-rich fraction warrants therapeutic potential against oxidative stress-related disorders.

  6. Bioprospecting the Curculigoside-Cinnamic Acid-Rich Fraction from Molineria latifolia Rhizome as a Potential Antioxidant Therapeutic Agent.

    PubMed

    Ooi, Der Jiun; Chan, Kim Wei; Sarega, Nadarajan; Alitheen, Noorjahan Banu; Ithnin, Hairuszah; Ismail, Maznah

    2016-01-01

    Increasing evidence from both experimental and clinical studies depicts the involvement of oxidative stress in the pathogenesis of various diseases. Specifically, disruption of homeostatic redox balance in accumulated body fat mass leads to obesity-associated metabolic syndrome. Strategies for the restoration of redox balance, potentially by exploring potent plant bioactives, have thus become the focus of therapeutic intervention. The present study aimed to bioprospect the potential use of the curculigoside-cinnamic acid-rich fraction from Molineria latifolia rhizome as an antioxidant therapeutic agent. The ethyl acetate fraction (EAF) isolated from M. latifolia rhizome methanolic extract (RME) contained the highest amount of phenolic compounds, particularly curculigoside and cinnamic acid. EAF demonstrated glycation inhibitory activities in both glucose- and fructose-mediated glycation models. In addition, in vitro chemical-based and cellular-based antioxidant assays showed that EAF exhibited high antioxidant activities and a protective effect against oxidative damage in 3T3-L1 preadipocytes. Although the efficacies of individual phenolics differed depending on the structure and concentration, a correlational study revealed strong correlations between total phenolic contents and antioxidant capacities. The results concluded that enriched phenolic contents in EAF (curculigoside-cinnamic acid-rich fraction) contributed to the overall better reactivity. Our data suggest that this bioactive-rich fraction warrants therapeutic potential against oxidative stress-related disorders. PMID:27322226

  7. In vitro antioxidant assay of medium chain fatty acid rich rice bran oil in comparison to native rice bran oil.

    PubMed

    Sengupta, Avery; Ghosh, Mahua; Bhattacharyya, D K

    2015-08-01

    The study aimed to evaluate the in vitro antioxidant activity of medium chain fatty acid (MCFA) rich-rice bran oils in comparison with native rice bran oil. Different in vitro methods were used to evaluate the free radical scavenging activity, metal chelation activity, reducing acitivity, ABTS radical scavenging activity, thiobarbituric acid (TBA) value and so on at different concentrations of the oils such as 10-100 μg/mL. Inhibition of lipid peroxidation was evaluated measuring thiobarbituric acid responsive substance (TBARS) and conjugated diene formation. All the oils showed potent antioxidant activity at 100 μg/mL concentration. TBARS formation and conjugated diene formation was lower with MCFA rich oils i.e. the inhibition of lipid peroxidation was more in MCFA rich oils than original rice bran oil. Caprylic acid rich rice bran oil showed maximum antioxidant activity in comparison to capric- and lauric acid rich rice bran oils. Overall the MCFA rich rice bran oils showed to be more potent antioxidant than rice bran oil due to their lower unsaturated fatty acid content. PMID:26243941

  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. Leucine supplementation improves skeletal muscle regeneration after cryolesion in rats.

    PubMed

    Pereira, Marcelo G; Baptista, Igor L; Carlassara, Eduardo O C; Moriscot, Anselmo S; Aoki, Marcelo S; Miyabara, Elen H

    2014-01-01

    This study was undertaken in order to provide further insight into the role of leucine supplementation in the skeletal muscle regeneration process, focusing on myofiber size and strength recovery. Young (2-month-old) rats were subjected or not to leucine supplementation (1.35 g/kg per day) started 3 days prior to cryolesion. Then, soleus muscles were cryolesioned and continued receiving leucine supplementation until 1, 3 and 10 days later. Soleus muscles from leucine-supplemented animals displayed an increase in myofiber size and a reduction in collagen type III expression on post-cryolesion day 10. Leucine was also effective in reducing FOXO3a activation and ubiquitinated protein accumulation in muscles at post-cryolesion days 3 and 10. In addition, leucine supplementation minimized the cryolesion-induced decrease in tetanic strength and increase in fatigue in regenerating muscles at post-cryolesion day 10. These beneficial effects of leucine were not accompanied by activation of any elements of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signalling pathway in the regenerating muscles. Our results show that leucine improves myofiber size gain and strength recovery in regenerating soleus muscles through attenuation of protein ubiquitination. In addition, leucine might have therapeutic effects for muscle recovery following injury and in some muscle diseases.

  10. 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.

  11. The leucine-rich repeat superfamily of synaptic adhesion molecules: LRRTMs and Slitrks.

    PubMed

    Ko, Jaewon

    2012-10-01

    Synapses are asymmetric intercellular junctions connected by multiple synaptic cell adhesion molecules (CAMs). Synaptic CAMs function in various stages of synaptogenesis - the process of synapse creation - encompassing synapse formation, maturation, refinement, plasticity, and elimination. The list of synaptic CAMs has rapidly grown, although their precise functions of most CAMs at synapses remain incomplete. Members of an emerging class of transmembrane proteins containing leucine-rich repeat (LRR) domains have received considerable recent research attention. In this minireview, I discuss recent findings on LRR-containing synaptic CAMs that impact synapse development and circuit formation, focusing on two families of LRR synaptic CAMs: leucine-rich transmembrane proteins (LRRTMs) and Slit and Trk-like family (Slitrks). Their basic biochemical properties, proposed functions at synapses, physiological significances, and open questions are summarized.

  12. 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...

  13. ESTIMATION OF BACTERIAL CELL NUMBERS IN HUMIC ACID-RICH SALT MARSH SEDIMENTS WITH PROBES DIRECTED TO 16S RIBOSOMAL DNA

    EPA Science Inventory

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membr...

  14. 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

  15. Conversion of cheese whey into a fucose- and glucuronic acid-rich extracellular polysaccharide by Enterobacter A47.

    PubMed

    Antunes, Sílvia; Freitas, Filomena; Alves, Vítor D; Grandfils, Christian; Reis, Maria A M

    2015-09-20

    Cheese whey was used as the sole substrate for the production of extracellular polysaccharides (EPS) by Enterobacter A47. An EPS concentration of 6.40 g L(-1) was reached within 3.2 days of cultivation, corresponding to a volumetric productivity of 2.00 g L(-1) d(-1). The produced EPS was mainly composed of glucuronic acid (29 mol%) and fucose (29 mol%), with lower contents of glucose and galactose (21 mol% each) and a total acyl groups content of 32 wt.%. The polymer had an average molecular weight of 1.8×10(6) Da, with a polydispersity index of 1.2, and an intrinsic viscosity of 8.0 dL g(-1). EPS aqueous solutions (1.0 wt.% in 0.01 M NaCl, at pH 8.0) presented a shear thinning behavior with a viscosity of the first Newtonian plateau approaching 0.1 Pas. This novel glucuronic acid-rich polymer possesses interesting rheological properties, which, together with its high content of glucuronic acid and fucose, two bioactive sugar monomers, confers it a great potential for use in high-value applications, such as cosmetics and pharmaceuticals. PMID:26119564

  16. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  17. Conversion of cheese whey into a fucose- and glucuronic acid-rich extracellular polysaccharide by Enterobacter A47.

    PubMed

    Antunes, Sílvia; Freitas, Filomena; Alves, Vítor D; Grandfils, Christian; Reis, Maria A M

    2015-09-20

    Cheese whey was used as the sole substrate for the production of extracellular polysaccharides (EPS) by Enterobacter A47. An EPS concentration of 6.40 g L(-1) was reached within 3.2 days of cultivation, corresponding to a volumetric productivity of 2.00 g L(-1) d(-1). The produced EPS was mainly composed of glucuronic acid (29 mol%) and fucose (29 mol%), with lower contents of glucose and galactose (21 mol% each) and a total acyl groups content of 32 wt.%. The polymer had an average molecular weight of 1.8×10(6) Da, with a polydispersity index of 1.2, and an intrinsic viscosity of 8.0 dL g(-1). EPS aqueous solutions (1.0 wt.% in 0.01 M NaCl, at pH 8.0) presented a shear thinning behavior with a viscosity of the first Newtonian plateau approaching 0.1 Pas. This novel glucuronic acid-rich polymer possesses interesting rheological properties, which, together with its high content of glucuronic acid and fucose, two bioactive sugar monomers, confers it a great potential for use in high-value applications, such as cosmetics and pharmaceuticals.

  18. Fatty acid rich effluent from acidogenic biohydrogen reactor as substrate for lipid accumulation in heterotrophic microalgae with simultaneous treatment.

    PubMed

    Venkata Mohan, S; Prathima Devi, M

    2012-11-01

    Acid-rich effluent generated from acidogenic biohydrogen production process was evaluated as substrate for lipid synthesis by integrating with heterotrophic cultivation of mixed microalgae. Experiments were performed both with synthetic volatile fatty acids (SVFA) and fermented fatty acids (FFA) from biohydrogen producing reactor. Fatty acid based platform evidenced significant influence on algal growth as well as lipid accumulation by the formation of triglycerides through fatty acid synthesis. Comparatively FFA documented higher biomass and lipid productivity (1.42mg/ml (wet weight); 26.4%) than SVFAs ((HAc+HBu+HPr), 0.60mg/ml; 23.1%). Lipid profiles varied with substrates and depicted 18 types of saturated and unsaturated fatty acids with wide fuel and food characteristics. The observed higher concentrations of Chl b over Chl a supports the biosynthesis of triacylglycerides. Microalgae diversity visualized the presence of lipid accumulating species viz., Scenedesmus sp. and Chlorella sp. Integration of microalgae cultivation with biohydrogen production showed lipid productivity for biodiesel production along with additional treatment.

  19. Growth response of Avena sativa in amino-acids-rich soils converted from phenol-contaminated soils by Corynebacterium glutamicum.

    PubMed

    Lee, Soo Youn; Kim, Bit-Na; Choi, Yong Woo; Yoo, Kye Sang; Kim, Yang-Hoon; Min, Jiho

    2012-04-01

    The biodegradation of phenol in laboratory-contaminated soil was investigated using the Gram-positive soil bacterium Corynebacterium glutamicum. This study showed that the phenol degradation caused by C. glutamicum was greatly enhanced by the addition of 1% yeast extract. From the toxicity test using Daphnia magna, the soil did not exhibit any hazardous effects after the phenol was removed using C. glutamicum. Additionally, the treatment of the phenolcontaminated soils with C. glutamicum increased various soil amino acid compositions, such as glycine, threonine, isoleucine, alanine, valine, leucine, tyrosine, and phenylalanine. This phenomenon induced an increase in the seed germination rate and the root elongation of Avena sativa (oat). This probably reflects that increased soil amino acid composition due to C. glutamicum treatment strengthens the plant roots. Therefore, the phenol-contaminated soil was effectively converted through increased soil amino acid composition, and additionally, the phenol in the soil environment was biodegraded by C. glutamicum.

  20. Leucine Supplementation Protects from Insulin Resistance by Regulating Adiposity Levels

    PubMed Central

    Binder, Elke; Bermúdez-Silva, Francisco J.; André, Caroline; Elie, Melissa; Romero-Zerbo, Silvana Y.; Leste-Lasserre, Thierry; Belluomo, llaria; Duchampt, Adeline; Clark, Samantha; Aubert, Agnes; Mezzullo, Marco; Fanelli, Flaminia; Pagotto, Uberto; Layé, Sophie; Mithieux, Gilles; Cota, Daniela

    2013-01-01

    Background Leucine supplementation might have therapeutic potential in preventing diet-induced obesity and improving insulin sensitivity. However, the underlying mechanisms are at present unclear. Additionally, it is unclear whether leucine supplementation might be equally efficacious once obesity has developed. Methodology/Principal Findings Male C57BL/6J mice were fed chow or a high-fat diet (HFD), supplemented or not with leucine for 17 weeks. Another group of HFD-fed mice (HFD-pairfat group) was food restricted in order to reach an adiposity level comparable to that of HFD-Leu mice. Finally, a third group of mice was exposed to HFD for 12 weeks before being chronically supplemented with leucine. Leucine supplementation in HFD-fed mice decreased body weight and fat mass by increasing energy expenditure, fatty acid oxidation and locomotor activity in vivo. The decreased adiposity in HFD-Leu mice was associated with increased expression of uncoupling protein 3 (UCP-3) in the brown adipose tissue, better insulin sensitivity, increased intestinal gluconeogenesis and preservation of islets of Langerhans histomorphology and function. HFD-pairfat mice had a comparable improvement in insulin sensitivity, without changes in islets physiology or intestinal gluconeogenesis. Remarkably, both HFD-Leu and HFD-pairfat mice had decreased hepatic lipid content, which likely helped improve insulin sensitivity. In contrast, when leucine was supplemented to already obese animals, no changes in body weight, body composition or glucose metabolism were observed. Conclusions/Significance These findings suggest that leucine improves insulin sensitivity in HFD-fed mice by primarily decreasing adiposity, rather than directly acting on peripheral target organs. However, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhans's physiology might help prevent type 2 diabetes development. Differently, metabolic benefit of leucine supplementation is lacking in

  1. 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).

  2. Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine). A highly conserved acidic calcium-binding extracellular-matrix protein.

    PubMed Central

    Damjanovski, S; Liu, F; Ringuette, M

    1992-01-01

    SPARC (Secreted Protein, Acidic, Rich in Cysteine) is expressed as a 1.6 kb mRNA in Xenopus laevis. On the basis of cDNA sequence analysis, Xenopus SPARC has a core Mr of 32643, with one potential N-glycosylation site. Western analysis of SPARC isolated from Xenopus long bone indicates that the mature protein has an Mr of 43,000. At the amino acid level, Xenopus SPARC has 78-79% sequence similarity to mouse, bovine and human SPARC. The least-conserved region is found within the N-terminal glutamic acid-rich domain, with the C-terminal Ca(2+)-binding domain being the most conserved. Adult Xenopus tissues show the same pattern of tissue-specific distribution of SPARC mRNAs as adult mouse. Images Fig. 1. Fig. 5. PMID:1736898

  3. 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.

  4. Light aerobic physical exercise in combination with leucine and/or glutamine-rich diet can improve the body composition and muscle protein metabolism in young tumor-bearing rats.

    PubMed

    Salomão, Emilianne Miguel; Gomes-Marcondes, Maria Cristina Cintra

    2012-12-01

    Nutritional supplementation with some amino acids may influence host's responses and also certain mechanism involved in tumor progression. It is known that exercise influences body weight and muscle composition. Previous findings from our group have shown that leucine has beneficial effects on protein composition in cachectic rat model as the Walker 256 tumor. The main purpose of this study was to analyze the effects of light exercise and leucine and/or glutamine-rich diet in body composition and skeletal muscle protein synthesis and degradation in young tumor-bearing rats. Walker tumor-bearing rats were subjected to light aerobic exercise (swimming 30 min/day) and fed a leucine-rich (3%) and/or glutamine-rich (4%) diet for 10 days and compared to healthy young rats. The carcasses were analyzed as total water and fat body content and lean body mass. The gastrocnemious muscles were isolated and used for determination of total protein synthesis and degradation. The chemical body composition changed with tumor growth, increasing body water and reducing body fat content and total body nitrogen. After tumor growth, the muscle protein metabolism was impaired, showing that the muscle protein synthesis was also reduced and the protein degradation process was increased in the gastrocnemius muscle of exercised rats. Although short-term exercise (10 days) alone did not produce beneficial effects that would reduce tumor damage, host protein metabolism was improved when exercise was combined with a leucine-rich diet. Only total carcass nitrogen and protein were recovered by a glutamine-rich diet. Exercise, in combination with an amino acid-rich diet, in particular, leucine, had effects beyond reducing tumoral weight such as improving protein turnover and carcass nitrogen content in the tumor-bearing host. PMID:22460363

  5. Effect of exercise training on leucine oxidation

    SciTech Connect

    Hendrix, M.K.; Layman, D.K.

    1986-03-01

    Oxidation of the BCAA leucine is increased during a bout of exhaustive exercise. The purpose of this study was to determine the effects of exercise training on leu oxidation during aerobic exercise. Female Sprague-Dawley rats were fed a commercial diet ad lib and divided into sedentary and two trained groups. Animals were trained to run on a treadmill with a 10/sup 0/ incline at 28 m/min for 5 wks for either 50 or 120 min/day. There were no differences in food intake or body weight. After a 12 hr fast, animals were run for 50 or 120 min and changes in leu catabolism determined by measurement of in vivo leu oxidation and activity of branched chain keto acid dehydrogenase (BCKAD). For measurement of leu oxidation, rats were injected IP with 4 ..mu..Ci 1-/sup 14/C-leu during the last 15 min of exercise, placed in glass metabolic chambers, and /sup 14/CO/sub 2/ collected in 1 N NaOH for 30 min periods. Leu oxidation was increased by 40% after 50 min of exercise and by 79% after 120 min of exercise. Five weeks of training reduced the rate of leu oxidation during an exercise bout. The activity of the BCKAD was not increased in the trained animals after either 50 or 120 min of exercise. These data indicate that the rate of leu oxidation during exercises is dependent on the duration of the exercise and that training will reduce the magnitude of this effect.

  6. 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.

  7. Fasting and postprandial phenylalanine and leucine kinetics in liver cirrhosis.

    PubMed

    Tessari, P; Inchiostro, S; Barazzoni, R; Zanetti, M; Orlando, R; Biolo, G; Sergi, G; Pino, A; Tiengo, A

    1994-07-01

    To investigate body protein turnover and the pathogenesis of increased concentration of plasma phenylalanine in liver cirrhosis, we have studied phenylalanine and leucine kinetics in cirrhotic (diabetic and nondiabetic) patients, and in normal subjects, both in the postabsorptive state and during a mixed meal, using combined intravenous and oral isotope infusions. Postabsorptive phenylalanine concentration and whole body rate of appearance (Ra) were approximately 40% greater (P < 0.05) in patients than in controls. Leucine concentrations were comparable, but intracellular leucine Ra was also increased (P < 0.05), suggesting increased whole body protein breakdown. Postprandial phenylalanine Ra was also greater (P < 0.05) in the patients. This difference was due to a diminished fractional splanchnic uptake of the dietary phenylalanine (approximately 40% lower in the cirrhotics vs. controls, P < or = 0.05). Postprandial leucine Ra was also increased in the patients, but splanchnic uptake of dietary leucine was normal. Thus both increased body protein breakdown and decreased splanchnic extraction of dietary phenylalanine can account for the increased phenylalanine concentrations in liver cirrhosis.

  8. Inhibition of leucine transport in Saccharomyces by S-adenosylmethionine.

    PubMed

    Law, R E; Ferro, A J

    1980-07-01

    S-Adenoxyl-L-methionine (SAM) inhibited leucine transport in Saccharomyces cerevisiae. By using a mutant defective in the active transport of SAM, we demonstrated that the inhibitory effect was exerted at an extracellular site. Cells preincubated wtih SAM for 120 min became refractory to its inhibitory effect, which was not a result of either the active transport or the metabolism of SAM. The quantitative recovery of labeled SAM from the incubation medium indicated that SAM, and not a metabolite, was the true inhibitory molecule. S-Adenosyl-L-homocysteine and S-adenosyl-L-ethionine also functioned as inhibitors of leucine transport, whereas S-adenosyl-D-methionine, S-adenosyl-D-homocystein, 5'-methylthioadenosine, 5'-dimethylthioadenosine, and adenosine lacked this property. Kinetic studies demonstrated that SAM was a competitive inhibitor of leucine transport.

  9. Inhibition of leucine transport in Saccharomyces by S-adenosylmethionine.

    PubMed Central

    Law, R E; Ferro, A J

    1980-01-01

    S-Adenoxyl-L-methionine (SAM) inhibited leucine transport in Saccharomyces cerevisiae. By using a mutant defective in the active transport of SAM, we demonstrated that the inhibitory effect was exerted at an extracellular site. Cells preincubated wtih SAM for 120 min became refractory to its inhibitory effect, which was not a result of either the active transport or the metabolism of SAM. The quantitative recovery of labeled SAM from the incubation medium indicated that SAM, and not a metabolite, was the true inhibitory molecule. S-Adenosyl-L-homocysteine and S-adenosyl-L-ethionine also functioned as inhibitors of leucine transport, whereas S-adenosyl-D-methionine, S-adenosyl-D-homocystein, 5'-methylthioadenosine, 5'-dimethylthioadenosine, and adenosine lacked this property. Kinetic studies demonstrated that SAM was a competitive inhibitor of leucine transport. PMID:6995442

  10. 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.

  11. 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.

  12. 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–...

  13. Comprehensive analysis of the homeodomain-leucine zipper IV transcription factor family in Cucumis sativus.

    PubMed

    Fu, Rao; Liu, Wei; Li, Qiang; Li, Jing; Wang, Lina; Ren, Zhonghai

    2013-07-01

    The class IV homeodomain-leucine zipper (HD-Zip IV) proteins are plant-specific transcriptional factors known to play crucial roles in plant growth and development. In this study, 11 cucumber (Cucumis sativus) HD-Zip IV genes were identified in the version 2 cucumber genome and found to be distributed unevenly across the chromosomes. The CsHDZIV (Cucumis sativus homeodomain-leucine zipper IV) gene family is smaller than in other studied species (except for rice) because of the absence of gene duplication events. Phylogenetic analysis showed that HD-Zip IV genes from cucumber, Arabidopsis, tomato, cotton, maize, and rice could be classified into five subgroups. All CsHDZIV genes appear to be derived from a basic module containing 11 exons in the coding region. Two conserved motifs of 21 and 19 nucleotides were found in the 3'-untranslated regions of six CsHDZIV genes, suggesting that post-transcriptional regulation may play a role in regulation of CsHDZIV genes. In addition, 6 of 11 CsHDZIV genes were found to undergo alternative splicing events. Reverse transcription PCR analysis showed that all CsHDZIV genes (except one) were expressed and showed preferential expression in reproductive organs.

  14. The apo-structure of the leucine sensor Sestrin2 is still elusive.

    PubMed

    Saxton, Robert A; Knockenhauer, Kevin E; Schwartz, Thomas U; Sabatini, David M

    2016-01-01

    Sestrin2 is a GATOR2-interacting protein that directly binds leucine and is required for the inhibition of mTORC1 under leucine deprivation, indicating that it is a leucine sensor for the mTORC1 pathway. We recently reported the structure of Sestrin2 in complex with leucine [Protein Data Bank (PDB) ID, 5DJ4] and demonstrated that mutations in the leucine-binding pocket that alter the affinity of Sestrin2 for leucine result in a corresponding change in the leucine sensitivity of mTORC1 in cells. A lower resolution structure of human Sestrin2 (PDB ID, 5CUF), which was crystallized in the absence of exogenous leucine, showed Sestrin2 to be in a nearly identical conformation as the leucine-bound structure. On the basis of this observation, it has been argued that leucine binding does not affect the conformation of Sestrin2 and that Sestrin2 may not be a sensor for leucine. We show that simple analysis of the reported "apo"-Sestrin2 structure reveals the clear presence of prominent, unmodeled electron density in the leucine-binding pocket that exactly accommodates the leucine observed in the higher resolution structure. Refining the reported apo-structure with leucine eliminated the large Fobs-Fcalc difference density at this position and improved the working and free R factors of the model. Consistent with this result, our own structure of Sestrin2 crystallized in the absence of exogenous leucine also contained electron density that is best explained by leucine. Thus, the structure of apo-Sestrin2 remains elusive. PMID:27649739

  15. The apo-structure of the leucine sensor Sestrin2 is still elusive

    PubMed Central

    Saxton, Robert A.; Knockenhauer, Kevin E.; Schwartz, Thomas U.; Sabatini, David M.

    2016-01-01

    Sestrin2 is a GATOR2 interacting protein that directly binds leucine and is required for the inhibition of mTORC1 under leucine deprivation, indicating that it is a leucine sensor for the mTORC1 pathway. We recently reported the structure of Sestrin2 in complex with leucine (PDB ID: 5DJ4), and demonstrated that mutations in the leucine-binding pocket alter the affinity of Sestrin2 for leucine and result in a corresponding change in the leucine sensitivity of mTORC1 in cells. A lower resolution structure of human Sestrin2 (PDB ID: 5CUF), which was crystallized in the absence of exogenous leucine, showed Sestrin2 to be in a nearly identical conformation as the leucine-bound structure. Based on this observation, it has been argued that leucine binding does not affect the conformation of Sestrin2 and thus that Sestrin2 may not be a sensor for leucine. Here, we show that simple analysis of the reported “apo”-Sestrin2 structure reveals the clear presence of prominent, unmodeled electron density in the leucine-binding pocket that exactly accommodates the leucine observed in the higher resolution structure. Refining the reported “apo”-structure with leucine eliminates the large FO-FC difference density at this position and improves the working and free R-factors of the model. Consistent with this, our own structure of Sestrin2 crystallized in the absence of exogenous leucine also contains electron density that is best explained by leucine. Thus, the structure of apo-Sestrin2 remains elusive. PMID:27649739

  16. The apo-structure of the leucine sensor Sestrin2 is still elusive.

    PubMed

    Saxton, Robert A; Knockenhauer, Kevin E; Schwartz, Thomas U; Sabatini, David M

    2016-09-20

    Sestrin2 is a GATOR2-interacting protein that directly binds leucine and is required for the inhibition of mTORC1 under leucine deprivation, indicating that it is a leucine sensor for the mTORC1 pathway. We recently reported the structure of Sestrin2 in complex with leucine [Protein Data Bank (PDB) ID, 5DJ4] and demonstrated that mutations in the leucine-binding pocket that alter the affinity of Sestrin2 for leucine result in a corresponding change in the leucine sensitivity of mTORC1 in cells. A lower resolution structure of human Sestrin2 (PDB ID, 5CUF), which was crystallized in the absence of exogenous leucine, showed Sestrin2 to be in a nearly identical conformation as the leucine-bound structure. On the basis of this observation, it has been argued that leucine binding does not affect the conformation of Sestrin2 and that Sestrin2 may not be a sensor for leucine. We show that simple analysis of the reported "apo"-Sestrin2 structure reveals the clear presence of prominent, unmodeled electron density in the leucine-binding pocket that exactly accommodates the leucine observed in the higher resolution structure. Refining the reported apo-structure with leucine eliminated the large Fobs-Fcalc difference density at this position and improved the working and free R factors of the model. Consistent with this result, our own structure of Sestrin2 crystallized in the absence of exogenous leucine also contained electron density that is best explained by leucine. Thus, the structure of apo-Sestrin2 remains elusive.

  17. 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.

  18. 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

  19. OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm

    PubMed Central

    Cai, Xiu-Ling

    2013-01-01

    Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control. PMID:23846875

  20. Enzyme-linked immunosorbent assay for leucine and methionine enkephalins

    SciTech Connect

    Zamboni, G.; Jones, C.A.; Hughes, J.

    1983-04-01

    An enzyme-linked immunosorbent assay for enkephalins was developed by coupling the peptides to a carrier molecule (bovine serum albumin) in order to allow the antibody-antigen reaction to take place in the solid phase. The assay was shown to be highly reproducible. Its sensitivity was 14 nmol/liter for leucine enkephalin and 27 nmol/liter for methionine enkephalin, which is similar to that obtained when the same antibodies were used in radioimmunoassay.

  1. Glutamine, arginine, and leucine signaling in the intestine.

    PubMed

    Marc Rhoads, J; Wu, Guoyao

    2009-05-01

    Glutamine and leucine are abundant constituents of plant and animal proteins, whereas the content of arginine in foods and physiological fluids varies greatly. Besides their role in protein synthesis, these three amino acids individually activate signaling pathway to promote protein synthesis and possibly inhibit autophagy-mediated protein degradation in intestinal epithelial cells. In addition, glutamine and arginine stimulate the mitogen-activated protein kinase and mammalian target of rapamycin (mTOR)/p70 (s6) kinase pathways, respectively, to enhance mucosal cell migration and restitution. Moreover, through the nitric oxide-dependent cGMP signaling cascade, arginine regulates multiple physiological events in the intestine that are beneficial for cell homeostasis and survival. Available evidence from both in vitro and in vivo animal studies shows that glutamine and arginine promote cell proliferation and exert differential cytoprotective effects in response to nutrient deprivation, oxidative injury, stress, and immunological challenge. Additionally, when nitric oxide is available, leucine increases the migration of intestinal cells. Therefore, through cellular signaling mechanisms, arginine, glutamine, and leucine play crucial roles in intestinal growth, integrity, and function.

  2. 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.

  3. 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…

  4. Effects of insulin on ovine fetal leucine kinetics and protein metabolism.

    PubMed Central

    Milley, J R

    1994-01-01

    Fetuses of eight pregnant ewes (114-117 d of gestation) were used to study whether fetal insulin concentration affects fetal protein accretion and, if so, whether such changes are caused by effects on protein synthesis or protein breakdown. Fetal leucine kinetics were measured by infusion of [1-14C]leucine during each of three protocols: (I) low vs. normal insulin concentration; (II) low vs. high insulin concentration; and (III) low vs. high insulin concentration during amino acid infusion to keep leucine concentration constant. Fetal leucine concentration (233 +/- 20 vs. 195 +/- 18 microM) and clearance (48.3 +/- 4.4 vs. 54.2 +/- 5.5 ml/kg per min) were the only aspects of fetal leucine kinetics that changed during protocol I. During protocol II, insulin infusion decreased fetal leucine concentration (222 +/- 22 vs. 175 +/- 22), decreased fetal leucine disposal (11.63 +/- 0.89 vs. 12.55 +/- 0.89 mumol/kg per min), increased leucine clearance (48.0 +/- 4.2 vs. 57.6 +/- 6.5 ml/kg per min), decreased leucine decarboxylation (1.77 +/- 0.17 vs. 2.04 +/- 0.21 mumol/kg per min), decreased nonoxidative leucine disposal (9.81 +/- 0.78 vs. 10.51 +/- 0.74 mumol/kg per min), decreased release of leucine from fetal protein (7.43 +/- 1.08 vs. 8.38 +/- 0.84 mumol/kg per min), but did not change the accretion of leucine into protein. In contrast, when leucine concentrations (205 +/- 25 vs. 189 +/- 23) were maintained (protocol III), insulin infusion did not change fetal leucine disposal, decarboxylation, or nonoxidative disposal although leucine clearance still rose (55.4 +/- 5.0 vs. 64.4 +/- 5.9 ml/kg/min). Fetal release of leucine from protein, however, decreased (7.46 +/- 0.83 vs. 8.57 +/- 0.71 mumol/kg per min) and the accretion of leucine into protein increased (3.27 +/- 0.30 vs. 1.80 +/- 0.32 mumol/kg/min). These findings show that insulin decreases fetal protein breakdown. If insulin-induced hypoaminoacidemia occurs, protein synthesis decreases so that no net accretion of

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia

    PubMed Central

    Rieu, Isabelle; Balage, Michèle; Sornet, Claire; Giraudet, Christophe; Pujos, Estelle; Grizard, Jean; Mosoni, Laurent; Dardevet, Dominique

    2006-01-01

    The present study was designed to assess the effects of dietary leucine supplementation on muscle protein synthesis and whole body protein kinetics in elderly individuals. Twenty healthy male subjects (70 ± 1 years) were studied before and after continuous ingestion of a complete balanced diet supplemented or not with leucine. A primed (3.6 μmol kg−1) constant infusion (0.06 μmol kg−1 min−1) of l-[1-13C]phenylalanine was used to determine whole body phenylalanine kinetics as well as fractional synthesis rate (FSR) in the myofibrillar fraction of muscle proteins from vastus lateralis biopsies. Whole body protein kinetics were not affected by leucine supplementation. In contrast, muscle FSR, measured over the 5-h period of feeding, was significantly greater in the volunteers given the leucine-supplemented meals compared with the control group (0.083 ± 0.008 versus 0.053 ± 0.009% h−1, respectively, P < 0.05). This effect was due only to increased leucine availability because only plasma free leucine concentration significantly differed between the control and leucine-supplemented groups. We conclude that leucine supplementation during feeding improves muscle protein synthesis in the elderly independently of an overall increase of other amino acids. Whether increasing leucine intake in old people may limit muscle protein loss during ageing remains to be determined. PMID:16777941

  10. 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.

  11. The expression of a mitochondria-localized glutamic acid-rich protein (MGARP/OSAP) is under the regulation of the HPG axis.

    PubMed

    Zhou, Mingxue; Wang, Yifeng; Qi, Shaoling; Wang, Jian; Zhang, Shuping

    2011-06-01

    The hypothalamic-pituitary-gonadal (HPG) axis exerts a profound effect on animal development, reproduction, and response to stress, and new insights into its complicated functional activities are continuously being made. In the present study, by using immunohistochemical studies and different mouse models (ovariectomy and ob/ob mice), we systemically analyzed the expression of a novel mitochondria-localized glutamic acid-rich protein (MGARP)/ovary-specific acid protein and demonstrated that MGARP is under the regulation of the HPG axis. MGARP is highly enriched in steroidogenic tissues and the visual system. Interestingly, its expression increases as mice develop. Early in development, MGARP is mainly detected in the retina and adrenal gland. At this early developmental stage, its expression is not detectable in the gonads, but its expression in the gonads dramatically increases during the first 2-4 wk after birth. Importantly, MGARP levels correlate with estrogen levels in the ovaries during the estrous cycle, and estrogen regulates the expression of MGARP in a tissue-specific manner and through a feedback regulatory mechanism. Functional inhibition of GnRH with an antagonist strongly reduces MGARP levels, and knockout of leptin (ob/ob) significantly reduces the MGARP expression in follicular granular cells. We proposed a model that elucidates the role MGARP plays in the HPG axis. Within the HPG axis loop, MGARP participates in hormone biosynthesis while being under the regulation of the hormones derived from the HPG axis.

  12. Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

    PubMed

    Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

    2010-05-01

    A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

  13. Functional characterization of a novel aspartic acid rich lipase, TALipC, from Trichosporon asahii MSR54: solvent-dependent enantio inversion during esterification of 1-phenylethanol.

    PubMed

    Kumari, Arti; Gupta, Rani

    2015-01-01

    A novel lipase gene TAlipC was isolated from Trichosporon asahii MSR54 and functionally expressed in Pichia pastoris. The protein was His-tagged and purified to homogeneity by affinity chromatography. Sequence comparison revealed a high homology with lipases from Cryptococcus sp. It had a GX type oxyanion hole and a GHSLG-type conserved penta-peptide similar to those in the lipases from Yarrowia lipolytica. The enzyme had optimal activity at pH 8 and 50 °C. It was specific for long chain fatty acid groups of p-nitrophenol esters and triacylglycerols, showing regio- and enantio-selectivity. It was activated by Mg(2+) ions (20 mM) and had a predicted Mg-binding domain at the aspartic acid-rich C-terminal. Solvent-based enantio- inversion was the key feature of the enzyme where it showed (S)-selectivity in 1,4-dioxane and 2-propanol and (R)-selectivity in hexane during chiral separation of (±)1-phenylethanol by esterification.

  14. Regular consumption of a silicic acid-rich water prevents aluminium-induced alterations of nitrergic neurons in mouse brain: histochemical and immunohistochemical studies.

    PubMed

    Foglio, E; Buffoli, B; Exley, C; Rezzani, R; Rodella, L F

    2012-08-01

    Silicon is not generally considered an essential nutrient for mammals and, to date, whether it has a biological role or beneficial effects in humans is not known. The results of a number of studies suggest that dietary silicon supplementation might have a protective effect both for limiting aluminium absorption across the gut and for the removal of systemic aluminium via the urine, hence, preventing potential accumulation of aluminium in the brain. Since our previous studies demonstrated that aluminium exposure reduces the number of nitrergic neurons, the aim of the present study was to compare the distribution and the morphology of NO-containing neurons in brain cortex of mice exposed to aluminium sulphate dissolved in silicic acid-rich or poor drinking water to assess the potential protective role of silicon against aluminium toxicity in the brain. NADPH-d histochemistry and nNOS immunohistochemistry showed that high concentrations of silicon in drinking water were able to minimize the impairment of the function of nitrergic neurons induced by aluminium administration. We found that silicon protected against aluminium-induced damage to the nitrergic system: in particular, we demonstrated that silicon maintains the number of nitrergic neurons and their expression of nitrergic enzymes at physiological levels, even after a 12 and 15 month exposure to aluminium.

  15. Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

    PubMed

    Cydzik, Marzena; Abdul-Wahid, Aws; Park, Soyeon; Bourdeau, Annie; Bowden, Katherine; Prodeus, Aaron; Kollara, Alexandra; Brown, Theodore J; Ringuette, Maurice J; Gariépy, Jean

    2015-08-01

    VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.

  16. 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 ...

  17. 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)

  18. 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 ...

  19. 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.

  20. 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

  1. The matricellular functions of small leucine-rich proteoglycans (SLRPs).

    PubMed

    Merline, Rosetta; Schaefer, Roland M; Schaefer, Liliana

    2009-12-01

    The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFalpha), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-beta, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.

  2. Dietary leucine requirement for juvenile large yellow croaker Pseudosciaena crocea (Richardson, 1846)

    NASA Astrophysics Data System (ADS)

    Li, Yan; Ai, Qinghui; Mai, Kangsen; Xu, Wei; Cheng, Zhenyan; He, Zhigang

    2010-12-01

    Dietary leucine requirement for juvenile large yellow croaker, Pseudosciaena crocea Richardson 1846 (initial body weight 6.0 g ± 0.1 g) was determined using dose-response method. Six isonitogenous (crude protein 43%) and isoenergetic (19 kJ g-1) practical diets containing six levels of leucine (Diets 1-6) ranging from 1.23% to 4.80% (dry matter) were made at about 0.7% increment of leucine. Equal amino acid nitrogen was maintained by replacing leucine with glutamic acid. Triplicate groups of 60 individuals were fed to apparent satiation by hand twice daily (05:00 and 17:30). The water temperature was 26-32°C, salinity 26-30 and dissolved oxygen approximately 7 mg L-1 during the experimental period. Final weight (FW) of large yellow croaker initially increased with increasing level of dietary leucine but then decreased at further higher level of leucine. The highest FW was obtained in fish fed diet with 3.30% Leucine (Diet 4). FW of fish fed the diet with 4.80% Leucine (Diet 6) was significantly lower than those fed Diet 4. However, no significant differences were observed between the other dietary treatments. Feed efficiency (FE) and whole body composition were independent of dietary leucine contents ( P > 0.05). The results indicated that leucine was essential for growth of juvenile large yellow croaker. On the basis of FW, the optimum dietary leucine requirement for juvenile large yellow croaker was estimated to be 2.92% of dry matter (6.79% of dietary protein).

  3. 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.

  4. 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...

  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. The intra-hippocampal leucine administration impairs memory consolidation and LTP generation in rats.

    PubMed

    Glaser, Viviane; Carlini, Valeria P; Gabach, Laura; Ghersi, Marisa; de Barioglio, Susana Rubiales; Ramirez, Oscar A; Perez, Mariela F; Latini, Alexandra

    2010-10-01

    Leucine accumulates in fluids and tissues of patients affected by maple syrup urine disease, an inherited metabolic disorder, predominantly characterized by neurological dysfunction. Although, a variable degree of cognition/psychomotor delay/mental retardation is found in a considerable number of individuals affected by this deficiency, the mechanisms underlying the neuropathology of these alterations are still not defined. Therefore, the aim of this study was to investigate the effect of acute intra-hippocampal leucine administration in the step-down test in rats. In addition, the leucine effects on the electrophysiological parameter, long-term potentiation generation, and on the activities of the respiratory chain were also investigated. Male Wistar rats were bilaterally administrated with leucine (80 nmol/hippocampus; 160 nmol/rat) or artificial cerebrospinal fluid (controls) into the hippocampus immediately post-training in the behavioral task. Twenty-four hours after training in the step-down test, the latency time was evaluated and afterwards animals were sacrificed for assessing the ex vivo biochemical measurements. Leucine-treated animals showed impairment in memory consolidation and a complete inhibition of long-term potentiation generation at supramaximal stimulation. In addition, a significant increment in complex IV activity was observed in hippocampus from leucine-administered rats. These data strongly indicate that leucine compromise memory consolidation, and that impairment of long-term potentiation generation and unbalance of the respiratory chain may be plausible mechanisms underlying the deleterious leucine effect on cognition.

  7. 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.

  8. 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 ...

  9. Basic brownfields

    SciTech Connect

    Jacobs, B.L.

    1997-12-31

    This article is a basic guide to the brownfields problem. It will define the problem and will attempt to identify the various causes thereof. It also will review federal brownfields initiatives and state brownfields reforms in Kentucky, Ohio, Pennsylvania, and West Virginia.

  10. 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)

  11. 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…

  12. 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…

  13. 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…

  14. 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…

  15. Dietary L-leucine improves the anemia in a mouse model for Diamond-Blackfan anemia.

    PubMed

    Jaako, Pekka; Debnath, Shubhranshu; Olsson, Karin; Bryder, David; Flygare, Johan; Karlsson, Stefan

    2012-09-13

    Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.

  16. Evolution of the Leucine Gene Cluster in Buchnera aphidicola: Insights from Chromosomal Versions of the Cluster

    PubMed Central

    Sabater-Muñoz, Beatriz; van Ham, Roeland C. H. J.; Moya, Andrés; Silva, Francisco J.; Latorre, Amparo

    2004-01-01

    In Buchnera aphidicola strains associated with the aphid subfamilies Thelaxinae, Lachninae, Pterocommatinae, and Aphidinae, the four leucine genes (leuA, -B, -C, and -D) are located on a plasmid. However, these genes are located on the main chromosome in B. aphidicola strains associated with the subfamilies Pemphiginae and Chaitophorinae. The sequence of the chromosomal fragment containing the leucine cluster and flanking genes has different positions in the chromosome in B. aphidicola strains associated with three tribes of the subfamily Pemphiginae and one tribe of the subfamily Chaitophorinae. Due to the extreme gene order conservation of the B. aphidicola genomes, the variability in the position of the leucine cluster in the chromosome may be interpreted as resulting from independent insertions from an ancestral plasmid-borne leucine gene. These findings do not support a chromosomal origin for the leucine genes in the ancestral B. aphidicola and do support a back transfer evolutionary scenario from a plasmid to the main chromosome. PMID:15090505

  17. 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

  18. Effect of leucine uptake on hepatic and skeletal muscle gene expression in rats: a microarray analysis

    PubMed Central

    Cheon, Wookwang

    2015-01-01

    [Purpose] This study was performed to explore the physiological functions of leucine by exploring genes with leucine-dependent variability using DNA microarray. [Methods] Sprague-Dawley rats (n = 20) were separated into a HPD (30% High Protein Diet, n = 10) group and a NPD (0% Non Protein Diet, n = 10) group and fed a protein diet for 2 weeks. At the end of the 2-week period, the rats were fasted for 12-16 hours, further separated into subgroups within the HPD (Saline, n = 5, Leucine, n = 5) and NPD (Saline, n = 5, Leucine, n = 5) groups and administered with a leucine solution. The liver and muscles were harvested after 2 hours for RNA extraction. RNA purification from the isolated muscles and target gene identification using DNA chip were performed. The target gene was determined based on the results of the DNA chip experiment, and mRNA expression of the target gene was analyzed using Real-Time PCR. [Results] In the skeletal muscle, 27 genes were upregulated while 52 genes were down regulated after leucine administration in the NPD group. In the liver, 160 genes were up-regulated while 126 were down-regulated. The per2 gene was one of the genes with leucine-dependent induction in muscles and liver. [Conclusion] This study was performed to explore the physiological functions of leucine, however, a large number of genes showed variability. Therefore, it was difficult to definitively identify the genes linked with a particular physiological function. Various nutritional effects of leucine were observed. High variability in cytokines, receptors, and various membrane proteins were observed, which suggests that leucine functions as more than a nutrient. The interpretation may depend on investigators’ perspectives, therefore, discussion with relevant experts and the BCAA (Branched-Chain Amino Acids) society may be needed for effective utilization of this data. PMID:26244133

  19. Education: The Basics. The Basics

    ERIC Educational Resources Information Center

    Wood, Kay

    2011-01-01

    Everyone knows that education is important, we are confronted daily by discussion of it in the media and by politicians, but how much do we really know about education? "Education: The Basics" is a lively and engaging introduction to education as an academic subject, taking into account both theory and practice. Covering the schooling system, the…

  20. Evolution of Class III Homeodomain–Leucine Zipper Genes in Streptophytes

    PubMed Central

    Floyd, Sandra K.; Zalewski, Christopher S.; Bowman, John L.

    2006-01-01

    Land plants underwent tremendous evolutionary change following the divergence of the ancestral lineage from algal relatives. Several important developmental innovations appeared as the embryophyte clade diversified, leading to the appearance of new organs and tissue types. To understand how these changes came about, we need to identify the fundamental genetic developmental programs that are responsible for growth, patterning, and differentiation and describe how these programs were modified and elaborated through time to produce novel morphologies. Class III homeodomain–leucine zipper (class III HD–Zip) genes, identified in the model plant Arabidopsis thaliana, provide good candidates for basic land plant patterning genes. We show that these genes may have evolved in a common ancestor of land plants and their algal sister group and that the gene family has diversified as land plant lineages have diversified. Phylogenetic analysis, expression data from nonflowering lineages, and evidence from Arabidopsis and other flowering plants indicate that class III HD–Zip genes acquired new functions in sporophyte apical growth, vascular patterning and differentiation, and leaf development. Modification of expression patterns that accompanied diversification of class III HD–Zip genes likely played an important role in the evolution of land plant form. PMID:16489224

  1. L-Leucine for gold nanoparticles synthesis and their cytotoxic effects evaluation.

    PubMed

    Berghian-Grosan, Camelia; Olenic, Liliana; Katona, Gabriel; Perde-Schrepler, Maria; Vulcu, Adriana

    2014-11-01

    This work reports the preparation of water-soluble leucine capped gold nanoparticles by two single-step synthesis methods. The first procedure involves a citrate reduction approach where the citrate is used as reducing agent and leucine as capping/stabilizing agent. Different sizes of gold nanoparticles, citrate reduced and stabilized by leucine, Leu-AuNPs-C, with the mean diameters in the range of 21-56 nm, were obtained by varying the macroscopic parameters such as: concentration of the gold precursor solution, Au (III):citrate molar ratio and leucine pH. In the second procedure, leucine acts both as reducing and stabilizing agent, allowing us to obtain spherical gold nanoparticles, Leu-AuNPs, with a majority of 80 % (with the mean diameter of 63 nm). This proves that leucine is an appropriate reductant for the formation of water-soluble and stable gold nanoparticles colloids. The characterization of the leucine coated gold nanoparticles was carried out by TEM, UV-Vis and FT-IR analysis. The cytotoxic effect of Leu-AuNPs-C and Leu-AuNPs was also evaluated. PMID:25092048

  2. Leucine supplementation improves leptin sensitivity in high-fat diet fed rats

    PubMed Central

    Yuan, Xue-Wei; Han, Shu-Fen; Zhang, Jian-Wei; Xu, Jia-Ying; Qin, Li-Qiang

    2015-01-01

    Background Several studies have reported the favorable effect of leucine supplementation on insulin resistance or insulin sensitivity. However, whether or not leucine supplementation improves leptin sensitivity remains unclear. Design Forty-eight male Sprague-Dawley rats were fed with either a high-fat diet (HFD) or HFD supplemented with 1.5, 3.0, and 4.5% leucine for 16 weeks. At the end of the experiment, serum leptin level was measured by ELISA, and leptin receptor (ObR) in the hypothalamus was examined by immunohistochemistry. The protein expressions of ObR and leptin-signaling pathway in adipose tissues were detected by western blot. Results No significant differences in body weight and food/energy intake existed among the four groups. Serum leptin levels were significantly lower, and ObR expression in the hypothalamus and adipose tissues was significantly higher in the three leucine groups than in the control group. These phenomena suggested that leptin sensitivity was improved in the leucine groups. Furthermore, the expressions of JAK2 and STAT3 (activated by ObR) were significantly higher, and that of SOCS3 (inhibits leptin signaling) was significantly lower in the three leucine groups than in the control group. Conclusions Leucine supplementation improves leptin sensitivity in rats on HFD likely by promoting leptin signaling. PMID:26115673

  3. 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

  4. Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization.

    PubMed Central

    Dang, C V; Barrett, J; Villa-Garcia, M; Resar, L M; Kato, G J; Fearon, E R

    1991-01-01

    The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c

  5. Leucine modulation of mitochondrial mass and oxygen consumption in skeletal muscle cells and adipocytes

    PubMed Central

    Sun, Xiaocun; Zemel, Michael B

    2009-01-01

    Background The effects of dairy on energy metabolism appear to be mediated, in part, by leucine and calcium which regulate both adipocyte and skeletal muscle energy metabolism. We recently demonstrated that leucine and calcitriol regulate fatty acid oxidation in skeletal muscle cells in vitro, with leucine promoting and calcitriol suppressing fatty acid oxidation. Moreover, leucine coordinately regulated adipocyte lipid metabolism to promote flux of lipid to skeletal muscle and regulate metabolic flexibility. We have now investigated the role of mitochondrial biogenesis in mediating these effects. Methods We tested the effect of leucine, calcitriol and calcium in regulation of mitochondrial mass using a fluorescence method and tested mitochondrial biogenesis regulatory genes as well mitochondrial component genes using real-time PCR. We also evaluated the effect of leucine on oxygen consumption with a modified perfusion system. Results Leucine (0.5 mM) increased mitochondrial mass by 30% and 53% in C2C12 myocytes and 3T3-L1 adipocytes, respectively, while calcitriol (10 nM) decreased mitochondrial abundance by 37% and 27% (p < 0.02). Leucine also stimulated mitochondrial biogenesis genes SIRT-1, PGC-1α and NRF-1 as well as mitochondrial component genes UCP3, COX, and NADH expression by 3–5 fold in C2C12 cells (p < 0.003). Adipocyte-conditioned medium reduced mitochondrial abundance (p < 0.001) and decreased UCP3 but increased PGC-1α expression in myocytes, suggesting a feedback stimulation of mitochondrial biogenesis. Similar data were observed in C2C12 myocytes co-cultured with adipocytes, with co-culture markedly suppressing mitochondrial abundance (p < 0.02). Leucine stimulated oxygen consumption in both C2C12 cells and adipocytes compared with either control or valine-treated cells. Transfection of C2C12 myocytes with SIRT-1 siRNA resulted in parallel suppression of SIRT-1 expression and leucine-induced stimulation of PGC-1α and NRF-1, indicating that SIRT

  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. 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

  8. 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.

  9. Molecular structure of leucine aminopeptidase at 2. 7- angstrom resolution

    SciTech Connect

    Burley, S.K. Brigham and Women's Hospital, Boston, MA ); David, P.R.; Lipscomb, W.N. ); Taylor, A. )

    1990-09-01

    The three-dimensional structure of bovine lens leucine aminopeptidase complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0-{angstrom} resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-{angstrom} resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry and is triangular in shape with a triangle edge length of 115 {angstrom} and maximal thickness of 90 {angstrom}. The monomers are crystallographically equivalent and each is folded into two unequal {alpha}/{beta} domains connected by an {alpha}-helix to give a comma-like shape with approximate maximal dimensions of 90 x 55 x 55 {angstrom}{sup 3}. The secondary structural composition is 40% {alpha}-helix and 19% {beta}-strand. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 {angstrom} and thickness 10 {angstrom}. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes.

  10. 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.

  11. Specific formation of negative ions from leucine and isoleucine molecules

    NASA Astrophysics Data System (ADS)

    Papp, Peter; Shchukin, Pavel; Matejčík, Štefan

    2010-01-01

    Dissociative electron attachment (DEA) to gas phase leucine (Leu) and isoleucine (Ile) molecules was studied using experimental and quantum-chemical methods. The relative partial cross sections for DEA have been measured using crossed electron/molecular beams technique. Supporting ab initio calculations of the structure, energies of neutral molecules, fragments, and negative ions have been carried out at G3MP2 and B3LYP levels in order to interpret the experimental data. Leu and Ile exhibit several common features. The negative ionic fragments from both molecules are formed in the electron energy range from 0 to approximately 14 eV via three resonances (1.2, 5.5, and 8 eV). The relative partial cross sections for DEA Leu and Ile are very similar. The dominant negative ions formed were closed shell negative ions (M-H)- (m/z=130) formed preferentially via low electron energy resonance of 1.23 eV. Additional negative ions with m/z=115, 114, 113, 112, 84, 82, 74, 45, 26, and 17 have been detected.

  12. 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

  13. 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

  14. Inflation Basics

    SciTech Connect

    Green, Dan

    2014-03-01

    inflation since 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.

  15. [Somatostatin does not modify 3-oxymethylglucose and leucine uptake by rat enterocytes].

    PubMed

    Crespo, C A; Martínez-Sapiña, J; Taboada, M C

    1988-09-01

    The effects of somatostatin on 3-oxymethylglucose (3-OMG) and leucine uptake by rat enterocytes were examined. Somatostatin did not decrease the 3-OMG enterocyte uptake. When the 3-OMG active transport was inhibited by phloridzin, Somatostatin presented no significant modifications. Somatostatin showed a slight decrease in 3-OMG release through the basolateral membrane, when such a release was inhibited with theophylline. Somatostatin did not modify the intestinal leucine uptake or its inhibition by methionine.

  16. 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

  17. Light dependence of [3H]leucine incorporation in the oligotrophic North Pacific ocean.

    PubMed

    Church, Matthew J; Ducklow, Hugh W; Karl, David M

    2004-07-01

    The influence of irradiance on bacterial incorporation of [(3)H]leucine was evaluated at Station ALOHA in the oligotrophic North Pacific subtropical gyre. Six experiments were conducted on three cruises to Station ALOHA to examine how [(3)H]leucine incorporation varied as a function of irradiance. Two experiments were also conducted to assess the photoautotrophic response to irradiance (based on photosynthetic uptake of [(14)C]bicarbonate) in both the upper and lower photic zones. Rates of [(3)H]leucine incorporation responded to irradiance in a photosynthesis-like manner, increasing sharply at low light and then saturating and sometimes declining with increasing light intensity. The influence of irradiance on bacterial growth was evaluated in both the well-lit (5 to 25 m) and dimly lit regions of the upper ocean (75 to 100 m) to determine whether the bacterial response to irradiance differed along the depth-dependent light gradient of the photic zone. [(3)H]leucine incorporation rates were analyzed with a photosynthesis-irradiance model for a quantitative description of the relationships between [(3)H]leucine incorporation and irradiance. Maximum rates of [(3)H]leucine incorporation in the upper photic zone increased 48 to 92% relative to those of dark-incubated samples, with [(3)H]leucine incorporation saturating at light intensities between 58 and 363 micromol of quanta m(-2) s(-1). Rates of [(3)H]leucine incorporation in the deep photic zone were photostimulated 53 to 114% and were susceptible to photoinhibition, with rates declining at light intensities of >100 micromol of quanta m(-2) s(-1). The results of these experiments revealed that sunlight directly influences bacterial growth in this open-ocean ecosystem.

  18. 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.

  19. Amino acid metabolism in the human fetus at term: leucine, valine, and methionine kinetics.

    PubMed

    van den Akker, Chris H P; Schierbeek, Henk; Minderman, Gardi; Vermes, Andras; Schoonderwaldt, Ernst M; Duvekot, Johannes J; Steegers, Eric A P; van Goudoever, Johannes B

    2011-12-01

    Human fetal metabolism is largely unexplored. Understanding how a healthy fetus achieves its fast growth rates could eventually play a pivotal role in improving future nutritional strategies for premature infants. To quantify specific fetal amino acid kinetics, eight healthy pregnant women received before elective cesarean section at term, continuous stable isotope infusions of the essential amino acids [1-13C,15N]leucine, [U-13C5]valine, and [1-13C]methionine. Umbilical blood was collected after birth and analyzed for enrichments and concentrations using mass spectrometry techniques. Fetuses showed considerable leucine, valine, and methionine uptake and high turnover rates. α-Ketoisocaproate, but not α-ketoisovalerate (the leucine and valine ketoacids, respectively), was transported at net rate from the fetus to the placenta. Especially, leucine and valine data suggested high oxidation rates, up to half of net uptake. This was supported by relatively low α-ketoisocaproate reamination rates to leucine. Our data suggest high protein breakdown and synthesis rates, comparable with, or even slightly higher than in premature infants. The relatively large uptakes of total leucine and valine carbon also suggest high fetal oxidation rates of these essential branched chain amino acids.

  20. 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

  1. Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

    NASA Astrophysics Data System (ADS)

    Talarmin, A.; van Wambeke, F.; Catala, P.; Courties, C.; Lebaron, P.

    2011-02-01

    The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l-1 h-1 (n=30). Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok) were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA) with high scatter (HNA-hs) and low scatter (HNA-ls) and low nucleic acid containing cells (LNA). Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell-1 h-1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30). In surface layers (from 0 m down to the deep chlorophyll depth, DCM), cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively). Nevertheless, on average HNA-hs (26%) and LNA (27%) equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell-1 h-1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The mixotrophic features of certain photosynthetic prokaryotes and the high contribution of LNA cells to leucine incorporation within the microbial

  2. 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

  3. 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

  4. Glutamine and leucine nitrogen kinetics and their relation to urea nitrogen in newborn infants.

    PubMed

    Parimi, Prabhu S; Devapatla, Srisatish; Gruca, Lourdes; O'Brien, Alicia M; Hanson, Richard W; Kalhan, Satish C

    2002-03-01

    Glutamine kinetics and its relation to transamination of leucine and urea synthesis were quantified in 16 appropriate-for-gestational-age infants, four small-for-gestational-age infants, and seven infants of diabetic mothers. Kinetics were measured between 4 and 5 h after the last feed (fasting) and in response to formula feeding using [5-(15)N]glutamine, [1-(13)C,(15)N]leucine, [(2)H(5)]phenylalanine, and [(15)N(2)]urea tracers. Leucine nitrogen and glutamine kinetics during fasting were significantly higher than those reported in adults. De novo synthesis accounted for approximately 85% of glutamine turnover. In response to formula feeding, a significant increase (P = 0.04) in leucine nitrogen turnover was observed, whereas a significant decrease (P = 0.002) in glutamine and urea rate of appearance was seen. The rate of appearance of leucine nitrogen was positively correlated (r(2) = 0.59, P = 0.001) with glutamine turnover. Glutamine flux was negatively correlated (r(2) = 0.39, P = 0.02) with the rate of urea synthesis. These data suggest that, in the human newborn, glutamine turnover is related to a high anaplerotic flux into the tricarboxylic acid cycle as a consequence of a high rate of protein turnover. The negative relationship between glutamine turnover and the irreversible oxidation of protein (urea synthesis) suggests an important role of glutamine as a nitrogen source for other synthetic processes and accretion of body proteins.

  5. 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

  6. Preferential utilization of exogenously supplied leucine for protein synthesis in estradiol-induced and uninduced cockerel liver explants.

    PubMed Central

    Gehrke, L; Ilan, J

    1983-01-01

    A cockerel liver explant system has been used to study protein synthesis and ribosome transit times. After a 2-hr preincubation of explant tissue in the presence of a large concentration of nonradioactive leucine, a small quantity of [3H]leucine was added and the kinetics of uptake of [3H]leucine into the intracellular acid-soluble leucine pool was compared to the incorporation of [3H]leucine into protein. Incorporation of [3H]leucine into protein reaches a linear rate almost immediately after addition of label, whereas the acid-soluble pool does not reach constant specific activity until much later. The length of time needed to reach a linear rate of incorporation of [3H]leucine into protein is approximately equal to the length of time needed to equilibrate nascent polypeptide chains with labeled precursor--that is, one average ribosome transit time. Therefore, it seems that the immediate precursor pool for protein synthesis reaches constant specific activity almost instantly after addition of [3H]leucine. The results indicate that at least part of the supply of leucine for protein synthesis is derived directly from the exogenous incubation medium and not from the intracellular acid-soluble amino acid pool. Images PMID:6574484

  7. Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems.

    PubMed

    Lichtenecker, Roman J; Weinhäupl, Katharina; Reuther, Lukas; Schörghuber, Julia; Schmid, Walther; Konrat, Robert

    2013-11-01

    The addition of labeled α-ketoisovalerate to the growth medium of a protein-expressing host organism has evolved into a versatile tool to achieve concomitant incorporation of specific isotopes into valine- and leucine- residues. The resulting target proteins represent excellent probes for protein NMR analysis. However, as the sidechain resonances of these residues emerge in a narrow spectral range, signal overlap represents a severe limitation in the case of high-molecular-weight NMR probes. We present a protocol to eliminate leucine labeling by supplying the medium with unlabeled α-ketoisocaproate. The resulting spectra of a model protein exclusively feature valine signals of increased intensity, confirming the method to be a first example of independent valine and leucine labeling employing α-ketoacid precursor compounds.

  8. X-ray scattering indicates that the leucine zipper is a coiled coil.

    PubMed Central

    Rasmussen, R; Benvegnu, D; O'Shea, E K; Kim, P S; Alber, T

    1991-01-01

    Dimerization of the bZIP class of eukaryotic transcriptional control proteins requires a sequence motif called the leucine zipper. We have grown two distinct crystal forms of a 33-amino acid peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4. This peptide is known to form a dimer of parallel helices in solution. X-ray scattering from both crystal forms shows reflections that are diagnostic of coiled coils. The most notable reflections occur at approximately 5.2 A resolution and correspond to the pitch of helices in coiled coils. There is no diffraction maximum near 5.4 A, the characteristic pitch of straight helices. Our results provide direct evidence that the leucine zipper of GCN4 is a coiled coil. Images PMID:1988953

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor.

    PubMed

    Yu, Xiaofei; Wang, Yuhao; Deng, Mi; Li, Yun; Ruhn, Kelly A; Zhang, Cheng Cheng; Hooper, Lora V

    2014-10-13

    Innate lymphoid cells (ILCs) are recently identified lymphocytes that limit infection and promote tissue repair at mucosal surfaces. However, the pathways underlying ILC development remain unclear. Here we show that the transcription factor NFIL3 directs the development of a committed bone marrow precursor that differentiates into all known ILC lineages. NFIL3 was required in the common lymphoid progenitor (CLP), and was essential for the differentiation of αLP, a bone marrow cell population that gives rise to all known ILC lineages. Clonal differentiation studies revealed that CXCR6(+) cells within the αLP population differentiate into all ILC lineages but not T- and B-cells. We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX. These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development.

  14. 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

  15. Increased synthesis of eicosanoids by human monocytes following leucine and methionine enkephalin administration

    SciTech Connect

    Wiederhold, M.D.; Ou, D.W.

    1986-03-05

    Regulation of eicosanoid biosynthesis by neuropeptides was investigated in human peripheral blood monocytes from normal donors. Metabolites of /sup 3/H-arachidonic acid (/sup 3/H-AA) were analyzed by thin layer and high pressure liquid chromatography following exposure to 0.2 ..mu..gm/ml and 2.0 ..mu..gm/ml of leucine (L-ENK) and methionine (M-ENK) enkephalin. Supernatants of cultured cells were analyzed. The data indicate that both leucine and methionine enkephalin can stimulate eicosanoid biosynthesis in human monocytes, and may indicate a possible regulatory mechanism between the central nervous system and the reticuloendothelial system.

  16. [Leucine and alanine aminopeptidase activity in the organs of cattle, sheep and swine].

    PubMed

    Goranov, Kh

    1982-01-01

    Studied was the activity of leucine-aminopeptidase and alanine-aminopeptidase in fresh tissue homogenates of liver, spleen, kidney, heart, pancreas, femoral muscle, stomach (rumen), small intestine, and lung taken from 8 cattle, sheep, and pigs. Both enzymes showed ubiquity. Leucine-aminopeptidase exhibited highest activity in the spleen of pigs and the kidney of sheep and cattle. The kidneys of all investigated animal species showed 10 to 15 times higher alanine-aminopeptidase activity than the remaining organs. This pointed to the relative ubiquity of the enzyme with special reference to kidneys.

  17. Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

    PubMed Central

    Quay, S C; Oxender, D L; Tsuyumu, S; Umbarger, H E

    1975-01-01

    Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears

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

    PubMed

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

    2015-05-08

    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.

  19. Differential effects of leucine supplementation in young and aged mice at the onset of skeletal muscle regeneration.

    PubMed

    Perry, Richard A; Brown, Lemuel A; Lee, David E; Brown, Jacob L; Baum, Jamie I; Greene, Nicholas P; Washington, Tyrone A

    2016-07-01

    Aging decreases the ability of skeletal muscle to respond to injury. Leucine has been demonstrated to target protein synthetic pathways in skeletal muscle thereby enhancing this response. However, the effect of aging on leucine-induced alterations in protein synthesis at the onset of skeletal muscle regeneration has not been fully elucidated. The purpose of this study was to determine if aging alters skeletal muscle regeneration and leucine-induced alterations in markers of protein synthesis. The tibialis anterior of young (3 months) and aged (24 months) female C57BL/6J mice were injected with either bupivacaine or PBS, and the mice were given ad libitum access to leucine-supplemented or normal drinking water. Protein and gene expression of markers of protein synthesis and degradation, respectively, were analyzed at three days post-injection. Following injury in young mice, leucine supplementation was observed to elevate only p-p70S6K. In aged mice, leucine was shown to elicit higher p-mTOR content with and without injury, and p-4EBP-1 content post-injury. Additionally in aged mice, leucine was shown to elicit higher content of relative p70S6K post-injury. Our study shows that leucine supplementation affects markers of protein synthesis at the onset of skeletal muscle regeneration differentially in young and aged mice. PMID:27327351

  20. 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

  1. 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

  2. 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 ...

  3. 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. ...

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

    PubMed

    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

    2013-10-01

    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.

  5. Nutritional and regulatory roles of leucine in muscle growth and fat reduction.

    PubMed

    Duan, Yehui; Li, Fengna; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Zhang, Yuzhe; Deng, Dun; Tang, Yulong; Feng, Zemeng; Wu, Guoyao; Yin, Yulong

    2015-01-01

    The metabolic roles for L-leucine, an essential branched-chain amino acid (BCAA), go far beyond serving exclusively as a building block for de novo protein synthesis. Growing evidence shows that leucine regulates protein and lipid metabolism in animals. Specifically, leucine activates the mammalian target of rapamycin (mTOR) signaling pathway, including the 70 kDa ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1) to stimulate protein synthesis in skeletal muscle and adipose tissue and to promote mitochondrial biogenesis, resulting in enhanced cellular respiration and energy partitioning. Activation of cellular energy metabolism favors fatty acid oxidation to CO2 and water in adipocytes, lean tissue gain in young animals, and alleviation of muscle protein loss in aging adults, lactating mammals, and food-deprived subjects. As a functional amino acid, leucine holds great promise to enhance the growth, efficiency of food utilization, and health of animals and humans.  PMID:25553480

  6. '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.

  7. 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...

  8. 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

  9. 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 ...

  10. 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...

  11. Increasing dietary leucine intake reduces diet-induced obesity and improves glucose and cholesterol metabolism in mice via multimechanisms.

    PubMed

    Zhang, Yiying; Guo, Kaiying; LeBlanc, Robert E; Loh, Daniella; Schwartz, Gary J; Yu, Yi-Hao

    2007-06-01

    Leucine, as an essential amino acid and activator of mTOR (mammalian target of rapamycin), promotes protein synthesis and suppresses protein catabolism. However, the effect of leucine on overall glucose and energy metabolism remains unclear, and whether leucine has beneficial effects as a long-term dietary supplement has not been examined. In the present study, we doubled dietary leucine intake via leucine-containing drinking water in mice with free excess to either a rodent chow or a high-fat diet (HFD). While it produced no major metabolic effects in chow-fed mice, increasing leucine intake resulted in up to 32% reduction of weight gain (P < 0.05) and a 25% decrease in adiposity (P < 0.01) in HFD-fed mice. The reduction of adiposity resulted from increased resting energy expenditure associated with increased expression of uncoupling protein 3 in brown and white adipose tissues and in skeletal muscle, while food intake was not decreased. Increasing leucine intake also prevented HFD-induced hyperglycemia, which was associated with improved insulin sensitivity, decreased plasma concentrations of glucagon and glucogenic amino acids, and downregulation of hepatic glucose-6-phosphatase. Additionally, plasma levels of total and LDL cholesterol were decreased by 27% (P < 0.001) and 53% (P < 0.001), respectively, in leucine supplemented HFD-fed mice compared with the control mice fed the same diet. The reduction in cholesterol levels was largely independent of leucine-induced changes in adiposity. In conclusion, increases in dietary leucine intake substantially decrease diet-induced obesity, hyperglycemia, and hypercholesterolemia in mice with ad libitum consumption of HFD likely via multiple mechanisms.

  12. Amino acid uptake by yeasts. IV. Effect of thiol reagents on L-leucine transport in Saccharomyces cerevisiae.

    PubMed

    Ramos, E H; De Bongioanni, L C; Wainer, S R; Stoppani, A O

    1983-06-10

    (1) N-Ethylmaleimide (a penetrating SH- reagent) inactivated L-[14C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with N-ethylmaleimide, N-ethylmaleimide concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With D-glucose-energized yeast, N-ethylmaleimide inhibited L-[14C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit L-[14C]leucine binding, except when the cells were preincubated with L-leucine. With the rho- respiratory-deficient mutant (energized cells), N-ethylmaleimide inhibited L-[14C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the N-ethylmaleimide effect as a function of L-[14C]leucine concentration showed a significant decrease of Jmax values of the high- (S1) and low- (S2) affinity amino acid transport systems, but KT values were not significantly modified. (3) When assayed in the presence of D-glucose, N-ethylmaleimide inhibition of D-glucose uptake and respiration contributed significantly to inactivation of L-[14C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of L-[14C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate L-[14C]leucine entrance, while p-chloromercuribenzoate, a slowly penetrating SH-reagent, inactivated it to a limited extent. When compared with the effect of N-ethylmaleimide, these negative results indicate that thiol groups of the L-[14C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Leucine transport in brush border membrane vesicles from freshwater insect larvae.

    PubMed

    Forcella, Matilde; Berra, Elisa; Giacchini, Roberto; Parenti, Paolo

    2006-11-01

    Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of V(max) (45.3 pmol.s(-1).mg proteins(-1)) was in E. danica, which, however, displayed the lowest affinity (K(m) 137 muM) when compared to the kinetic parameters of other taxa. The V(max) and K(m) values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects.

  18. Leucine meal increases glutamine and total nitrogen release from forearm muscle.

    PubMed Central

    Aoki, T T; Brennan, M F; Fitzpatrick, G F; Knight, D C

    1981-01-01

    To assess the consequences of elevated branched chain amino acid levels on alanine, glutamine, and ammonia metabolism in muscle, L-leucine meals (14.7 g) were consumed by six normal postabsorptive individuals. Bilateral forearm studies were performed, and the dominant arm was subjected to 15 min of light exercise, using a calibrated dynamometer, beginning 45 min after the ingestion of the meal. Large uptakes of leucine were seen across both forearm muscle beds within 30 min of the meal. After exercise, blood flow in the dominant arm increased from 3.1 +/- 0.4 to 5.2 +/- 0.9 ml/100 ml forearm per minute (mean +/- SEM, P less than 0.005). Glutamine flux out of the dominant forearm increased threefold after the ingestion of the leucine meal and increased eightfold over base line after exercise. Less marked changes (significant only at 90 min) in the nonexercised, nondominant arm were also seen. Alanine flux out of the dominant forearm muscle bed increased modestly at 75 and 90 min. No significant change in ammonia flux across either forearm muscle bed was noted. Unexpectedly, large and significant net nitrogen loss from both forearm muscle beds was documented. Thus, following the ingestion of a leucine meal and light exercise, the primary means by which excess nitrogen is routed out of muscle is via glutamine formation and release with alanine and ammonia pathways playing relatively minor roles. More importantly, the ingestion of significant amounts of leucine by normal subjects, presumably in optimal nitrogen balance, results in a net loss of nitrogen from muscle. PMID:7320199

  19. 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.

  20. 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

  1. 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

  2. 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…

  3. A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif.

    PubMed

    Martínez-Turiño, Sandra; Hernández, Carmen

    2011-05-10

    Two small viral proteins (DGBp1 and DGBp2) have been proposed to act in a concerted manner to aid intra- and intercellular trafficking of carmoviruses though the distribution of functions and mode of action of each protein partner are not yet clear. Here we have confirmed the requirement of the DGBps of Pelargonium flower break virus (PFBV), p7 and p12, for pathogen movement. Studies focused on p12 have shown that it associates to cellular membranes, which is in accordance to its hydrophobic profile and to that reported for several homologs. However, peculiarities that distinguish p12 from other DGBps2 have been found. Firstly, it contains a leucine zipper-like motif which is essential for virus infectivity in plants. Secondly, it has an unusually long and basic N-terminal region that confers RNA binding activity. The results suggest that PFBV p12 may differ mechanistically from related proteins and possible roles of PFBV DGBps are discussed.

  4. 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

  5. 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.

  6. Dietary Leucine - An Environmental Modifier of Insulin Resistance Acting on Multiple Levels of Metabolism

    PubMed Central

    Macotela, Yazmin; Emanuelli, Brice; Bång, Anneli M.; Espinoza, Daniel O.; Boucher, Jeremie; Beebe, Kirk; Gall, Walter; Kahn, C. Ronald

    2011-01-01

    Environmental factors, such as the macronutrient composition of the diet, can have a profound impact on risk of diabetes and metabolic syndrome. In the present study we demonstrate how a single, simple dietary factor—leucine—can modify insulin resistance by acting on multiple tissues and at multiple levels of metabolism. Mice were placed on a normal or high fat diet (HFD). Dietary leucine was doubled by addition to the drinking water. mRNA, protein and complete metabolomic profiles were assessed in the major insulin sensitive tissues and serum, and correlated with changes in glucose homeostasis and insulin signaling. After 8 weeks on HFD, mice developed obesity, fatty liver, inflammatory changes in adipose tissue and insulin resistance at the level of IRS-1 phosphorylation, as well as alterations in metabolomic profile of amino acid metabolites, TCA cycle intermediates, glucose and cholesterol metabolites, and fatty acids in liver, muscle, fat and serum. Doubling dietary leucine reversed many of the metabolite abnormalities and caused a marked improvement in glucose tolerance and insulin signaling without altering food intake or weight gain. Increased dietary leucine was also associated with a decrease in hepatic steatosis and a decrease in inflammation in adipose tissue. These changes occurred despite an increase in insulin-stimulated phosphorylation of p70S6 kinase indicating enhanced activation of mTOR, a phenomenon normally associated with insulin resistance. These data indicate that modest changes in a single environmental/nutrient factor can modify multiple metabolic and signaling pathways and modify HFD induced metabolic syndrome by acting at a systemic level on multiple tissues. These data also suggest that increasing dietary leucine may provide an adjunct in the management of obesity-related insulin resistance. PMID:21731668

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

    PubMed

    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

  8. 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.

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

    PubMed Central

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

    2015-01-01

    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

  10. LRRCE: a leucine-rich repeat cysteine capping motif unique to the chordate lineage

    PubMed Central

    Park, Hosil; Huxley-Jones, Julie; Boot-Handford, Ray P; Bishop, Paul N; Attwood, Teresa K; Bella, Jordi

    2008-01-01

    Background The small leucine-rich repeat proteins and proteoglycans (SLRPs) form an important family of regulatory molecules that participate in many essential functions. They typically control the correct assembly of collagen fibrils, regulate mineral deposition in bone, and modulate the activity of potent cellular growth factors through many signalling cascades. SLRPs belong to the group of extracellular leucine-rich repeat proteins that are flanked at both ends by disulphide-bonded caps that protect the hydrophobic core of the terminal repeats. A capping motif specific to SLRPs has been recently described in the crystal structures of the core proteins of decorin and biglycan. This motif, designated as LRRCE, differs in both sequence and structure from other, more widespread leucine-rich capping motifs. To investigate if the LRRCE motif is a common structural feature found in other leucine-rich repeat proteins, we have defined characteristic sequence patterns and used them in genome-wide searches. Results The LRRCE motif is a structural element exclusive to the main group of SLRPs. It appears to have evolved during early chordate evolution and is not found in protein sequences from non-chordate genomes. Our search has expanded the family of SLRPs to include new predicted protein sequences, mainly in fishes but with intriguing putative orthologs in mammals. The chromosomal locations of the newly predicted SLRP genes would support the large-scale genome or gene duplications that are thought to have occurred during vertebrate evolution. From this expanded list we describe a new class of SLRP sequences that could be representative of an ancestral SLRP gene. Conclusion Given its exclusivity the LRRCE motif is a useful annotation tool for the identification and classification of new SLRP sequences in genome databases. The expanded list of members of the SLRP family offers interesting insights into early vertebrate evolution and suggests an early chordate evolutionary

  11. 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

  12. 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.

  13. 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

  14. Regulation of Transaminase C Synthesis in Escherichia coli: Conditional Leucine Auxotrophy

    PubMed Central

    McGilvray, Derek; Umbarger, H. E.

    1974-01-01

    The regulation of synthesis of the valine-alanine-α-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 α-acetohydroxy acid synthase activity, causing α-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

  15. 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

  16. Characteristics and Development of Leucine Transport Activity in the Scutellum of Germinating Barley Grain 1

    PubMed Central

    Sopanen, Tuomas; Uuskallio, Marjukka; Nyman, Seija; Mikola, Juhani

    1980-01-01

    Scutella separated from grains of Himalaya barley after germination for 3 days rapidly took up l-leucine from aerated incubation media; with 1 millimolar leucine the rate varied between 4 and 14 micromoles per gram per hour and the pH optimum was at 3.5 to 5, both depending on buffer composition and prewashing time. The rate of the uptake increased with increasing concentration of leucine in a complex manner, which could be interpreted as multiphasic kinetics with apparent Km values of 3.4 and 15.5 millimolar below and above 3 millimolar leucine, respectively. The uptake took place against a concentration difference (highest estimated ratio 270: 1) and was strongly inhibited by dinitrophenol. Uptake was apparently due to active transport requiring metabolic energy. The development of the uptake activity during germination was studied using Pirkka barley. A low activity was present in the scutella of ungerminated grains. It began to increase after 6 hours imbibition, and the increase was biphasic, the major changes occurring during days 0 to 3 and 4 to 6. The total increase was about 20-fold. The regulation of the development was studied by allowing separated embryos to germinate on agar gel. The increase of uptake activity was strongly inhibited by inhibitors of RNA or protein synthesis. Increase did not require the presence of the embryo proper, and was not affected by gibberellic or abscisic acid. Removal of the endosperm greatly accelerated the increase of uptake activity, and the presence of 5 or 20 millimolar glutamine counteracted the removal of the endosperm. The results suggest that the availability of glutamine or amino acids in general in the endosperm may regulate the development or the activity of the transport system. PMID:16661169

  17. The SAGA histone acetyltransferase complex regulates leucine uptake through the Agp3 permease in fission yeast.

    PubMed

    Takahashi, Hidekazu; Sun, Xiaoying; Hamamoto, Makiko; Yashiroda, Yoko; Yoshida, Minoru

    2012-11-01

    Metabolic responses of unicellular organisms are mostly acute, transient, and cell-autonomous. Regulation of nutrient uptake in yeast is one such rapid response. High quality nitrogen sources such as NH(4)(+) inhibit uptake of poor nitrogen sources, such as amino acids. Both transcriptional and posttranscriptional mechanisms operate in nutrient uptake regulation; however, many components of this system remain uncharacterized in the fission yeast, Schizosaccharomyces pombe. Here, we demonstrate that the Spt-Ada-Gcn acetyltransferase (SAGA) complex modulates leucine uptake. Initially, we noticed that a branched-chain amino acid auxotroph exhibits a peculiar adaptive growth phenotype on solid minimal media containing certain nitrogen sources. In fact, the growth of many auxotrophic strains is inhibited by excess NH(4)Cl, possibly through nitrogen-mediated uptake inhibition of the corresponding nutrients. Surprisingly, DNA microarray analysis revealed that the transcriptional reprogramming during the adaptation of the branched-chain amino acid auxotroph was highly correlated with reprogramming observed in deletions of the SAGA histone acetyltransferase module genes. Deletion of gcn5(+) increased leucine uptake in the prototrophic background and rendered the leucine auxotroph resistant to NH(4)Cl. Deletion of tra1(+) caused the opposite phenotypes. The increase in leucine uptake in the gcn5Δ mutant was dependent on an amino acid permease gene, SPCC965.11c(+). The closest budding yeast homolog of this permease is a relatively nonspecific amino acid permease AGP3, which functions in poor nutrient conditions. Our analysis identified the regulation of nutrient uptake as a physiological function for the SAGA complex, providing a potential link between cellular metabolism and chromatin regulation.

  18. BASIC Tools: Structured Programming Techniques in BASIC.

    ERIC Educational Resources Information Center

    Moyer, Patrick C.

    1985-01-01

    Structured programing is an attempt to formalize the logic and structure of computer programs. Examples of structured programing techniques in BASIC are provided. Two major disadvantages of this style of programing for the personal user are noted. (JN)

  19. Amino Acid Availability and Age Affect the Leucine Stimulation of Protein Synthesis and eIF4F Formation in Muscle

    PubMed Central

    Escobar, Jeffery; Frank, Jason W.; Suryawan, Agus; Nguyen, Hanh V.; Davis, Teresa A.

    2009-01-01

    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 due to the decrease in plasma amino acids (AA). In the current study, 7- and 26-day-old pigs were fasted overnight and infused with leucine (0 or 400 µmol· kg−1· h−1) for 120 min to raise leucine within the postprandial range. The leucine was infused in the presence or absence of a replacement AA mixture (without leucine) to maintain baseline plasma AA levels. AA administration prevented the leucine-induced reduction in plasma AA in both age groups. At 7 days, leucine infusion alone increased eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) phosphorylation, decreased inactive 4E-BP1·eIF4E complex abundance, and increased active eIF4G·eIF4E complex formation in skeletal muscle; leucine infusion with replacement AA also stimulated these, as well as S6K1, rpS6, and eIF4G phosphorylation. At 26 days, leucine infusion alone increased 4E-BP1 phosphorylation and decreased the inactive 4E-BP1·eIF4E complex only; leucine with AA also stimulated these, as well as S6K1 and rpS6 phosphorylation. Muscle protein synthesis was increased in 7-day-old (+60%) and 26-day-old (+40%) pigs infused with leucine and replacement AA, but not with leucine alone. Thus, the ability of leucine to stimulate eIF4F formation and protein synthesis in skeletal muscle is dependent on AA availability and age. PMID:17878223

  20. Incorporation of l-[14C]leucine into egg proteins by liver slices from cod

    PubMed Central

    Plack, P. A.; Fraser, N. W.

    1971-01-01

    1. Liver slices from cod (Gadus morhua L.) were incubated with l-[14C]leucine and the incorporation of label into total protein, precipitated with trichloroacetic acid, and into egg proteins, precipitated with an antibody after addition of carrier egg proteins, was measured. 2. Liver slices from immature male or female cod, and from male fish with developing testes, did not incorporate significant amounts of l-[14C]leucine into egg proteins, whereas with slices from female cod with developing ovaries the rate of incorporation into egg proteins was 8% of the rate of incorporation into total protein. 3. Liver slices from immature male or female fish that had received an intramuscular injection of oestradiol benzoate (1mg/kg) 5–8 days previously incorporated l-[14C]leucine into egg proteins at about 26% of the rate of incorporation into total protein. 4. Incorporation into total protein and into egg proteins was inhibited by puromycin, and 1.2 and 0.13μg of puromycin/mg of tissue protein, respectively, gave 50% inhibition. PMID:16742749

  1. Sunlight Effects on the Osmotrophic Uptake of DMSP-Sulfur and Leucine by Polar Phytoplankton

    PubMed Central

    Ruiz-González, Clara; Galí, Martí; Sintes, Eva; Herndl, Gerhard J.; Gasol, Josep M.; Simó, Rafel

    2012-01-01

    Even though the uptake and assimilation of organic compounds by phytoplankton has been long recognized, very little is still known about its potential ecological role in natural marine communities and whether it varies depending on the light regimes the algae experience. We combined measurements of size-fractionated assimilation of trace additions of 3H-leucine and 35S-dimethylsulfoniopropionate (DMSP) with microautoradiography to assess the extent and relevance of osmoheterotrophy in summer phytoplankton assemblages from Arctic and Antarctic waters, and the role of solar radiation on it was further investigated by exposing samples to different radiation spectra. Significant assimilation of both substrates occurred in the size fraction containing most phytoplankton (>5 µm), sunlight exposure generally increasing 35S-DMSP-sulfur assimilation and decreasing 3H-leucine assimilation. Microautoradiography revealed that the capacity to take up both organic substrates seemed widespread among different polar algal phyla, particularly in pennate and centric diatoms, and photosynthetic dinoflagellates. Image analysis of the microautoradiograms showed for the first time interspecific variability in the uptakes of 35S-DMSP and 3H-leucine by phytoplankton depending on the solar spectrum. Overall, these results suggest that the role of polar phytoplankton in the utilization of labile dissolved organic matter may be significant under certain conditions and further confirm the relevance of solar radiation in regulating heterotrophy in the pelagic ocean. PMID:23029084

  2. Gentamicin and leucine inhalable powder: what about antipseudomonal activity and permeation through cystic fibrosis mucus?

    PubMed

    Russo, Paola; Stigliani, Mariateresa; Prota, Lucia; Auriemma, Giulia; Crescenzi, Carlo; Porta, Amalia; Aquino, Rita P

    2013-01-20

    The aim of this study was to evaluate the permeation properties of gentamicin (G) in a novel dry powder form for inhalation through an artificial mucus model. Moreover, since respiratory infections sustained by Pseudomonas are a major cause of sickness and death in CF patients, the susceptibility of P. aeruginosa to engineered G powders was investigated. Micronized G and G/leucine (85:15) formulations were produced by co-spray-drying, using process parameters and conditions previously set. Powders were characterized in terms of yield, drug content and aerodynamic profiles, analyzed by Andersen Cascade Impactor. Different mucus models were prepared, showing composition and viscosity similar to those of the native CF mucus. To investigate the impact on drug permeation, Franz-type vertical diffusion cells were used; the powders were applied directly on a synthetic membrane with or without the interposition of the artificial mucus layer. In buffer, gentamicin showed a diffusion controlled release; the presence of leucine reduced powder wettability and, consequently, the permeation rate. Otherwise, mucus delayed drug permeation from both G and G/leucine formulations, with a faint influence of the aminoacid. Antimicrobial tests revealed that G/leu engineered particles are able to preserve the antipseudomonal activity, even in presence of the mucus.

  3. 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

  4. Molecular Characterization of the Leucine Cluster in Buchnera sp. Strain PSY, a Primary Endosymbiont of the Aphid Pemphigus spyrothecae

    PubMed Central

    Sabater-Muñoz, Beatriz; Gómez-Valero, Laura; van Ham, Roeland C. H. J.; Silva, Francisco J.; Latorre, Amparo

    2002-01-01

    Buchnera strains from most aphid subfamilies studied to date have been found to carry the leucine gene cluster (leuA, -B, -C, and -D) on a plasmid, an organization unique among bacteria. Here, however, we demonstrate a classical chromosomal location of the cluster in Buchnera sp. strain PSY from the aphid Pemphigus spyrothecae (subfamily Pemphiginae). The genes that flank leuABCD in Buchnera sp. strain PSY appear to be adjacent in the genome of Buchnera sp. strain APS, a strain carrying a leucine plasmid. We propose that the presence of a leucine plasmid predates the diversification of symbiotic Buchnera and that the chromosomal location observed in Buchnera sp. strain PSY arose by a transfer of the leucine genes from a plasmid to the chromosome. PMID:11976137

  5. Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae

    PubMed Central

    Dziedzic, Slawomir A.; Caplan, Allan B.

    2012-01-01

    Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Δatg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Δatg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Δatg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death. PMID:22361650

  6. Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo

    PubMed Central

    Sheen, Joon-Ho; Zoncu, Roberto; Kim, Dohoon; Sabatini, David M.

    2011-01-01

    SUMMARY Autophagy is of increasing interest as a target for cancer therapy. We find that leucine deprivation causes the caspase-dependent apoptotic death of melanoma cells because it fails to appropriately activate autophagy. Hyperactivation of the RAS-MEK pathway, which is common in melanoma, prevents leucine deprivation from inhibiting mTORC1, the main repressor of autophagy under nutrient-rich conditions. In an in vivo tumor xenograft model, the combination of a leucine-free diet and an autophagy inhibitor synergistically suppresses the growth of human melanoma tumors and triggers widespread apoptosis of the cancer cells. Together, our study represents proof of principle that anti-cancer effects can be obtained with a combination of autophagy inhibition and strategies to deprive tumors of leucine. PMID:21575862

  7. 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

  8. 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

  9. 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)

  10. 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 ...

  11. 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

  12. 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.

  13. 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

  14. Whose Basics? Whose Competencies?

    ERIC Educational Resources Information Center

    Squires, Robert

    Among the advocates of the "Back to Basics" trend there seems to be little concensus as to what exactly constitutes the "basics." It is clear, however, that what most people mean by "basics" is mechanical skills, punctuation, spelling and grammar. The task of teachers of language is to foster an understanding of how language can be and has been…

  15. 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);…

  16. 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)

  17. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    PubMed

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.

  18. A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle

    PubMed Central

    Combaret, Lydie; Dardevet, Dominique; Rieu, Isabelle; Pouch, Marie-Noëlle; Béchet, Daniel; Taillandier, Daniel; Grizard, Jean; Attaix, Didier

    2005-01-01

    We tested the hypothesis that skeletal muscle ubiquitin–proteasome-dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome-dependent proteolysis in incubated muscles from 8- and 22-month-old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22-month-old rats compared with 8-month-old animals, while the rate of ubiquitination was not altered. Furthermore, the down-regulation of in vitro proteasome-dependent proteolysis that prevailed in the postprandial state in 8-month-old rats was defective in 22-month-old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine-supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome-dependent proteolysis in 22-month-old animals, by down-regulating both rates of ubiquitination and proteasome activities. In Experiment 2, we verified that dietary leucine supplementation had long-lasting effects by comparing 8- and 22-month-old rats that were fed either a leucine-supplemented diet or an alanine-supplemented diet for 10 days. The inhibited in vitro proteolysis was maintained in the postprandial state in the 22-month-old rats fed the leucine-supplemented diet. Moreover, elevated mRNA levels for ubiquitin, 14-kDa ubiquitin-conjugating enzyme E2, and C2 and X subunits of the 20S proteasome that were characteristic of aged muscle were totally suppressed in 22-month-old animals chronically fed the leucine-supplemented diet, demonstrating an in vivo effect. Thus the defective postprandial down-regulation of in vitro proteasome-dependent proteolysis in 22-month-old rats was restored in animals chronically fed a leucine-supplemented diet. PMID:16195315

  19. 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

  20. Effects of acetyl-DL-leucine in patients with cerebellar ataxia: a case series.

    PubMed

    Strupp, Michael; Teufel, Julian; Habs, Maximilian; Feuerecker, Regina; Muth, Carolin; van de Warrenburg, Bart P; Klopstock, Thomas; Feil, Katharina

    2013-10-01

    No existing medication has yet been shown to convincingly improve cerebellar ataxia. Therefore, the identification of new drugs for its symptomatic treatment is desirable. The objective of this case series was to evaluate the efficacy of treatment of cerebellar ataxia with the amino acid acetyl-DL-leucine (Tanganil). Thirteen patients (eight males, median age 51 years) with degenerative cerebellar ataxia of different etiologies (SCA1/2, ADCA, AOA, SAOA) were treated with acetyl-DL-leucine (5 g/day) without titration for 1 week. Motor function was evaluated by changes in the Scale for the Rating and Assessment of Ataxia (SARA) and in the Spinocerebellar Ataxia Functional Index (SCAFI) during treatment compared to a baseline examination. Quality of life (EuroQol-5D-3L) and side effects were also assessed. Mean total SARA decreased remarkably (p = 0.002) from a baseline of 16.1 ± 7.1 to 12.8 ± 6.8 (mean ± SD) on medication. There were also significant improvements in sub-scores for gait (p = 0.022), speech (p = 0.007), finger-chase (p = 0.042), nose-finger-test (p = 0.035), rapid-alternating-movements (p = 0.002) and heel-to-shin (p = 0.018). Furthermore, patients showed better performance in the SCAFI consisting of the 8-m-walking-time (8 MW, p = 0.003), 9-Hole-Peg-Test of the dominant hand (9HPTD, p = 0.011) and the PATA rate (p = 0.005). Quality of life increased during treatment (p = 0.003). No side effects were reported. In conclusion, acetyl-DL-leucine significantly improved ataxic symptoms without side effects and therefore showed a good risk-benefit profile. These findings need to be confirmed in placebo-controlled trials.

  1. Accumulation of D- vs. L-isomers of alanine and leucine in rat prostatic adenocarcinoma

    SciTech Connect

    Conti, P.S.; Schmall, B.; Bigler, R.E.; Zanzonico, P.B.; Kleinert, E.; Whitmore, W.F. Jr.

    1985-05-01

    It has been reported that tumor tissue may accumulate some D-amino acids preferentially over the L-isomers. In order to investigate the potential use of carbon-11 labeled amino acid isomers for in vivo tumor studies with positron emission tomography in patients, the tissue distributions of alanine and leucine, substrates for the A-type and L-type amino acid transport systems, respectively, were studied in Copenhagen rates bearing the Dunning R3327G prostatic adenocarcinoma. The authors have previously reported differences in the accumulation of A-type vs. L-type amino acids in rat prostatic adenocarcinoma and normal tissues. All compounds were labeled with C-14 in the carboxyl position with specific activities of 30.0-56.6 mCi/mmol. Higher levels of C-14 activity (Relative Concentration (RC)=dpm found per gm tissue + dpm inject per gm animal mass) were observed in tumor tissue using D-alanine (0.71) compared to L- (0.21) or DL-alanine (0.27) at 45 min post-injection. While tumor/prostate and tumor/liver ratios were above 2 for all three substrates, tumor/blood and tumor/muscle were above one for only the D-isomer. Comparisons made with D-, L-, and DL-leucine also demonstrated a higher level of RC in tumor tissue with the D-isomer (0.84) vs. the L-(0.66) and DL-leucine (0.63). In this case, however, tumor/blood, tumor/prostate, and tumor/muscle ratios were above one for all three substrates, while tumor/liver ratios were below one. These results support the observation of a preferential accumulation of D-amino acids in tumor tissue over the natural L-isomers. Observed differences in the accumulation of the isomers in normal tissues are discussed.

  2. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    PubMed Central

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  3. 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

  4. 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

  5. Crystal Structure of FadA Adhesin from Fusobacterium nucleatum Reveals a Novel Oligomerization Motif, the Leucine Chain

    SciTech Connect

    Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori; Ikegami, Akihiko; Shoham, Menachem; Han, Yiping W.

    2009-04-07

    Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 {angstrom} crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel {alpha}-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the 'leucine chain.' The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.

  6. Toxicity of fungicides to natural bacterial communities in wetland water and sediment measured using leucine incorporation and potential denitrification.

    PubMed

    Milenkovski, Susann; Bååth, Erland; Lindgren, Per-Eric; Berglund, Olof

    2010-02-01

    We assessed potential toxicity of fungicides to natural bacterial communities from a constructed wetland, located in southern Sweden, and compared the sensitivity of two endpoints indicating bacterial activity, leucine incorporation, and potential denitrification, in detecting toxicity. The effects of eight fungicides (benomyl, carbendazim, carboxin, captan, cycloheximide, fenpropimorph, propiconazole, and thiram), two bactericides (bronopol and chlortetracycline) as controls, and one reference compound (3,5-dichlorophenol), were tested in a water-sediment microcosm set-up. Leucine incorporation was measured in both the water and sediment column, while potential denitrification was measured for the entire microcosm. The bactericides and the reference compound gave sigmoid concentration-response curves for both endpoints in all but one case. The fungicides thiram, captan, and benomyl, and to a lesser extent fenpropimorph and propiconazole had quantifiable toxic effects on leucine incorporation, with EC(50) values ranging from 3 to 70 mg l(-1), while carbendazim, carboxin, and cycloheximide had little effect at the investigated concentrations. Only thiram and captan inhibited potential denitrification; the other fungicides showed no quantifiable effect. A greater toxic effect on leucine incorporation was recorded for bacterial communities associated with the water column, compared to the sediment column, for all tested compounds. Leucine incorporation was the more sensitive method for toxicity assessment of bacterial communities, and also allowed for a rapid and simple way of comparing exposure in the sediment and water column, making it an attractive standard method for community based toxicological assays in aquatic environments.

  7. Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins.

    PubMed

    Yamazaki, N; Fry, S C; Darvill, A G; Albersheim, P

    1983-07-01

    A bioassay to measure the incorporation of [(14)C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [(14)C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [(14)C]leucine uptake. The effectiveness of the wall fragments in inhibiting [(14)C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.

  8. Identification of valine/leucine/isoleucine and threonine/alanine/glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation.

    PubMed Central

    Bock-Möbius, I; Brune, M; Wittinghofer, A; Zimmermann, H; Leberman, R; Dauvergne, M T; Zimmermann, S; Brandmeier, B; Rösch, P

    1991-01-01

    Adenylate kinase from two types of Escherichia coli strains, a wild-type and a leucine-auxotrophic strain, was purified. On the one hand, growing the leucine-auxotrophic bacteria on a medium containing deuterated leucine yielded E. coli adenylate kinase with all leucine residues deuterated. On the other hand, by growing the wild-type bacteria on deuterated medium with phenylalanine, threonine and isoleucine present as protonated specimens, 80% randomly deuterated enzyme with protonated phenylalanine, threonine and isoleucine residues could be prepared. Use of these proteins enabled identification of the spin systems of these amino acid residues in the n.m.r. spectra of the protein. PMID:1991031

  9. 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.

  10. 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…

  11. 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…

  12. 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…

  13. 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)

  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 Stuff Series.

    ERIC Educational Resources Information Center

    Bain, Linda, Ed.; And Others

    1981-01-01

    The Basic Stuff project is an effort to include more general concepts such as the effects of exercise, the learning of a new skill, and psychological factors influencing performance. The Basic Stuff Series attempts to summarize for teachers appropriate concepts and teaching methods. (JN)

  16. Basic Skills for Life.

    ERIC Educational Resources Information Center

    Department for Education and Employment, London (England).

    This booklet describes existing and planned government initiatives to address the long-standing issue of low achievement in basic literacy and numeracy skills in England. The booklet begins with a brief explanation of the economic and social importance of basic functional literacy and numeracy skills. Part 1 is a discussion of English government…

  17. 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…

  18. 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…

  19. 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…

  20. L-(1-/sup 11/C)leucine: routine synthesis by enzymatic resolution

    SciTech Connect

    Barrio, J.R.; Keen, R.E.; Ropchan, J.R.; MacDonald, N.S.; Baumgartner, F.J.; Padgett, H.C.; Phelps, M.E.

    1983-06-01

    L-(1-/sup 11/C)leucine, suitable for the determination of cerebal protein synthesis rates in man using positron emission tomography, has been synthesized using a modified Bucherer-Strecker reaction sequence. The isolation of the pure L-amino acid isomer from the enantiomeric mixture, initially obtained using either an open or closed reaction vessel, was achieved using a D-amino acid oxidase/catalase enzyme complex immobilized on a Sepharose support. The O/sub 2/ required by the D-amino acid oxidase as the hydrogen acceptor was supplied by catalase. The L-(1-/sup 11/C)leucine was obtained with a radiochemical purity of >99% and with a radiochemical yield of 25%. Using a remote, semiautomated synthesis system, typical production time was 30 to 40 min after preparation of H/sup 11/CN. The use of immobilized enzymes for rapid and effective resolution of amino acid enantiomers eliminates the possibility of protein contamination and assures the production of a sterile, pyrogen-free product.

  1. Leucine is a major regulator of muscle protein synthesis in neonates.

    PubMed

    Columbus, Daniel A; Fiorotto, Marta L; Davis, Teresa A

    2015-02-01

    Approximately 10% of infants born in the United States are of low birth weight. Growth failure during the neonatal period is a common occurrence in low birth weight infants due to their inability to tolerate full feeds, concerns about advancing protein supply, and high nutrient requirements for growth. An improved understanding of the nutritional regulation of growth during this critical period of postnatal growth is vital for the development of strategies to improve lean gain. Past studies with animal models have demonstrated that muscle protein synthesis is increased substantially following a meal and that this increase is due to the postprandial rise in amino acids as well as insulin. Both amino acids and insulin act independently to stimulate protein synthesis in a mammalian target of rapamycin-dependent manner. Further studies have elucidated that leucine, in particular, and its metabolites, α-ketoisocaproic acid and β-hydroxy-β-methylbutyrate, have unique anabolic properties. Supplementation with leucine, provided either parenterally or enterally, has been shown to enhance muscle protein synthesis in neonatal pigs, making it an ideal candidate for stimulating growth of low birth weight infants.

  2. 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.

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

    PubMed

    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

  4. Structure and function of homodomain-leucine zipper (HD-Zip) proteins

    PubMed Central

    Elhiti, Mohamed

    2009-01-01

    Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors unique to plants and are encoded by more than 25 genes in Arabidopsis thaliana. Based on sequence analyses these proteins have been classified into four distinct groups: HD-Zip I–IV. HD-Zip proteins are characterized by the presence of two functional domains; a homeodomain (HD) responsible for DNA binding and a leucine zipper domain (Zip) located immediately C-terminal to the homeodomain and involved in protein-protein interaction. Despite sequence similarities HD-ZIP proteins participate in a variety of processes during plant growth and development. HD-Zip I proteins are generally involved in responses related to abiotic stress, abscisic acid (ABA), blue light, de-etiolation and embryogenesis. HD-Zip II proteins participate in light response, shade avoidance and auxin signalling. Members of the third group (HD-Zip III) control embryogenesis, leaf polarity, lateral organ initiation and meristem function. HD-Zip IV proteins play significant roles during anthocyanin accumulation, differentiation of epidermal cells, trichome formation and root development. PMID:19649178

  5. 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

  6. 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

  7. Small leucine-rich repeat proteoglycans in corneal inflammation and wound healing.

    PubMed

    Frikeche, Jihane; Maiti, George; Chakravarti, Shukti

    2016-10-01

    The small leucine rich repeat proteoglycans are major components of the cornea. Lumican, keratocan, decorin, biglycan and osteoglycin are present throughout the adult corneal stroma, and fibromodulin in the peripheral limbal area. In the cornea literature these proteoglycan have been reviewed as structural, collagen fibril-regulating proteins of the cornea. However, these proteoglycans are members of the leucine-rich-repeat superfamily, and share structural similarities with pathogen recognition toll-like receptors. Emerging studies are showing that these have a range of interactions with cell surface receptors, chemokines, growth factors and pathogen associated molecular patterns and are able to regulate host immune response, inflammation and wound healing. This review discusses what is known about their innate immune-related role directly in the cornea, and studies outside the field that find interesting links with innate immune and wound healing responses that are likely to be relevant to the ocular surface. In addition, the review discusses phenotypes of mice with targeted deletion of proteoglycan genes and genetic variants associated with human pathologies.

  8. A leucine zipper motif determines different functions in a DNA replication protein.

    PubMed Central

    Garcia de Viedma, D; Giraldo, R; Rivas, G; Fernández-Tresguerres, E; Diaz-Orejas, R

    1996-01-01

    RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors. Images PMID:8631313

  9. ValidNESs: a database of validated leucine-rich nuclear export signals.

    PubMed

    Fu, Szu-Chin; Huang, Hsuan-Cheng; Horton, Paul; Juan, Hsueh-Fen

    2013-01-01

    ValidNESs (http://validness.ym.edu.tw/) is a new database for experimentally validated leucine-rich nuclear export signal (NES)-containing proteins. The therapeutic potential of the chromosomal region maintenance 1 (CRM1)-mediated nuclear export pathway and disease relevance of its cargo proteins has gained recognition in recent years. Unfortunately, only about one-third of known CRM1 cargo proteins are accessible in a single database since the last compilation in 2003. CRM1 cargo proteins are often recognized by a classical NES (leucine-rich NES), but this signal is notoriously difficult to predict from sequence alone. Fortunately, a recently developed prediction method, NESsential, is able to identify good candidates in some cases, enabling valuable hints to be gained by in silico prediction, but until now it has not been available through a web interface. We present ValidNESs, an integrated, up-to-date database holding 221 NES-containing proteins, combined with a web interface to prediction by NESsential.

  10. Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.

    PubMed

    Jebelli, Joseph; Piers, Thomas; Pocock, Jennifer

    2015-01-01

    Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique - described here - is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis(13,14). Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer's or Parkinson's disease.

  11. 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

  12. 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

  13. Ultrafast Reorientational Dynamics of Leucine at the Air-Water Interface.

    PubMed

    Donovan, Michael A; Yimer, Yeneneh Y; Pfaendtner, Jim; Backus, Ellen H G; Bonn, Mischa; Weidner, Tobias

    2016-04-27

    Ultrafast dynamics of protein side chains are involved in important biological processes such as ligand binding, protein folding, and hydration. In addition, the dynamics of a side chain can report on local environments within proteins. While protein side chain dynamics have been probed for proteins in solution with nuclear magnetic resonance and infrared methods for decades, information about side chain dynamics at interfaces is lacking. At the same time, the dynamics and motions of side chains can be particularly important for interfacial binding and protein-driven surface manipulation. We here demonstrate that ultrafast reorientation dynamics of leucine amino acids at interfaces can be recorded in situ and in real time using polarization- and time-resolved pump-probe sum frequency generation (SFG). Combined with molecular dynamics simulations, time-resolved SFG was used to probe the reorientation of the isopropyl methyl groups of l-leucine at the air-water interface. The data show that the methyl units reorient diffusively at an in plane rate of Dφ = 0.07 rad(2)/ps and an out of plane rate of Dθ = 0.05 rad(2)/ps. PMID:27057584

  14. Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans.

    PubMed

    McEwan, Paul A; Scott, Paul G; Bishop, Paul N; Bella, Jordi

    2006-08-01

    The family of small leucine-rich repeat proteins and proteoglycans (SLRPs) contains several extracellular matrix molecules that are structurally related by a protein core composed of leucine-rich repeats (LRRs) flanked by two conserved cysteine-rich regions. The small proteoglycan decorin is the archetypal SLRP. Decorin is present in a variety of connective tissues, typically "decorating" collagen fibrils, and is involved in important biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. Several SLRPs are known to regulate collagen fibrillogenesis and there is evidence that they may share other biological functions. We have recently determined the crystal structure of the protein core of decorin, the first such determination of a member of the SLRP family. This structure has highlighted several correlations: (1) SLRPs have similar internal repeat structures; (2) SLRP molecules are far less curved than an early model of decorin based on the three-dimensional structure of ribonuclease inhibitor; (3) the N-terminal and C-terminal cysteine-rich regions are conserved capping motifs. Furthermore, the structure shows that decorin dimerizes through the concave surface of its LRR domain, which has been implicated previously in its interaction with collagen. We have established that both decorin and opticin, another SLRP, form stable dimers in solution. Conservation of residues involved in decorin dimerization suggests that the mode of dimerization for other SLRPs will be similar. Taken together these results suggest the need for reevaluation of currently accepted models of SLRP interaction with their ligands.

  15. The role of leucine in weight loss diets and glucose homeostasis.

    PubMed

    Layman, Donald K

    2003-01-01

    Debate about the optimum balance of macronutrients for adult weight maintenance or weight loss continues to expand. Often this debate centers on the relative merits or risks of carbohydrates vs. fats; however, there is increasing interest in the optimal level of dietary protein for weight loss. Diets with a reduced ratio of carbohydrates/protein are reported to be beneficial for weight loss, although diet studies appear to lack a fundamental hypothesis to support higher protein intakes. Presently, needs for dietary proteins are established by the recommended daily allowance (RDA) as the minimum level of protein necessary to maintain nitrogen balance. The RDA define the primary use of amino acids as substrates for synthesis of body proteins. There is emerging evidence that additional metabolic roles for some amino acids require plasma and intracellular levels above minimum needs for protein synthesis. The branched-chain amino acid leucine is an example of an amino acid with numerous metabolic roles that function in proportion with cellular concentration. This review provides an overview of the current understanding of metabolic roles of leucine and proposes a metabolic framework to evaluate the merits of a higher protein diet for weight loss.

  16. BASIC: Updating a Familiar Language.

    ERIC Educational Resources Information Center

    Eyman, David H.

    1988-01-01

    Discusses reasons for learning to program in BASIC, various versions of BASIC, BASIC compilers, and adherence to proposed standards. Brief reviews of six BASIC software packages are included. (12 references) (MES)

  17. Differential dose response of mTOR signaling to oral administration of leucine in skeletal muscle and liver of rats.

    PubMed

    Yoshizawa, Fumiaki; Mochizuki, Shinji; Sugahara, Kunio

    2013-01-01

    Phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1) in the rat liver increased in proportion to the amount of leucine administered, ranging from 0.169 to 1.35 g/kg of body weight. In the skeletal muscle, phosphorylation of these factors reached a plateau at 0.675 g/kg of body weight. The sensitivity of mammalian target of rapamycin (mTOR) signaling to leucine in the skeletal muscle appeared to be higher than that in the liver.

  18. 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)

  19. Basic poster discussion: summary.

    PubMed

    Widdicombe, John

    2011-06-01

    Twelve posters were presented in the section on Basic Research. They were discussed in a session chaired by Paul Davenport (Gainesville, US) and Marian Kollarik (Boston, US), with each poster presenter first briefly describing his/her poster.

  20. 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.

  1. 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- ...

  2. Wth Basic Art Materials

    ERIC Educational Resources Information Center

    Herberholz, Barbara

    2010-01-01

    In this article, the author presents a checklist of basic materials for two-dimensional activities that are necessary for an elementary-school art program. She also provides a few tips on how to use them.

  3. 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

  4. 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...

  5. Leucine stimulates PPARβ/δ-dependent mitochondrial biogenesis and oxidative metabolism with enhanced GLUT4 content and glucose uptake in myotubes.

    PubMed

    Schnuck, Jamie K; Sunderland, Kyle L; Gannon, Nicholas P; Kuennen, Matthew R; Vaughan, Roger A

    2016-01-01

    Leucine stimulates anabolic and catabolic processes in skeletal muscle, however little is known about the effects of leucine on peroxisome proliferator-activated receptor (PPAR) activity. This work characterized the effects of 24-h leucine treatment on metabolic parameters and protein expression in cultured myotubes. Leucine significantly increased PPARβ/δ expression as well as markers of mitochondrial biogenesis, leading to significantly increased mitochondrial content and oxidative metabolism in a PPARβ/δ-dependent manner. However, leucine-treated cells did not display significant alterations in uncoupling protein expression or oxygen consumed per relative mitochondrial content suggesting leucine-mediated increases in oxidative metabolism are a function of increased mitochondrial content and not altered mitochondrial efficiency. Leucine treatment also increased GLUT4 content and glucose uptake as well as PPARγ and FAS expression leading to increased total lipid content. Leucine appears to activate PPAR activity leading to increased mitochondrial biogenesis and elevated substrate oxidation, while simultaneously promoting substrate/lipid storage and protein synthesis. PMID:27345255

  6. Use of secondary isotope effects and varying pH to investigate the mode of binding of inhibitory amino aldehydes by leucine aminopeptidase

    SciTech Connect

    Andersson, L.; MacNeela, J.; Wolfenden, R.

    1985-01-15

    Ki values for leucine aldehyde, a competitive inhibitor of leucine aminopeptidase, vary with pH in a manner compatible with the binding of uncharged inhibitors. The pH dependence of kcat/Km suggests likewise that the substrate leucine p-nitroanilide is productively bound as the uncharged species. Comparison of pKa values of the model compounds aminoacetone and aminoacetal indicates that the equilibrium constant for hydration of amino aldehydes is reduced by a factor of about 2 when a proton is lost from the alpha-ammonium group near pH 8. Effects of deuterium substitution at C-1 on equilibrium binding of leucine aldehyde were determined with immobilized enzymes and inhibitors doubly labeled with radioisotopes. The observed isotope effect (KD/KH) is approximately unity, suggesting that leucine aldehyde combines with the enzyme as an oxygen adduct, not as the intact aldehyde.

  7. 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

  8. 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.

  9. 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

  10. Structure of the OsSERK2 leucine-rich repeat extracellular domain

    PubMed Central

    McAndrew, Ryan; Pruitt, Rory N.; Kamita, Shizuo G.; Pereira, Jose Henrique; Majumdar, Dipali; Hammock, Bruce D.; Adams, Paul D.; Ronald, Pamela C.

    2014-01-01

    Somatic embryogenesis receptor kinases (SERKs) are leucine-rich repeat (LRR)-containing integral membrane receptors that are involved in the regulation of development and immune responses in plants. It has recently been shown that rice SERK2 (OsSERK2) is essential for XA21-mediated resistance to the pathogen Xanthomonas oryzae pv. oryzae. OsSERK2 is also required for the BRI1-mediated, FLS2-mediated and EFR-mediated responses to brassinosteroids, flagellin and elongation factor Tu (EF-Tu), respectively. Here, crystal structures of the LRR domains of OsSERK2 and a D128N OsSERK2 mutant, expressed as hagfish variable lymphocyte receptor (VLR) fusions, are reported. These structures suggest that the aspartate mutation does not generate any significant conformational change in the protein, but instead leads to an altered interaction with partner receptors. PMID:25372696

  11. 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

  12. Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment.

    PubMed

    Brandt, Ronny; Cabedo, Marc; Xie, Yakun; Wenkel, Stephan

    2014-06-01

    The Arabidopsis (Arabidopsis thaliana L.) genome encodes for four distinct classes of homeodomain leucine-zipper (HD-ZIP) transcription factors (HD-ZIPI to HD-ZIPIV), which are all organized in multi-gene families. HD-ZIP transcription factors act as sequence-specific DNA-binding proteins that are able to control the expression level of target genes. While HD-ZIPI and HD-ZIPII proteins are mainly associated with environmental responses, HD-ZIPIII and HD-ZIPIV are primarily known to act as patterning factors. Recent studies have challenged this view. It appears that several of the different HD-ZIP families interact genetically to align both morphogenesis and environmental responses, most likely by modulating phytohormone-signaling networks.

  13. 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

  14. VUV photofragmentation of protonated leucine-enkephalin peptide dimer below ionization energy

    NASA Astrophysics Data System (ADS)

    Milosavljević, Aleksandar R.; Cerovski, Viktor Z.; Ranković, Miloš Lj.; Canon, Francis; Nahon, Laurent; Giuliani, Alexandre

    2014-03-01

    The experimental investigation of 5-8 eV photons induced dissociation of the leucine-enkephalin (Leu-Enk) peptide dimer, performed by coupling a linear ion trap with a synchrotron beamline, in combination with the tandem mass spectrometry, has been reported. The present work extends the existing results on Leu-Enk VUV-induced dissociation to lower sub-ionization photon energy range. The measured tandem mass spectra show that even at the photon energies below the ionization threshold, VUV irradiation of the protonated Leu-Enk dimer precursor can lead to a rich fragmentation pattern, including peptide sequence ions and neutral losses. The photodissociation yields of selected ionic fragments reveal the absorption bands at about 6.7-7.1 eV (185-175 nm). The experimental results have been supported by theoretical description of the [2Leu-Enk + H]+ precursors, optimized at B3LYP/6-31+G( d, p) level of DFT.

  15. 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

  16. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs.

    PubMed

    Rinaldi, Gabriel; Morales, Maria E; Alrefaei, Yousef N; Cancela, Martín; Castillo, Estela; Dalton, John P; Tort, José F; Brindley, Paul J

    2009-10-01

    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 S. 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 SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 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 >or=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. 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

  18. Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

    PubMed Central

    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 signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P 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 32P-orthophosphate. The 32P-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 (32P 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

  19. The O(2) binding pocket of myohemerythrin: role of a conserved leucine.

    PubMed

    Xiong, J; Phillips, R S; Kurtz, D M; Jin, S; Ai, J; Sanders-Loehr, J

    2000-07-25

    A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.

  20. 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 ...

  1. 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…

  2. The effects of adding leucine to pre and postexercise carbohydrate beverages on acute muscle recovery from resistance training.

    PubMed

    Stock, Matt S; Young, John C; Golding, Lawrence A; Kruskall, Laura J; Tandy, Richard D; Conway-Klaassen, Janice M; Beck, Travis W

    2010-08-01

    The present study examined the effects of adding leucine to pre and postexercise carbohydrate beverages on selected markers of muscle damage, delayed-onset muscle soreness (DOMS), and squat performance for up to 72 hours after lower-body resistance training. Seventeen resistance trained men (mean +/- SD age 22.9 +/- 2.9 years) and 3 resistance trained women (mean +/- SD age 21.6 +/- 2.6 years) performed 6 sets of squats to fatigue using 75% of the 1 repetition maximum. Each subject consumed a carbohydrate beverage 30 minutes before and immediately after exercise with or without the addition of 22.5 mgxkg (45 mgxkg total) of leucine in a randomized, double-blind fashion. Serum creatine kinase (CK), lactate dehydrogenase (LDH), and DOMS were analyzed immediately before (TIME1), 24 (TIME2), 48 (TIME3), and 72 (TIME4) hours after exercise. The subjects repeated the squat protocol at TIME4 to test recovery. No differences were observed between groups for squat performance, defined as the total number of repetitions performed during 6 sets of squats, for both TIME1 and TIME4. The addition of leucine did not significantly decrease CK and LDH activity or DOMS. These results suggested that adding leucine to carbohydrate beverages did not affect acute muscle recovery and squat performance during both initial testing and during a subsequent exercise bout 72 hours later in resistance trained subjects.

  3. 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...

  4. 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...

  5. Chronic leucine supplementation improves lipid metabolism in C57BL/6J mice fed with a high-fat/cholesterol diet

    PubMed Central

    Jiao, Jun; Han, Shu-Fen; Zhang, Wei; Xu, Jia-Ying; Tong, Xing; Yin, Xue-Bin; Yuan, Lin-Xi; Qin, Li-Qiang

    2016-01-01

    Background Leucine supplementation has been reported to improve lipid metabolism. However, lipid metabolism in adipose tissues and liver has not been extensively studied for leucine supplementation in mice fed with a high-fat/cholesterol diet (HFCD). Design C57BL/6J mice were fed a chow diet, HFCD, HFCD supplemented with 1.5% leucine (HFCD+1.5% Leu group) or 3% leucine (HFCD+3% Leu group) for 24 weeks. The body weight, peritoneal adipose weight, total cholesterol (TC), triglyceride in serum and liver, and serum adipokines were analyzed. In addition, expression levels of proteins associated with hepatic lipogenesis, adipocyte lipolysis, and white adipose tissue (WAT) browning were determined. Results Mice in the HFCD group developed obesity and deteriorated lipid metabolism. Compared with HFCD, leucine supplementation lowered weight gain and TC levels in circulation and the liver without changing energy intake. The decrease in body fat was supported by histological examination in the WAT and liver. Furthermore, serum levels of proinflammatory adipokines, such as leptin, IL-6, and tumor necrosis factor-alpha, were significantly decreased by supplemented leucine. At the protein level, leucine potently decreased the hepatic lipogenic enzymes (fatty acid synthase and acetyl-coenzyme A carboxylase) and corresponding upstream proteins. In epididymal WAT, the reduced expression levels of two major lipases by HFCD, namely phosphorylated hormone-sensitive lipase and adipose triglyceride lipase, were reversed when leucine was supplemented. Uncoupling protein 1, β3 adrenergic receptors, peroxisome proliferator-activated receptor g coactivator-1α, and fibroblast growth factor 21 were involved in the thermogenic program and WAT browning. Leucine additionally upregulated their protein expression in both WAT and interscapular brown adipose tissue. Conclusion This study demonstrated that chronic leucine supplementation reduced the body weight and improved the lipid profile of

  6. The atu and liu clusters are involved in the catabolic pathways for acyclic monoterpenes and leucine in Pseudomonas aeruginosa.

    PubMed

    Aguilar, J A; Zavala, A N; Díaz-Pérez, C; Cervantes, C; Díaz-Pérez, A L; Campos-García, J

    2006-03-01

    Evidence suggests that the Pseudomonas aeruginosa PAO1 gnyRDBHAL cluster, which is involved in acyclic isoprenoid degradation (A. L. Díaz-Pérez, N. A. Zavala-Hernández, C. Cervantes, and J. Campos-García, Appl. Environ. Microbiol. 70:5102-5110, 2004), corresponds to the liuRABCDE cluster (B. Hoschle, V. Gnau, and D. Jendrossek, Microbiology 151:3649-3656, 2005). A liu (leucine and isovalerate utilization) homolog cluster was found in the PAO1 genome and is related to the catabolism of acyclic monoterpenes of the citronellol family (AMTC); it was named the atu cluster (acyclic terpene utilization), consisting of the atuCDEF genes and lacking the hydroxymethyl-glutaryl-coenzyme A (CoA) lyase (HMG-CoA lyase) homolog. Mutagenesis of the atu and liu clusters showed that both are involved in AMTC and leucine catabolism by encoding the enzymes related to the geranyl-CoA and the 3-methylcrotonyl-CoA pathways, respectively. Intermediary metabolites of the acyclic monoterpene pathway, citronellic and geranic acids, were accumulated, and leucine degradation rates were affected in both atuF and liuD mutants. The alpha subunit of geranyl-CoA carboxylase and the alpha subunit of 3-methylcrotonyl-CoA carboxylase (alpha-MCCase), encoded by the atuF and liuD genes, respectively, were both induced by citronellol, whereas only the alpha-MCCase subunit was induced by leucine. Both citronellol and leucine also induced a LacZ transcriptional fusion at the liuB gene. The liuE gene encodes a probable hydroxy-acyl-CoA lyase (probably HMG-CoA lyase), an enzyme with bifunctional activity that is essential for both AMTC and leucine degradation. P. aeruginosa PAO1 products encoded by the liuABCD cluster showed a higher sequence similarity (77.2 to 79.5%) with the probable products of liu clusters from several Pseudomonas species than with the atuCDEF cluster from PAO1 (41.5%). Phylogenetic studies suggest that the atu cluster from P. aeruginosa could be the result of horizontal transfer

  7. 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

  8. Small-molecule inhibition of c-MYC:MAX leucine zipper formation is revealed by ion mobility mass spectrometry.

    PubMed

    Harvey, Sophie R; Porrini, Massimiliano; Stachl, Christiane; MacMillan, Derek; Zinzalla, Giovanna; Barran, Perdita E

    2012-11-28

    The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-ZipΔDT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 ± 9.3 Å(2) and one compact with a CCS of 982 ± 6.6 Å(2); similar values are observed for the two forms of c-MYC-ZipΔDT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 Å(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.

  9. Beneficial effects of l-leucine and l-valine on arrhythmias, hemodynamics and myocardial morphology in rats.

    PubMed

    Mitręga, Katarzyna; Zorniak, Michał; Varghese, Benoy; Lange, Dariusz; Nożynski, Jerzy; Porc, Maurycy; Białka, Szymon; Krzemiński, Tadeusz F

    2011-09-01

    Branched chain amino acids (BCAA) have been shown to have a general protective effect on the heart in different animal models as well as in humans. However, so far no attempt has been made to specifically elucidate their influence on arrhythmias. Our study was performed to evaluate whether an infusion of either l-leucine or l-valine in a dose of 1mgkg(-1)h(-1) 10min before a 7-min period of left anterior descending artery occlusion followed by 15min of reperfusion, had an effect on arrhythmias measured during the reperfusion phase in the ischemia- and reperfusion-induced arrhythmias model in rats in vivo. The effect of the infusion of these substances on mean arterial blood pressure was monitored throughout the experiment. Both of the tested amino acids exhibited significant antiarrhythmic properties. l-Leucine reduced the duration of ventricular fibrillation (P<0.05) and l-valine decreased the duration of ventricular fibrillation (P<0.001) and ventricular tachycardia (P<0.05). The two amino acids were generally hypotensive. l-Valine lowered blood pressure in all phases of the experiment (P<0.05) while l-leucine lowered this parameter mainly towards the end of occlusion and reperfusion (P<0.05). In addition, 30min infusion of the amino acids in the used dose did not produce any apparent adverse histological changes that were remarkably different from control. In summary, the results of our study suggest that l-leucine and l-valine in the dose that was used attenuates arrhythmias and are hypotensive in their influence. Our findings lend support to the many ongoing investigations into the benefit of the application of l-leucine and l-valine in cardiology like their addition to cardioplegic solutions.

  10. 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

  11. 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

  12. 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.

  13. The Structure and Enzyme Characteristics of a Recombinant Leucine Aminopeptidase rLap1 from Aspergillus sojae and Its Application in Debittering.

    PubMed

    Huang, Wei-Qian; Zhong, Li-Fen; Meng, Zhi-Zhong; You, Zi-Juan; Li, Jia-Zhou; Luo, Xiao-Chun

    2015-09-01

    A leucine aminopeptidase Lap1 was cloned from Aspergillus sojae GIM3.30. The truncated Lap1 without a signal peptide was over-expressed in P. pastoris, and the enzymatic characteristics of recombinant Lap1 (rLap1) were tested. The rLap1 was about 36.7 kDa with an optimal pH 8.0 and optimal temperature 50 °C for substrate Leu-p-nitroanilide and it sustained 50 % activity after 1 h incubation at 50 °C. The activity of rLap1 was significantly inhibited by EDTA, whereas Co(2+), Mn(2+), and Ca(2+) ions, but not Zn(2+) ions, restored its activity. rLap1 showed the highest activity against Arg-pNA and then Leu-, Lys-, Met-, and Phe-pNA. The 3D structure of rLap1 showed it had a conserved functional charge/dipole complex and a hydrogen bond network of Zn2-D179-S228-Q177-D229-S158 around its active center. An acidic Asp residue was found at the bottom of the substrate binding pocket, which explains its preference for basic N-terminal amino acid substrates such as Arg and Lys. rLap1 improved the degree of hydrolysis of casein and soy protein hydrolysates and also decreased their bitterness, indicating its potential utility in food production.

  14. 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;…

  15. 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.…

  16. 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,…

  17. 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)

  18. Developing Basic Electronics Aptitudes.

    ERIC Educational Resources Information Center

    Lakeshore Technical Coll., Cleveland, WI.

    This curriculum guide provides materials for basic training in electrical and electronic theory to enable participants to analyze circuits and use test equipment to verify electrical operations and to succeed in the beginning electrical and electronic courses in the Lakeshore Technical College (Wisconsin) electronics programs. The course includes…

  19. 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…

  20. 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.…

  1. River Bank Basics.

    ERIC Educational Resources Information Center

    Zar-Kessler, Arnold

    1982-01-01

    Discusses how the science faculty at one rural Massachusetts school responded to the state mandate requiring basic competency testing for all students. The approach taken (includes unit on Connecticut River) does not call for major changes in science course format, only in broadening definition of the responsibilities of science teaching.…

  2. 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…

  3. 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,…

  4. 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…

  5. 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…

  6. 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…

  7. Basic Experiments in Telecommunications.

    ERIC Educational Resources Information Center

    Andresen, S. G.

    Presented is a set of laboratory experiments developed to provide students with demonstrations and hands-on experiences with a variety of basic communications methods. These experiments may be used with students who have training in engineering, as well as those with social sciences who have no engineering background. Detailed exercises dealing…

  8. 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,…

  9. 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,…

  10. Basic Math I.

    ERIC Educational Resources Information Center

    Mercer County Community Coll., Trenton, NJ.

    This document offers instructional materials for a 60-hour course on basic math operations involving decimals, fractions, and proportions as applied in the workplace. The course, part of a workplace literacy project developed by Mercer County Community College (New Jersey) and its partners, contains the following: course outline; 17 lesson…

  11. 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.

  12. 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…

  13. 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…

  14. Flattening basic blocks.

    SciTech Connect

    Utke, J.; Mathematics and Computer Science

    2006-01-01

    The application of cross country elimination strategies requires access to the computational graph or at least subgraphs for certain scopes, e.g. a basic block. Under the presence of aliased variables the construction of these (sub)graphs encounters ambiguities. We propose an algorithm to construct ambiguity free subgraphs.

  15. Lippincott Basic Reading Program.

    ERIC Educational Resources Information Center

    Monterey Peninsula Unified School District, Monterey, CA.

    This program, included in "Effective Reading Programs...," serves 459 students in grades 1-3 at 15 elementary schools. The program employs a diagnostic-prescriptive approach to instruction in a nongraded setting through the use of the Lippincott Basic Reading program. When a child enters the program, he is introduced to a decoding process that…

  16. Basic Self-Esteem.

    ERIC Educational Resources Information Center

    Kaeter, Margaret

    1993-01-01

    Training employees in basic skills necessitates sensitivity to their self-esteem. This can be achieved if the organizational culture supports training, the program is voluntary, it uses the group's strengths, it challenges them on an adult level, it does not resemble traditional schooling, and it builds in quick success. (SK)

  17. 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. ...

  18. 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…

  19. 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…

  20. 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…

  1. 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…

  2. 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,…

  3. 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…

  4. 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…

  5. 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…

  6. 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…

  7. Sara Basic Course.

    ERIC Educational Resources Information Center

    Thayer, James E.; Maraby, Julien

    The basic plan of this course in Sara is modeled after "An Experimental Course in Hausa" (FSI 1965). The course uses short cycles consisting of mimicry followed by conversations built on the same vocabulary and syntactic pattern. The format has been condensed and altered. The course contains 95 cycles and would require approximately 50 hours to…

  8. Ketoisocaproic acid, a metabolite of leucine, suppresses insulin-stimulated glucose transport in skeletal muscle cells in a BCAT2-dependent manner.

    PubMed

    Moghei, Mahshid; Tavajohi-Fini, Pegah; Beatty, Brendan; Adegoke, Olasunkanmi A J

    2016-09-01

    Although leucine has many positive effects on metabolism in multiple tissues, elevated levels of this amino acid and the other branched-chain amino acids (BCAAs) and their metabolites are implicated in obesity and insulin resistance. While some controversies exist about the direct effect of leucine on insulin action in skeletal muscle, little is known about the direct effect of BCAA metabolites. Here, we first showed that the inhibitory effect of leucine on insulin-stimulated glucose transport in L6 myotubes was dampened when other amino acids were present, due in part to a 140% stimulation of basal glucose transport (P < 0.05). Importantly, we also showed that α-ketoisocaproic acid (KIC), an obligatory metabolite of leucine, stimulated mTORC1 signaling but suppressed insulin-stimulated glucose transport (-34%, P < 0.05) in an mTORC1-dependent manner. The effect of KIC on insulin-stimulated glucose transport was abrogated in cells depleted of branched-chain aminotransferase 2 (BCAT2), the enzyme that catalyzes the reversible transamination of KIC to leucine. We conclude that although KIC can modulate muscle glucose metabolism, this effect is likely a result of its transamination back to leucine. Therefore, limiting the availability of leucine, rather than those of its metabolites, to skeletal muscle may be more critical in the management of insulin resistance and its sequelae. PMID:27488662

  9. 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.

  10. Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Uptake Kinetics and Intracellular Isotope Dilution

    PubMed Central

    Jørgensen, Niels O. G.

    1992-01-01

    Incorporation of [3H]leucine and [3H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of 3H and 14C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [3H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [3H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 1016 and 6.6 × 1016 cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production. PMID:16348807

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Prolonged infusion of amino acids increases leucine oxidation in fetal sheep.

    PubMed

    Maliszewski, Anne M; Gadhia, Monika M; O'Meara, Meghan C; Thorn, Stephanie R; Rozance, Paul J; Brown, Laura D

    2012-06-15

    Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids. PMID:22454287

  17. 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

  18. Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis.

    PubMed

    Deng, Chuanhuan; Sun, Jiufeng; Li, Xuerong; Wang, Lexun; Hu, Xuchu; Wang, Xiaoyun; Chen, Wenjun; Lv, Xiaoli; Liang, Chi; Li, Wenfang; Huang, Yan; Li, Ran; Wu, Zhongdao; Yu, Xinbing; Xu, Jin

    2012-10-01

    Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2 μM and 10.7 μM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

  19. 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.

  20. 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.

  1. Comprehensive analysis and expression profile of the homeodomain leucine zipper IV transcription factor family in tomato.

    PubMed

    Gao, Yanna; Gao, Shenghua; Xiong, Cheng; Yu, Gang; Chang, Jiang; Ye, Zhibiao; Yang, Changxian

    2015-11-01

    Homeodomain leucine zipper IV (HD-ZIP IV) proteins are plant-specific transcription factors that play important roles in development of epidermal cell layers and cuticle formation. The functions of two HD-ZIP IV family genes, CD2 and Wo, have been well characterized in tomato (Solanum lycopersicum). CD2 and Wo are involved in cuticle biosynthesis and trichome formation, respectively. In this study, we identified 13 novel tomato HD-ZIP IV (SlHDZIV) genes. We analyzed the structures, chromosome locations, phylogeny, protein motifs, and expression profiles of these SlHDZIV genes. Gene structure analysis revealed that a module of 11 exons and 10 introns existed in the SlHDZIV genes. These genes were asymmetrically distributed on chromosomes, except on chromosome 4 and 5. Segmental duplication possibly contributed to the expansion of tomato HD-ZIP IV genes. The expression profiles of these genes revealed their broad expression pattern and high expression in young leaves and flowers. Each gene responded to more than one of different phytohormones [abscisic acid, ethephon, 4-(indolyl)-butyric acid, jasmonic acid, salicylic acid, gibberellic acid, and 6-benzylaminopurine] and four abiotic stress treatments (cold, heat, salt, and drought). This study provided significant insights into the diverse roles of SlHDZIV genes in tomato growth and development.

  2. 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

  3. 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

  4. 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.

  5. The Leucine-rich Pentatricopeptide-Repeat Containing Protein Regulates Mitochondrial Transcription

    PubMed Central

    Sondheimer, Neal; Fang, Ji-Kang; Polyak, Erzsebet; Falk, Marni; Avadhani, Narayan G.

    2010-01-01

    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

  6. 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

  7. Leucine zipper, down regulated in cancer-1 gene expression in prostate cancer

    PubMed Central

    Salemi, Michele; Barone, Nunziata; La Vignera, Sandro; Condorelli, Rosita A.; Recupero, Domenico; Galia, Antonio; Fraggetta, Filippo; Aiello, Anna Maria; Pepe, Pietro; Castiglione, Roberto; Vicari, Enzo; Calogero, Aldo E.

    2016-01-01

    Numerous genetic alterations have been implicated in the development of prostate cancer (PCa). DNA and protein microarrays have enabled the identification of genes associated with apoptosis, which is important in PCa development. Despite the molecular mechanisms are not entirely understood, inhibition of apoptosis is a critical pathophysiological factor that contributes to the onset and progression of PCa. Leucine zipper, down-regulated in cancer 1 (LDOC-1) is a known regulator of the nuclear factor (NF)-mediated pathway of apoptosis through the inhibition of NF-κB. The present study investigated the expression of the LDOC-1 gene in LNCaP, PC-3, PNT1A and PNT2 prostate cell lines by reverse transcription-quantitative polymerase chain reaction. In addition LDOC-1 protein expression in normal prostate tissues and PCa was studied by immunohistochemistry. LDOC-1 messenger RNA resulted overexpressed in LNCaP and PC-3 PCa cell lines compared with the two normal prostate cell lines PNT1A and PNT2. The results of immunohistochemistry demonstrated a positive cytoplasmic LDOC-1 staining in all PCa and normal prostate samples, whereas no nuclear staining was observed in any sample. Furthermore, a more intense signal was evidenced in PCa samples. LDOC-1 gene overexpression in PCa suggests an activity of LDOC-1 in PCa cell lines. PMID:27698860

  8. 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

  9. Temperature dependent characteristics of intestinal glycyl-L-leucine dipeptidase in boreal zone fish.

    PubMed

    Gelman, A; Kuz'mina, V; Drabkin, V; Glatman, L

    2003-10-01

    Three kinds of boreal zone fish were investigated for gastrointestinal glycyl-L-leucine (GL) dipeptide cleaving activity as a function of feeding stage and seasonal changes. The enzyme activity tested in the perch (Perca fluviatilis L.) intestine increased steadily during digestion and rapidly disappeared after completion. The temperature characteristics and the seasonal changes in dipeptide cleaving activity in pike perch (Stizostedion lucioperca L.) and bream (Abramis brama L.) were studied. In summer, the maximal activities in the pike perch and the bream were found at temperatures of 40 and 30 degrees C, respectively. In winter, the temperature of maximal activity in pike perch fell to only 30 degrees C, whereas no changes were observed in bream. The activation energies in bream and pike perch were several times lower in winter than in summer. Seasonal changes in the dipeptide cleaving activity at low temperature relative to that at the temperature of maximal activity were found. At high temperatures, the stability of the enzyme decreases in winter and increases in summer, but in the presence of a substrate the thermal stability of the enzyme increases both in winter and in summer. In our experiments, we found that in these fish, GL dipeptidase was unstable at 0 and -10 degrees C.

  10. Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223

    PubMed Central

    Shen, Yanfei; Wang, Fanghua; Lan, Dongming; Liu, Yuanyuan; Yang, Bo; Wang, Yonghua

    2011-01-01

    Experiments were carried out to investigate the effects of various factors on the activity and conformation of recombinant leucine aminopeptidase of Bacillus kaustophilus CCRC 11223 (BkLAP) and potential utilization of BkLAP in the hydrolysis of anchovy protein. Optimal temperature and pH of BkLAP were 70 °C and 8.0 in potassium-phosphate buffer, respectively, and the activity was strongly stimulated by Ni2+, followed by Mn2+ and Co2+. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of BkLAP to different extents and further induce the changes in enzymatic activity. The secondary structure of BkLAP was slightly modified by Ni2+ at the concentration of 1×10−4 M, however, significant changes on the secondary structures of the enzyme were observed when Hg2+ was added to the concentration of 1×10−4 M. The potential application of BkLAP was evaluated through combination with the commercial or endogenous enzyme to hydrolysis the anchovy protein. Results showed that combining the BkLAP with other enzymes could significantly increase the degree of hydrolysis and amino acid component of hydrolysate. In this regard, BkLAP is a potential enzyme that can be used in the protein hydrolysate industry. PMID:22174620

  11. 3-D Self-Assembling Leucine Zipper Hydrogel With Tunable Properties For Tissue Engineering

    PubMed Central

    Huang, Chun-Chieh; Ravindran, Sriram; Yin, Ziying; George, Anne

    2014-01-01

    Peptide-based engineered hydrogel scaffolds present several advantages over traditional protein or polymeric hydrogels by imparting more robust control over hydrogel properties. In this manuscript, we report the synthesis and characterization of a leucine zipper (LZ) based self assembling hydrogel for use in tissue engineering applications. Although, LZ hydrogels posses several advantages, the stability of these hydrogels has always been elusive. In this study, we have standardized the procedure for creating a stable LZ hydrogel. Pore-size was tunable by altering the peptide concentration from 7% to 12% by weight. In order to create a microenvironment for cell adhesion, the LZ polypeptide was functionalized by the incorporation of the cell attachment RGD domain. In vivo implantation of the LZ scaffolds in a mouse model showed absence of foreign body reaction to the scaffold. In vivo experiments with human marrow stem cells (HMSCs) in immunocompromised mice showed the biological property of the hydrogel to promote cell attachment, proliferation and its ability to support neovascularization. Our results show for the first time, that it is possible to generate a functional and stable LZ scaffold that can be used in vivo for tissue engineering applications. PMID:24713184

  12. Alterations in the Helicoverpa armigera Midgut Digestive Physiology after Ingestion of Pigeon Pea Inducible Leucine Aminopeptidase

    PubMed Central

    Lomate, Purushottam R.; Jadhav, Bhakti R.; Giri, Ashok P.; Hivrale, Vandana K.

    2013-01-01

    Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory. PMID:24098675

  13. Leucine zipper, down regulated in cancer-1 gene expression in prostate cancer

    PubMed Central

    Salemi, Michele; Barone, Nunziata; La Vignera, Sandro; Condorelli, Rosita A.; Recupero, Domenico; Galia, Antonio; Fraggetta, Filippo; Aiello, Anna Maria; Pepe, Pietro; Castiglione, Roberto; Vicari, Enzo; Calogero, Aldo E.

    2016-01-01

    Numerous genetic alterations have been implicated in the development of prostate cancer (PCa). DNA and protein microarrays have enabled the identification of genes associated with apoptosis, which is important in PCa development. Despite the molecular mechanisms are not entirely understood, inhibition of apoptosis is a critical pathophysiological factor that contributes to the onset and progression of PCa. Leucine zipper, down-regulated in cancer 1 (LDOC-1) is a known regulator of the nuclear factor (NF)-mediated pathway of apoptosis through the inhibition of NF-κB. The present study investigated the expression of the LDOC-1 gene in LNCaP, PC-3, PNT1A and PNT2 prostate cell lines by reverse transcription-quantitative polymerase chain reaction. In addition LDOC-1 protein expression in normal prostate tissues and PCa was studied by immunohistochemistry. LDOC-1 messenger RNA resulted overexpressed in LNCaP and PC-3 PCa cell lines compared with the two normal prostate cell lines PNT1A and PNT2. The results of immunohistochemistry demonstrated a positive cytoplasmic LDOC-1 staining in all PCa and normal prostate samples, whereas no nuclear staining was observed in any sample. Furthermore, a more intense signal was evidenced in PCa samples. LDOC-1 gene overexpression in PCa suggests an activity of LDOC-1 in PCa cell lines.

  14. 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

  15. 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

  16. Physiologically relevant factors influence tau phosphorylation by leucine-rich repeat kinase 2.

    PubMed

    Hamm, Matthew; Bailey, Rachel; Shaw, Gerry; Yen, Shu-Hui; Lewis, Jada; Giasson, Benoit I

    2015-10-01

    Hyperphosphorylation and aggregation of tau are observed in multiple neurodegenerative diseases termed tauopathies. Tau has also been implicated in the pathogenesis of Parkinson's disease (PD) and parkinsonisms. Some PD patients with mutations in the leucine-rich repeat kinase 2 (LRRK2) gene exhibit tau pathology. Mutations in LRRK2 are a major risk factor for PD, but LRRK2 protein function remains unclear. The most common mutation, G2019S, is located in the kinase domain of LRRK2 and enhances kinase activity in vitro. This suggests that the kinase activity of LRRK2 may underlie its cellular toxicity. Recently, in vitro studies have suggested a direct interaction between tubulin-bound tau and LRRK2 that results in tau phosphorylation at one identified site. Here we present data suggesting that microtubules (MTs) enhance LRRK2-mediated tau phosphorylation at three different epitopes. We also explore the effect of divalent cations as catalytic cofactors for G2019S LRRK2-mediated tau phosphorylation and show that manganese does not support kinase activity but inhibits the efficient ability of magnesium to catalyze LRRK2-mediated phosphorylation of tau. These results suggest that cofactors such as MTs and cations in the cellular milieu have an important impact on LRRK2-tau interactions and resultant tau phosphorylation.

  17. 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

  18. Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase.

    PubMed

    Dounce, A L; Allen, P Z

    1987-08-15

    The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.

  19. 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

  20. Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP.

    PubMed

    Gómez-Suaga, Patricia; Luzón-Toro, Berta; Churamani, Dev; Zhang, Ling; Bloor-Young, Duncan; Patel, Sandip; Woodman, Philip G; Churchill, Grant C; Hilfiker, Sabine

    2012-02-01

    Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson's disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets. PMID:22012985

  1. Sputum Leucine-Rich Alpha-2 Glycoprotein as a Marker of Airway Inflammation in Asthma

    PubMed Central

    Honda, Hiromi; Fujimoto, Minoru; Miyamoto, Shintaro; Ishikawa, Nobuhisa; Serada, Satoshi; Hattori, Noboru; Nomura, Shintaro; Kohno, Nobuoki; Yokoyama, Akihito; Naka, Tetsuji

    2016-01-01

    Background Asthma is a chronic inflammatory disease of airways, but an ideal biomarker that accurately reflects ongoing airway inflammation has not yet been established. The aim of this study was to examine the potential of sputum leucine-rich alpha-2 glycoprotein (LRG) as a new biomarker for airway inflammation in asthma. Methods We obtained induced sputum samples from patients with asthma (N = 64) and healthy volunteers (N = 22) and measured LRG concentration by sandwich enzyme-linked immunosorbent assay (ELISA). Ovalbumin (OVA)-induced asthma model mice were used to investigate the mechanism of LRG production during airway inflammation. The LRG concentrations in the bronchoalveolar lavage fluid (BALF) obtained from mice were determined by ELISA and mouse lung sections were stained with anti-LRG antibody and periodic acid-Schiff (PAS) reagent. Results Sputum LRG concentrations were significantly higher in patients with asthma than in healthy volunteers (p = 0.00686). Consistent with patients’ data, BALF LRG levels in asthma model mice were significantly higher than in control mice (p = 0.00013). Immunohistochemistry of lung sections from asthma model mice revealed that LRG was intensely expressed in a subpopulation of bronchial epithelial cells, which corresponded with PAS-positive mucus producing cells. Conclusion These findings suggest that sputum LRG is a promising biomarker of local inflammation in asthma. PMID:27611322

  2. [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.

  3. Two-state conformational equilibrium in the Par-4 leucine zipper domain.

    PubMed

    Schwalbe, Martin; Dutta, Kaushik; Libich, David S; Venugopal, Hariprasad; Claridge, Jolyon K; Gell, David A; Mackay, Joel P; Edwards, Patrick J B; Pascal, Steven M

    2010-08-15

    Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-suppressive protein. A highly conserved heptad repeat sequence at the Par-4 C-terminus suggests the presence of a leucine zipper (LZ). This C-terminal region is essential for Par-4 self-association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par-4 at neutral pH. Further, we have investigated the properties of the Par-4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment-dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium.

  4. 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.

  5. Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

    PubMed

    Lomate, Purushottam R; Jadhav, Bhakti R; Giri, Ashok P; Hivrale, Vandana K

    2013-01-01

    Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

  6. mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1

    PubMed Central

    Manzoni, Claudia; Mamais, Adamantios; Roosen, Dorien A.; Dihanich, Sybille; Soutar, Marc P. M.; Plun-Favreau, Helene; Bandopadhyay, Rina; Hardy, John; Tooze, Sharon A.; Cookson, Mark R.; Lewis, Patrick A.

    2016-01-01

    Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase. PMID:27731364

  7. Controlled localization of functionally active proteins to inclusion bodies using leucine zippers.

    PubMed

    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.

  8. 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

  9. Production of leucine amino peptidase in lab scale bioreactors using Streptomyces gedanensis.

    PubMed

    Rahulan, Raji; Dhar, Kiran S; Madhavan Nampoothiri, K; Pandey, Ashok

    2011-09-01

    Studies were conducted on the production of leucine amino peptidase (LAP) by Streptomyces gedanensis to ascertain the performance of the process in shake flask, parallel fermenter and 5-L fermenter utilizing soy bean meal as the carbon source. Experiments were conducted to analyze the effects of aeration and agitation rate on cell growth and LAP production. The results unveiled that an agitation rate of 300 rpm, 50% dissolved oxygen (DO) upholding and 0.15 vvm strategies were the optimal for the enzyme production, yielding 22.72 ± 0.11 IU/mL LAP in parallel fermenter which was comparable to flask level (24.65 ± 0.12 IU/mL LAP) fermentation. Further scale-up, in 5-L fermenter showed 50% DO and 1 vvm aeration rate was the best, producing optimum and the production was 20.09 ± 0.06 IU/mL LAP. The information obtained could be useful to design a strategy to improve a large-scale bioreactor cultivation of cells and production of LAP. PMID:21733679

  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. Cellular pattern formation by SCRAMBLED, a leucine-rich repeat receptor-like kinase in Arabidopsis.

    PubMed

    Kwak, Su-Hwan; Schiefelbein, John

    2008-02-01

    The appropriate specification of distinct cell types is important for generating the proper tissues and bodies of multicellular organisms. In the root epidermis of Arabidopsis, cell fate determination is accomplished by a transcriptional regulatory circuit that is influenced by positional signaling. A leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), has been shown to be responsible for the position-dependent aspect of this epidermal pattern. In a recent report, we find that SCM affects the transcriptional regulatory network by down-regulating the WEREWOLF (WER) MYB gene expression in a set of epidermal cells located in a specific position. We also find that SCM and the SCM-related SRF1 and SRF3 are not required for embryonic epidermal patterning and that SRF1 and SRF3 do not act redundantly with SCM. This suggests that distinct positional signaling mechanisms exist for embryonic and post-embryonic epidermal patterning. In this addendum, we discuss the implications of our recent findings and extend our working model for epidermal cell pattering.

  12. A glucocorticoid-induced leucine-zipper protein, GILZ, inhibits adipogenesis of mesenchymal cells

    PubMed Central

    Shi, Xingming; Shi, Weibin; Li, Qingnan; Song, Buer; Wan, Mei; Bai, Shuting; Cao, Xu

    2003-01-01

    Mesenchymal stem cells have the potential to differentiate into various cell lineages, including adipocytes and osteoblasts. The induction of adipocyte differentiation by glucocorticoids (GCs) not only causes the accumulation of fat cells in bone marrow, but also depletes the supply of osteoblasts for new bone formation, thus leading to osteoporosis. We have shown that a GC-induced leucine-zipper protein (GILZ) antagonizes adipocyte differentiation. GILZ binds to a tandem repeat of CCAAT/enhancer-binding protein (C/EBP) binding sites in the promoter of the gene encoding peroxisome-proliferator-activated receptor-γ2 (PPAR-γ2), and inhibits its transcription as a sequence-specific transcriptional repressor. We have also shown that ectopic expression of GILZ blocks GC-induced adipocyte differentiation. Furthermore, adipogenic marker genes (for example, those encoding PPAR-γ2, C/EBP-α, lipoprotein lipase and adipsin) are also inhibited by GILZ. Our results reveal a novel GC antagonistic mechanism that has potential therapeutic applications for the inhibition of GC-induced adipocyte differentiation. PMID:12671681

  13. 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

  14. A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins.

    PubMed

    Tugarinov, Vitali; Venditti, Vincenzo; Marius Clore, G

    2014-01-01

    A methyl-detected 'out-and-back' NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile(δ1), Leu(δ) and Val(γ) (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of (13)C(α), (13)Cβ and (13)CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.

  15. Non-empirical analysis of the nature of the inhibitor active-site interactions in leucine aminopeptidase

    NASA Astrophysics Data System (ADS)

    Grembecka, J.; K ȩdzierski, P.; Sokalski, W. A.

    1999-11-01

    Non-empirical analysis of the physical nature of the intermolecular interactions between several leucine aminopeptidase inhibitors and various constituents of the enzyme active site has been performed using a direct version of the hybrid variation-perturbation decomposition of SCF and MP2 interaction energies. The interaction energy terms obtained at different theory levels have been correlated with experimentally measured activities of the inhibitors, indicating that the more advanced the quantum-chemical method and, the larger the active-site model, the better is the correlation between calculated and measured binding energies. The electrostatic multipole term constitutes the dominant contribution in the total interaction energy, whereas Zn 2+488 and Lys +262 enzyme residues play the crucial role in the binding of these inhibitors by leucine aminopeptidase.

  16. 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.

  17. 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.

  18. Basic genetics for dermatologists.

    PubMed

    Kumaran, Muthu Sendhil; De, Dipankar

    2013-01-01

    During the past few decades, advances in the field of molecular genetics have enriched us in understanding the pathogenesis of diseases, their identification, and appropriate therapeutic interventions. In the last 20 years, genetic basis of more than 350 monogenic skin diseases have been elucidated and is counting. The widespread use of molecular genetics as a tool in diagnosis is not practiced routinely due to genetic heterogenicity, limited access and low sensitivity. In this review, we have presented the very basics of genetics so as to enable dermatologists to have working understanding of medical genetics.

  19. 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

  20. 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.

  1. 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.

  2. Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity.

    PubMed

    Yamamoto, Taichi; Ryan, Robert O

    2006-09-01

    The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.

  3. Crystal and molecular structure of bestatin and its implications regarding substrate binding to the active site of leucine aminopeptidase

    SciTech Connect

    Ricci, J.S. Jr.; Bousvaros, A.; Taylor, A.

    1982-07-30

    The X-ray crystal structure of bestatin, ((2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl)-L-leucine (C/sub 16/H/sub 24/N/sub 2/O/sub 4/), has been determined. Four molecules of bestatin crystallize with four molecules of 2-methyl-2,4-pentanediol (MPD) and eight molecules of water in the space group P2/sub 1/2/sub 1/2/sub 1/. Unit cell dimensions are a = 6.653 (1), b = 15.150 (3), and c = 27.309 (4) angstrom. The final R was 8.5%, based on 2871 independent structure amplitudes. The MPD was found to be disordered. In addition to the usual functional groups needed for binding to leucine aminopeptidase, bestatin includes a tetrahedral carbon, C(8), as might be found in the putative transition-state intermediate. The structure indicates that the nonpolar side chains are oppositely disposed and separated by approx.10 angstrom. The peptide bond is trans. There is no H bonding between OH on C(8) and the adjacent carbonyl. These data suggest possible modes of binding of this transition-state analogue to leucine aminopeptidase.

  4. (19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.

    PubMed

    Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A

    2003-10-01

    The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Hemoglobin Abraham Lincoln, β32 (B14) Leucine → Proline AN UNSTABLE VARIANT PRODUCING SEVERE HEMOLYTIC DISEASE

    PubMed Central

    Honig, George R.; Green, David; Shamsuddin, Mir; Vida, Loyda N.; Mason, R. George; Gnarra, David J.; Maurer, Helen S.

    1973-01-01

    An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t½ using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[14C]leucine indicated a t½ of 7.2 days. When stroma-free hemolysates were heated at 50°C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37°C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant β-chain by precipitation with p-hydroxymercuribenzoate. Purification of the β-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for β32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at β31 to impair heme stabilization. Images PMID:4352462

  10. 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

  11. Hemoglobin Abraham Lincoln, beta32 (B14) leucine leads to proline. An unstable variant producing severe hemolytic disease.

    PubMed

    Honig, G R; Green, D; Shamsuddin, M; Vida, L N; Mason, R G; Gnarra, D J; Maurer, H S

    1973-07-01

    An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t(1/2) using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[(14)C]leucine indicated a t(1/2) of 7.2 days. When stroma-free hemolysates were heated at 50 degrees C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37 degrees C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant beta-chain by precipitation with p-hydroxymercuribenzoate. Purification of the beta-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for beta32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at beta31 to impair heme stabilization.

  12. 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.

  13. 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.

  14. Maternal post-absorptive leucine kinetics during late pregnancy in US women with HIV taking antiretroviral therapy: a cross-sectional pilot study

    PubMed Central

    Cade, W. Todd; Singh, Gautam K.; Holland, Mark R.; Reeds, Dominic N.; Overton, E. Turner; Cibulka, Nancy; Bahow, Karen; Presti, Rachel; Stephens, Andrea; Cahill, Alison G.

    2015-01-01

    Background Despite the success of combination antiretroviral therapy (cART) for the prevention of mother to child transmission of HIV, infants exposed to cART in utero frequently are born smaller and have mild cardiac abnormalities. The mechanisms responsible for lower birth weight and cardiac abnormalities in children exposed to cART are unclear but could be related to dysregulation of maternal amino acid metabolism during pregnancy. Previous data in HIV(−) women have shown a relationship between abnormal maternal protein metabolism during pregnancy and low infant birth weight and animal data demonstrate a relationship between altered maternal protein metabolism and increased risk for offspring cardiovascular abnormalities. Objective The objectives of this study were to: characterize post-absorptive maternal leucine kinetics during late pregnancy and examine the relationships between maternal leucine kinetics and offspring birth weight and cardiac function. Design Post-absorptive maternal leucine kinetics (evaluated by using stable isotope tracer methodology) in 16 HIV(+) women receiving cART and 14 HIV(−) US women during the 3rd trimester of pregnancy were compared. Relationships between post-absorptive maternal leucine kinetics, cardiac function (echocardiography) and birth weight were statistically examined. Results Maternal plasma leucine concentration (HIV(−): 82.8 ± 10.7 vs. HIV(+): 72.3 ± 13.5 μM, p=0.06) and leucine oxidation rate (HIV(−): 6.1 ± 1.6 vs. HIV(+): 4.9 ± 1.8 μmol/kgBW/min, p=0.03) were lower in HIV+ women compared to controls. Total leucine turnover rate, non-oxidative leucine disposal rate and post-absorptive maternal glucose and palmitate kinetics did not differ between groups. Left ventricular fractional shortening tended to be lower in children born to HIV(+) compared to controls (HIV(−): 42 ± 1 vs. HIV+: 36 ± 5 %, p=0.08) and associated with lower maternal plasma leucine concentration (r= 0.43, p=0.08). Conclusions

  15. 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

    Bandyopadhyay, Sushobhana; Dey, Isha; Suresh, Megalakshmi; Sundaram, Geetanjali

    2014-06-01

    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 G(2)/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 G(2)/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 G(2)/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 G(2)/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 G(2)/M transition.

  16. 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.

  17. 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

  18. Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

    NASA Astrophysics Data System (ADS)

    Talarmin, A.; van Wambeke, F.; Catala, P.; Courties, C.; Lebaron, P.

    2010-08-01

    Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31) pmol leu l-1 h-1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok) were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA) and the high nucleic acid content cells (HNA), with high size and low size (HNA-hs and HNA-ls, respectively). LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31). Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM), HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9-54.3×10-21 mol leu cell-1 h-1). At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5-55×10-21 mol leu cell-1 h-1). We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of certain photosynthetic prokaryotes and the non-negligible activity of LNA cells all over Mediterranean were reinforced.

  19. 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.

  20. 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.

  1. BasicODT

    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

  2. Basic obstetric pharmacology.

    PubMed

    Zhao, Yang; Hebert, Mary F; Venkataramanan, Raman

    2014-12-01

    Pregnancy is associated with a variety of physiological changes that can alter the pharmacokinetics and pharmacodynamics of several drugs. However, limited data exists on the pharmacokinetics and pharmacodynamics of the majority of the medications used in pregnancy. In this article, we first describe basic concepts (drug absorption, bioavailability, distribution, metabolism, elimination, and transport) in pharmacokinetics. Then, we discuss several physiological changes that occur during pregnancy that theoretically affect absorption, distribution, metabolism, and elimination. Further, we provide a brief review of the literature on the clinical pharmacokinetic studies performed in pregnant women in recent years. In general, pregnancy increases the clearance of several drugs and correspondingly decreases drug exposure during pregnancy. Based on current drug exposure measurements during pregnancy, alterations in the dose or dosing regimen of certain drugs are essential during pregnancy. More pharmacological studies in pregnant women are needed to optimize drug therapy in pregnancy.

  3. Atomic Basic Blocks

    NASA Astrophysics Data System (ADS)

    Scheler, Fabian; Mitzlaff, Martin; Schröder-Preikschat, Wolfgang

    Die Entscheidung, einen zeit- bzw. ereignisgesteuerten Ansatz für ein Echtzeitsystem zu verwenden, ist schwierig und sehr weitreichend. Weitreichend vor allem deshalb, weil diese beiden Ansätze mit äußerst unterschiedlichen Kontrollflussabstraktionen verknüpft sind, die eine spätere Migration zum anderen Paradigma sehr schwer oder gar unmöglich machen. Wir schlagen daher die Verwendung einer Zwischendarstellung vor, die unabhängig von der jeweils verwendeten Kontrollflussabstraktion ist. Für diesen Zweck verwenden wir auf Basisblöcken basierende Atomic Basic Blocks (ABB) und bauen darauf ein Werkzeug, den Real-Time Systems Compiler (RTSC) auf, der die Migration zwischen zeit- und ereignisgesteuerten Systemen unterstützt.

  4. L-leucine, L-methionine, and L-phenylalanine share a Na(+)/K (+)-dependent amino acid transporter in shrimp hepatopancreas.

    PubMed

    Duka, Ada; Ahearn, Gregory A

    2013-08-01

    Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J

  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. Small leucine-rich proteoglycans in atherosclerotic lesions: novel targets of chronic statin treatment?

    PubMed Central

    Marzoll, Andrea; Melchior-Becker, Ariane; Cipollone, Francesco; Fischer, Jens W

    2011-01-01

    Abstract Small leucine-rich proteoglycans (SLRPs), such as decorin and biglycan, regulate the assembly and turnover of collagenous matrix. The aim of the study was to analyse the effect of chronic rosuvastatin treatment on decorin, biglycan and the collagen matrix in ApoE-deficient mice. Twenty-week-old male ApoE-deficient mice received normal chow or 20 mg rosuvastatin/kg × day for 32 weeks. Subsequently, matrix composition was analysed by histochemistry and immunostaining at the aortic root and in innominate arteries of ApoE deficient mice as well as in human carotid endarterectomy specimens. Immunoblotting of proteoglycans was performed from aortic extracts of ApoE-deficient mice. Immunohistochemistry and immunoblotting revealed strongly increased decorin and biglycan deposition in atherosclerotic plaques at the aortic root and in innominate arteries. In contrast, versican and perlecan expression was not changed by rosu-vastatin. Furthermore, matrix metalloproteinase 2 and gelatinolytic activity were decreased in response to rosuvastatin and a condensed collagen-rich matrix was formed. In carotid endarterectomy specimens of statin-treated patients increased decorin and biglycan accumulation was detected as well. Drug treatment did not change low-density lipoprotein (LDL) plasma levels in ApoE-deficient mice and did not significantly affect lipid retention at the aortic root level as demonstrated by oil-red O staining and immunohistochemistry of LDL. Long-term treatment with rosuvastatin caused pronounced remodelling of atherosclerotic plaque matrix characterized specifically by enrichment with SLRPs and formation of a condensed collagen matrix. Therefore, decorin and biglycan might represent novel targets of statin treatment that contribute to a stable plaque phenotype. PMID:20015203

  7. Glutamine and Leucine Provide Enhanced Protective Immunity Against Mucosal Infection with Herpes Simplex Virus Type 1

    PubMed Central

    Uyangaa, Erdenebileg; Lee, Hern-Ku

    2012-01-01

    Besides their role as building blocks of protein, there are growing evidences that some amino acids have roles in regulating key metabolic pathways that are necessary for maintenance, growth, reproduction, and immunity. Here, we evaluated the modulatory functions of several amino acids in protective immunity against mucosal infection of herpes simplex virus type 1 (HSV-1). We found that glutamine (Gln) and leucine (Leu) showed enhanced protective immunity to HSV-1 mucosal infection when two administration of Gln and single administration of Leu per day, but not when administered in combinations. Ameliorated clinical signs of HSV-1 challenged mice by the intraperitoneal administration of Gln and Leu were closely associated with viral burden and IFN-γ production in the vaginal tract at 2 and 4 days post-infection. In addition, the enhanced production of vaginal IFN-γ appeared to be caused by NK and HSV-1 antigen-specific Th1-type CD4+ T cells recruited into vaginal tract of mice treated with Gln and Leu, which indicates that IFN-γ, produced by NK and Th1-type CD4+ T cells, may be critical to control the outcome of diseases caused by HSV-1 mucosal infection. Collectively, our results indicate that intraperitoneal administration of Gln and Leu following HSV-1 mucosal infection could provide beneficial effects for the modulation of protective immunity, but dosage and frequency of administration should be carefully considered, because higher frequency and overdose of Gln and Leu, or their combined treatment, showed detrimental effects to protective immunity. PMID:23213313

  8. Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

    PubMed Central

    Tibbles, B. J.; Harris, J. M.

    1996-01-01

    Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41. PMID:16535246

  9. Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin.

    PubMed

    Min, T; Ergenekan, C E; Eidsness, M K; Ichiye, T; Kang, C

    2001-03-01

    Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.

  10. The small leucine-rich proteoglycan BGN accumulates in CADASIL and binds to NOTCH3

    PubMed Central

    Zhang, Xiaojie; Lee, Soo Jung; Young, Marian F.; Wang, Michael M.

    2015-01-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited form of cerebral small vessel disease caused by mutations in conserved residues of NOTCH3. Affected arteries of CADASIL feature fibrosis and accumulation of NOTCH3. A variety of collagen subtypes (types I, III, IV, and VI) have been identified in fibrotic CADASIL vessels. Biglycan (BGN) and decorin (DCN), are Class I members of the small leucine-rich proteoglycan (SLRP) family that regulate collagen fibril size. Because DCN has been shown to deposit in arteries in cerebral small vessel disease, we tested whether BGN accumulates in arteries of CADASIL brains. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain. BGN protein was localized to all three layers of arteries (intima, media, and adventitia). Substantially more immunoreactivity was observed in CADASIL brains compared to controls. Immunoblotting of brain lysates showed a 4-fold increase in CADASIL brains (compared to controls). Messenger RNA encoding BGN was also increased in CADASIL and was localized by in situ hybridization to all three vascular layers in CADASIL. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. In conclusion, BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen. PMID:25578324

  11. Effect of leucine 13-motilin (KW5139) on early gastric stasis after pylorus-preserving pancreatoduodenectomy.

    PubMed Central

    Matsunaga, H; Tanaka, M; Naritomi, G; Yokohata, K; Yamaguchi, K; Chijiwa, K

    1998-01-01

    OBJECTIVE: To test a hypothesis that exogenously administered motilin would improve early gastric stasis after pylorus-preserving pancreatoduodenectomy (PPPD). SUMMARY BACKGROUND DATA: Prolonged gastric stasis is a frequent complication after PPPD. We demonstrated that this might at least in part be attributable to delayed recovery of phase III activity of the gastric migrating motor complex due to low concentrations of plasma motilin caused by resection of the duodenum. METHODS: Ten patients with a mean age of 54 years (range, 33-70) who underwent PPPD were studied. An assembly of manometric tubes was placed in the gastric antrum and jejunum (neoduodenum) at surgery. A gastrostomy tube was added for drainage and volume measurements of the gastric juice. After baseline recording, saline as a placebo was given intravenously on day 14 and 0.5 microg/kg of KW5139 (leucine-13 motilin) was given on days 17 and 18 every 2 hours, 6 times a day. The daily volume of gastric juice output and a gastric motility index were measured. RESULTS: The mean period until the first appearance of phase III activity in the stomach was 41 +/- 2 days. The injection of saline did not change the gastric motility index (7.3 +/- 1.1 to 7.1 +/- 1.3 mmHg; p = 0.72). In contrast, motilin resulted in a significant increase in the gastric motility index (7.5 +/- 1.0 to 17.7 +/- 2.0 mmHg; p < 0.001). The saline injection produced no change in the daily gastric juice output (1175 +/- 140 to 1393 +/- 193 mL; p = 0.09). Motilin significantly decreased the gastric juice output (1387 +/- 157 to 934 +/- 142 mL; p = 0.01). CONCLUSIONS: These data indicate that KW5139 is a safe and effective prokinetic drug for the treatment of early gastric stasis after PPPD. PMID:9563538

  12. The bHLH Transcription Factor bHLH104 Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis

    PubMed Central

    Zhang, Jie; Liu, Bing; Li, Mengshu; Feng, Dongru; Jin, Honglei; Wang, Peng; Liu, Jun; Xiong, Feng; Wang, Jinfa; Wang, Hong-Bin

    2015-01-01

    Iron (Fe) is an indispensable micronutrient for plant growth and development. The regulation of Fe homeostasis in plants is complex and involves a number of transcription factors. Here, we demonstrate that a basic helix-loop-helix (bHLH) transcription factor, bHLH104, belonging to the IVc subgroup of bHLH family, acts as a key component positively regulating Fe deficiency responses. Knockout of bHLH104 in Arabidopsis thaliana greatly reduced tolerance to Fe deficiency, whereas overexpression of bHLH104 had the opposite effect and led to accumulation of excess Fe in soil-grown conditions. The activation of Fe deficiency-inducible genes was substantially suppressed by loss of bHLH104. Further investigation showed that bHLH104 interacted with another IVc subgroup bHLH protein, IAA-LEUCINE RESISTANT3 (ILR3), which also plays an important role in Fe homeostasis. Moreover, bHLH104 and ILR3 could bind directly to the promoters of Ib subgroup bHLH genes and POPEYE (PYE) functioning in the regulation of Fe deficiency responses. Interestingly, genetic analysis showed that loss of bHLH104 could decrease the tolerance to Fe deficiency conferred by the lesion of BRUTUS, which encodes an E3 ligase and interacts with bHLH104. Collectively, our data support that bHLH104 and ILR3 play pivotal roles in the regulation of Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE expression. PMID:25794933

  13. Basic concepts of epigenetics

    PubMed Central

    Mazzio, Elizabeth A

    2012-01-01

    Through epigenetic modifications, specific long-term phenotypic consequences can arise from environmental influence on slowly evolving genomic DNA. Heritable epigenetic information regulates nucleosomal arrangement around DNA and determines patterns of gene silencing or active transcription. One of the greatest challenges in the study of epigenetics as it relates to disease is the enormous diversity of proteins, histone modifications and DNA methylation patterns associated with each unique maladaptive phenotype. This is further complicated by a limitless combination of environmental cues that could alter the epigenome of specific cell types, tissues, organs and systems. In addition, complexities arise from the interpretation of studies describing analogous but not identical processes in flies, plants, worms, yeast, ciliated protozoans, tumor cells and mammals. This review integrates fundamental basic concepts of epigenetics with specific focus on how the epigenetic machinery interacts and operates in continuity to silence or activate gene expression. Topics covered include the connection between DNA methylation, methyl-CpG-binding proteins, transcriptional repression complexes, histone residues, histone modifications that mediate gene repression or relaxation, histone core variant stability, H1 histone linker flexibility, FACT complex, nucleosomal remodeling complexes, HP1 and nuclear lamins. PMID:22395460

  14. 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

  15. [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

  16. A new method to measure muscle protein synthesis in humans by endogenously introduced d9-leucine and using blood for precursor enrichment determination.

    PubMed

    Tran, Lee; Masters, Haley; Roust, Lori R; Katsanos, Christos S

    2015-08-01

    Enrichment from the easily accessible blood amino acid pool is commonly used as precursor enrichment to calculate rates of muscle protein fractional synthesis in relevant human studies in lieu of the less accessible muscle fluid amino acid pool. However, the accuracy of this approach depends largely on the extent to which there is low discrepancy in free amino acid enrichment between blood and muscle. Steady-state gradient (i.e., ratio) of amino acid enrichment between blood and muscle fluid in the basal state and in response to amino acid infusion were determined in five healthy subjects, and in association with two separate tracers: d9-leucine, introduced endogenously by the metabolism of d10-leucine (i.e., l-[2,3,3,4,5,5,5,6,6,6-(2)H10]leucine) infused in blood, and (13)C6-phenylalanine introduced/infused in blood. The blood-to-muscle fluid amino acid enrichment ratio was lower (P < 0.05) for d9-leucine compared to (13)C6-phenylalanine both before (1.5 ± 0.1 vs. 2.5 ± 0.1) and during (1.1 ± 0.1 vs. 1.2 ± 0.1) amino acid infusion. Importantly, the decrease in this ratio in association with the amino acid infusion was considerably less for the d9-leucine than the (13)C6-phenylalanine (-0.38 ± 0.03 vs. -1.29 ± 0.07; P < 0.05). In conclusion, blood d9-leucine enrichment introduced endogenously by intravenous infusion of d10-leucine provides a closer estimate of the muscle fluid amino acid enrichment, and its associated changes, than blood phenylalanine enrichment to calculate rates of muscle protein synthesis in humans. PMID:26243214

  17. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) by recombinant Escherichia coli expressing leucine metabolism-related enzymes derived from Clostridium difficile.

    PubMed

    Saika, Azusa; Watanabe, Yoriko; Sudesh, Kumar; Tsuge, Takeharu

    2014-06-01

    An obligate anaerobic bacterium Clostridium difficile has a unique metabolic pathway to convert leucine to 4-methylvalerate, in which 4-methyl-2-pentenoyl-CoA (4M2PE-CoA) is an intermediate of this pathway. 4M2PE-CoA is also able to be converted to 3-hydroxy-4-methylvalerate (3H4MV), a branched side chain monomer unit, for synthesis of polyhydroxyalkanoate (PHA) copolymer. In this study, to synthesize 3H4MV-containing PHA copolymer from leucine, the leucine metabolism-related enzymes (LdhA and HadAIBC) derived from C. difficile and PHA biosynthesis enzymes (PhaPCJAc and PhaABRe) derived from Aeromonas caviae and Ralstonia eutropha were co-expressed in the codon usage-improved Escherichia coli. Under microaerobic culture conditions, this E. coli was able to synthesize P(3HB-co-12.2 mol% 3H4MV) from glucose with the supplementation of 1 g/L leucine. This strain also produced P(3HB-co-12.6 mol% 3H4MV) using the culture supernatant of leucine overproducer E. coli strain NS1391 as the medium for PHA production, achieving 3H4MV copolymer synthesis only from glucose. Furthermore, we tested the feasibility of the 3H4MV copolymer synthesis in E. coli strain NS1391 from glucose. The recombinant E. coli NS1391 was able to synthesize P(3HB-co-3.0 mol% 3H4MV) from glucose without any leucine supplementation. This study demonstrates the potential of the new metabolic pathway for 3H4MV synthesis using leucine metabolism-related enzymes from C. difficile.

  18. A new method to measure muscle protein synthesis in humans by endogenously introduced d9-leucine and using blood for precursor enrichment determination

    PubMed Central

    Tran, Lee; Masters, Haley; Roust, Lori R; Katsanos, Christos S

    2015-01-01

    Enrichment from the easily accessible blood amino acid pool is commonly used as precursor enrichment to calculate rates of muscle protein fractional synthesis in relevant human studies in lieu of the less accessible muscle fluid amino acid pool. However, the accuracy of this approach depends largely on the extent to which there is low discrepancy in free amino acid enrichment between blood and muscle. Steady-state gradient (i.e., ratio) of amino acid enrichment between blood and muscle fluid in the basal state and in response to amino acid infusion were determined in five healthy subjects, and in association with two separate tracers: d9-leucine, introduced endogenously by the metabolism of d10-leucine (i.e., l-[2,3,3,4,5,5,5,6,6,6-2H10]leucine) infused in blood, and 13C6-phenylalanine introduced/infused in blood. The blood-to-muscle fluid amino acid enrichment ratio was lower (P < 0.05) for d9-leucine compared to 13C6-phenylalanine both before (1.5 ± 0.1 vs. 2.5 ± 0.1) and during (1.1 ± 0.1 vs. 1.2 ± 0.1) amino acid infusion. Importantly, the decrease in this ratio in association with the amino acid infusion was considerably less for the d9-leucine than the 13C6-phenylalanine (−0.38 ± 0.03 vs. −1.29 ± 0.07; P < 0.05). In conclusion, blood d9-leucine enrichment introduced endogenously by intravenous infusion of d10-leucine provides a closer estimate of the muscle fluid amino acid enrichment, and its associated changes, than blood phenylalanine enrichment to calculate rates of muscle protein synthesis in humans. PMID:26243214

  19. Activation of the mTOR pathway by the amino acid (L)-leucine in the 5q- syndrome and other ribosomopathies.

    PubMed

    Boultwood, Jacqueline; Yip, Bon Ham; Vuppusetty, Chaitanya; Pellagatti, Andrea; Wainscoat, James S

    2013-01-01

    Patients with the 5q- syndrome and Diamond-Blackfan anemia (DBA) suffer from a severe macrocytic anemia. The 5q- syndrome and DBA are disorders of aberrant ribosome biogenesis (ribosomopathies) and haploinsufficiency of the ribosomal protein genes RPS14 and RPS19, respectively, underlies the anemia found in these disorders. Erythroblasts obtained from patients with the 5q- syndrome and DBA show impaired mRNA translation and this defect in translation may represent a potential therapeutic target in these ribosomopathies. There are some indications that the amino acid l-leucine, a translation enhancer, may have some efficacy in this group of disorders. Recent studies have shown that l-leucine treatment of zebrafish and murine models of the 5q- syndrome and DBA results in a marked improvement in the anemia. l-leucine treatment of RPS14-deficient and RPS19-deficient erythroblasts and erythroblasts from patients with the 5q- syndrome has been shown to result in an increase in cell proliferation, erythroid differentiation and mRNA translation in culture. l-leucine has been shown to improve hemoglobin levels and transfusion independence in a patient with DBA. l-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls cell growth and mRNA translation. There is evidence to suggest that the promotion of translation via the mTOR pathway by l-leucine is the mechanism that underlies the enhanced erythroid progenitor cell growth and differentiation observed in animal and cellular models of the 5q- syndrome and DBA treated with this amino acid. These data support the rationale for clinical trials of l-leucine as a therapeutic agent for the 5q- syndrome and DBA. PMID:23031788

  20. Basic science of pain.

    PubMed

    DeLeo, Joyce A

    2006-04-01

    The origin of the theory that the transmission of pain is through a single channel from the skin to the brain can be traced to the philosopher and scientist René Descartes. This simplified scheme of the reflex was the beginning of the development of the modern doctrine of reflexes. Unfortunately, Descartes' reflex theory directed both the study and treatment of pain for more than 330 years. It is still described in physiology and neuroscience textbooks as fact rather than theory. The gate control theory proposed by Melzack and Wall in 1965 rejuvenated the field of pain study and led to further investigation into the phenomena of spinal sensitization and central nervous system plasticity, which are the potential pathophysiologic correlates of chronic pain. The processing of pain takes place in an integrated matrix throughout the neuroaxis and occurs on at least three levels-at peripheral, spinal, and supraspinal sites. Basic strategies of pain control monopolize on this concept of integration by attenuation or blockade of pain through intervention at the periphery, by activation of inhibitory processes that gate pain at the spinal cord and brain, and by interference with the perception of pain. This article discusses each level of pain modulation and reviews the mechanisms of action of opioids and potential new analgesics. A brief description of animal models frames a discussion about recent advances regarding the role of glial cells and central nervous system neuroimmune activation and innate immunity in the etiology of chronic pain states. Future investigation into the discovery and development of novel, nonopioid drug therapy may provide needed options for the millions of patients who suffer from chronic pain syndromes, including syndromes in which the pain originates from peripheral nerve, nerve root, spinal cord, bone, muscle, and disc.

  1. Leucine-rich repeat kinase 2-sensitive Na+/Ca2+ exchanger activity in dendritic cells.

    PubMed

    Yan, Jing; Almilaji, Ahmad; Schmid, Evi; Elvira, Bernat; Shimshek, Derya R; van der Putten, Herman; Wagner, Carsten A; Shumilina, Ekaterina; Lang, Florian

    2015-05-01

    Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations

  2. The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

    SciTech Connect

    Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

    2014-10-25

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins

  3. The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

    DOE PAGES

    Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

    2014-10-25

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scatteringmore » (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins« less

  4. Inhibitory Effect of Solar Radiation on Thymidine and Leucine Incorporation by Freshwater and Marine Bacterioplankton

    PubMed Central

    Sommaruga, R.; Obernosterer, I.; Herndl, G. J.; Psenner, R.

    1997-01-01

    We studied the effect of solar radiation on the incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) and [(sup14)C]leucine ([(sup14)C]Leu) by bacterioplankton in a high mountain lake and the northern Adriatic Sea. After short-term exposure (3 to 4 h) of natural bacterial assemblages to sunlight just beneath the surface, the rates of incorporation of [(sup3)H]TdR and [(sup14)C]Leu were reduced at both sites by up to (symbl)70% compared to those for the dark control. Within the solar UV radiation (290 to 400 nm), the inhibition was caused exclusively by UV-A radiation (320 to 400 nm). However, photosynthetically active radiation (PAR) (400 to 700 nm) contributed almost equally to this effect. Experiments with samples from the high mountain lake showed that at a depth of 2.5 m, the inhibition was caused almost exclusively by UV-A radiation. At a depth of 8.5 m, where chlorophyll a concentrations were higher than those in the upper water column, the rates of incorporation of [(sup3)H]TdR were higher in those samples exposed to full sunlight or to UV-A plus PAR than in the dark control. In laboratory experiments with artificial UV light, the incorporation of [(sup3)H]TdR and [(sup14)C]Leu by mixed bacterial lake cultures was also inhibited mainly by UV-A. In contrast, in the presence of the green alga Chlamydomonas geitleri at a chlorophyll a concentration of 2.5 (mu)g liter(sup-1), inhibition by UV radiation was significantly reduced. These results suggest that there may be complex interactions among UV radiation, heterotrophic bacteria, and phytoplankton and their release of extracellular organic carbon. Our findings indicate that the wavelengths which caused the strongest inhibition of TdR and Leu incorporation by bacterioplankton in the water column were in the UV-A range. However, it may be premature to extrapolate this effect to estimates of bacterial production before more precise information on how solar radiation affects the transport of TdR and Leu into the cell

  5. Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b.

    PubMed

    Deejing, Somkid; Yoshimune, Kazuaki; Lumyong, Saisamorn; Moriguchi, Mitsuaki

    2005-07-01

    A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90 degrees C for up to 1 h. Its optimal pH and temperature were observed to be 7.6-7.8 and 60 degrees C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K(m) and V(max) values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 micromol min(-1) mg(-1) protein and 1,149 micromol min(-1) mg(-1) protein, respectively. The turnover rate (k(cat)) and catalytic efficiency (k(cat)/ K(m)) for Leu-p-NA and LeuGlyGly were 10,179 s(-1) and 49,543 s(-1) and 15,470 mM(-1 ) s(-1) and 1981.7 mM(-1 ) s(-1), respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, beta-mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co(2+) .

  6. (19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.

    PubMed

    Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A

    2003-10-01

    The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein. PMID:12956607

  7. Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach.

    PubMed

    Zhu, Xiaoxia; Barman, Arghya; Ozbil, Mehmet; Zhang, Tingting; Li, Shanghao; Prabhakar, Rajeev

    2012-02-01

    In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1-Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H(2)O and -OH) have been investigated. In addition, the effects of the substrate (L-leucine-p-nitroanilide → L-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1-Zn2 and Mg1-Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with L-leucine-p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for L-leucyl-p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0-19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1-Zn2, Mg1-Zn2, and Mg1-Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases.

  8. Serum cystine aminopeptidase and leucine aminopeptidase activity in women with benign and malignant uterine and ovarian tumors.

    PubMed

    Blum, M; Sirota, P

    1977-09-01

    The serum enzymatic activities of cystine aminopeptidase and leucine aminopeptidase were measured in a group of 113 patients of whom 90 had benign uterine or ovarian tumors, and 23 had cancer of the endometrium or ovary. Thirty healthy nonpregnant women and 260 women at different stages of normal pregnancy served as control groups. The presence of pregnancy-specific enzymes in women with uterine or ovarian tumors showed once again that similar processes occur during pregnancy and malignancy. When ovarian or uterine malignancy is suspected on clinical examination, determination of the activities of these enzymes in serum may be of diagnostic value.

  9. The Effects of Leucine, Zinc, and Chromium Supplements on Inflammatory Events of the Respiratory System in Type 2 Diabetic Rats

    PubMed Central

    Kolahian, Saeed; Sadri, Hassan; Shahbazfar, Amir Ali; Amani, Morvarid; Mazadeh, Anis; Mirani, Mehdi

    2015-01-01

    Diabetes mellitus is a major cause of serious micro- and macrovascular diseases that affect nearly every system in the body, including the respiratory system. Non-enzymatic protein glycation due to hyperglycaemic stress has fundamental implications due to the large capillary network and amount of connective tissue in the lung. The current study was designed to determine whether leucine, zinc, and chromium supplementations influence the function and histological structure of the respiratory tract in a rat model of type 2 diabetes. Seventy-seven rats were divided into eleven groups, consisting of 7 animals each. One group served as negative control and insulin and glibenclamide were used as positive control drugs. Thus, eight groups received the nutritional supplements alone or in combination with each other. Nutritional supplements and glibenclamide were added to the drinking water and neutral protamine Hagedorn insulin was subcutaneously injected during the 4 weeks of treatment period. The induction of type 2 diabetes in the rats caused an infiltration of mononuclear cells and edema in the submucosa of the trachea and lung, severe fibrosis around the vessels and airways, and perivascular and peribronchial infiltration of inflammatory cells and fibrin. In the diabetic group, the total inflammation score and Reid index significantly increased. Diabetes induction significantly reduced the total antioxidant status and elevated the lipid peroxidation products in the serum, lung lavage and lung tissue of the diabetic animals. Treatment with nutritional supplements significantly decreased the histopathological changes and inflammatory indices in the diabetic animals. Supplementation of diabetic rats with leucine, zinc, and chromium, alone and in combination, significantly increased the total antioxidant status and lipid peroxidation level in the diabetic animals. The nutritional supplements improved the enzymatic antioxidant activity of catalase, glutathione peroxidase

  10. A novel mutation of a leucine residue in coil 1A of keratin 9 in epidermolytic palmoplantar keratoderma.

    PubMed

    Endo, H; Hatamochi, A; Shinkai, H

    1997-07-01

    Keratin 9 mutation was examined in a Japanese kindred of epidermolytic palmoplantar keratoderma (EPPK), which is a dominantly inherited autosomal disorder of keratinization characterized by diffuse thickening of the palms and soles and by epidermolytic hyperkeratosis histologically. We report herein a novel mutation, a C --> G transversion at nucleotide position 541 that converts a leucine residue (CTC) to a valine (GTC) at codon 159. As in all other reported cases of keratin 9 mutation in EPPK, this mutation lies within the highly conserved coil 1A of the rod domain, which is considered to play a role in the correct alignment of the coiled-coil molecules.

  11. The Effects of Leucine, Zinc, and Chromium Supplements on Inflammatory Events of the Respiratory System in Type 2 Diabetic Rats.

    PubMed

    Kolahian, Saeed; Sadri, Hassan; Shahbazfar, Amir Ali; Amani, Morvarid; Mazadeh, Anis; Mirani, Mehdi

    2015-01-01

    Diabetes mellitus is a major cause of serious micro- and macrovascular diseases that affect nearly every system in the body, including the respiratory system. Non-enzymatic protein glycation due to hyperglycaemic stress has fundamental implications due to the large capillary network and amount of connective tissue in the lung. The current study was designed to determine whether leucine, zinc, and chromium supplementations influence the function and histological structure of the respiratory tract in a rat model of type 2 diabetes. Seventy-seven rats were divided into eleven groups, consisting of 7 animals each. One group served as negative control and insulin and glibenclamide were used as positive control drugs. Thus, eight groups received the nutritional supplements alone or in combination with each other. Nutritional supplements and glibenclamide were added to the drinking water and neutral protamine Hagedorn insulin was subcutaneously injected during the 4 weeks of treatment period. The induction of type 2 diabetes in the rats caused an infiltration of mononuclear cells and edema in the submucosa of the trachea and lung, severe fibrosis around the vessels and airways, and perivascular and peribronchial infiltration of inflammatory cells and fibrin. In the diabetic group, the total inflammation score and Reid index significantly increased. Diabetes induction significantly reduced the total antioxidant status and elevated the lipid peroxidation products in the serum, lung lavage and lung tissue of the diabetic animals. Treatment with nutritional supplements significantly decreased the histopathological changes and inflammatory indices in the diabetic animals. Supplementation of diabetic rats with leucine, zinc, and chromium, alone and in combination, significantly increased the total antioxidant status and lipid peroxidation level in the diabetic animals. The nutritional supplements improved the enzymatic antioxidant activity of catalase, glutathione peroxidase

  12. The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association

    SciTech Connect

    Wei, Zhuang; Zou, Xinle; Wang, Hongzhong; Lei, Jigang; Wu, Yuan; Liao, Kan

    2015-01-16

    Highlight: • The N-terminal leucine-zipper motif in PTRF/cavin-1 determines caveolar association. • Different cellular localization of PTRF/cavin-1 influences its serine 389 and 391 phosphorylation state. • PTRF/cavin-1 regulates cell motility via its caveolar association. - Abstract: PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AA 235–251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1{sup −/−} mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1.

  13. Leucine acts in the brain to suppress food intake but does not function as a physiological signal of low dietary protein.

    PubMed

    Laeger, Thomas; Reed, Scott D; Henagan, Tara M; Fernandez, Denise H; Taghavi, Marzieh; Addington, Adele; Münzberg, Heike; Martin, Roy J; Hutson, Susan M; Morrison, Christopher D

    2014-08-01

    Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 μg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.

  14. The Akt/mTOR pathway: Data comparing young and aged mice with leucine supplementation at the onset of skeletal muscle regeneration.

    PubMed

    Perry, Richard A; Brown, Lemuel A; Lee, David E; Brown, Jacob L; Baum, Jamie I; Greene, Nicholas P; Washington, Tyrone A

    2016-09-01

    The data described herein is related to the article "Differential Effects of Leucine Supplementation in Young and Aged Mice at the Onset of Skeletal Muscle Regeneration" [1]. Aging is associated with a decreased ability of skeletal muscle to regenerate following injury. Leucine supplementation has been extensively shown, in young subjects, to promote protein synthesis during regeneration; however, the effects of leucine supplementation on the Akt/mTOR pathway in aged mice at the onset of muscle regeneration are not fully elucidated. In this article, we present data on the Akt/mTOR protein synthesis pathway at the onset of muscle regeneration in young and aged C57BL/6J mice that are and are not receiving leucine supplementation. More specifically, protein content of total Akt, mTOR, p70S6K and 4EBP-1 are presented. Additionally, we provide relative (phosphorylated:total) protein content comparisons of these targets as they present themselves in young and aged mice who have neither been injured nor received leucine supplementation. Lastly, markers of atrophy (FoxO1/O3, MuRF-1, Atrogin-1) are also reported in these young and aged control groups. PMID:27617277

  15. In vivo and in vitro effects of growth hormone on the incorporation of ( UC)leucine into protein of liver and muscle of the eel

    SciTech Connect

    Inui, Y.; Ishioka, H.

    1985-08-01

    In vivo administration of ovine GH (2 micrograms/g body wt) increased ( UC)leucine incorporation into protein of the liver, skeletal muscle, and opercular muscle of hypophysectomized eels. Addition of ovine GH into the medium slightly increased ( UC)leucine incorporation into protein of liver slices during 5 hr in vitro incubation, but did not affect protein synthesis from ( UC)leucine in opercular muscle. In vivo pretreatment with ovine GH 48 hr prior to tissue preparation clearly increased ( UC)leucine incorporation into protein of liver slices in vitro. However, no statistically significant change was observed for in vitro incorporation of ( UC)leucine into protein of opercular muscle of hypophysectomized eels which had been previously treated with ovine GH. These results indicate that ovine GH has a protein anabolic action in the liver and muscle of the eel and that compared to mammals a rather long lag period is needed to elicit such protein anabolic actions of GH in these animals.

  16. Fluctuations between stabilizing and destabilizing electrostatic contributions of ion pairs in conformers of the c-Myc-Max leucine zipper.

    PubMed

    Kumar, S; Nussinov, R

    2000-12-01

    In solution proteins often exhibit backbone and side-chain flexibility. Yet electrostatic interactions in proteins are sensitive to motions. Hence, here we study the contribution of ion pairs toward protein stability in a range of conformers which sample the conformational space in solution. Specifically, we focus on the electrostatic contributions of ion pairs to the stability of each of the conformers in the NMR ensemble of the c-Myc-Max leucine zipper and to their average energy minimized structure. We compute the electrostatic contributions of inter- and intra-helical ion pairs and of an ion pair network. We find that the electrostatic contributions vary considerably among the 40 NMR conformers. Each ion pair, and the network, fluctuates between being stabilizing and being destabilizing. This fluctation reflects the variability in the location of the ion pairing residues and in the geometric orientation of these residues, both with respect to each other and with respect to other charged groups in the rest of the protein. Ion pair interactions in the c-Myc-Max leucine zipper in solution depend on the protein conformer which is analyzed. Hence, the overall stabilizing (or destabilizing) contribution of an ion pair is conformer population-dependent. This study indicates that free energy calculations performed using the continuum electrostatics methodology are sensitive to protein conformational details.

  17. 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

  18. Chiral HPLC Separation and Modeling of Four Stereomers of DL-Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column.

    PubMed

    Alajmi, Mohammed F; Hussain, Afzal; Suhail, Mohd; Mukhtar, Sofi Danish; Sahoo, Dibya Ranjan; Asnin, Leonid; Ali, Imran

    2016-09-01

    Chiral high-performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column are described. The mobile phase applied was ammonium acetate (10 mM)-methanol-acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0-2.3 and 5.6-14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π-π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in unknown matrices. Chirality 28:642-648, 2016. © 2016 Wiley Periodicals, Inc. PMID:27474783

  19. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

    PubMed Central

    McAdam, R A; Weisbrod, T R; Martin, J; Scuderi, J D; Brown, A M; Cirillo, J D; Bloom, B R; Jacobs, W R

    1995-01-01

    Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen. PMID:7868221

  20. Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

    PubMed Central

    Blagoveshchenskaya, Anastasiya D.; Hewitt, Eric W.; Cutler, Daniel F.

    1999-01-01

    One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents. PMID:10564285

  1. Identification, molecular characterization, and chromosomal localization of the cDNA encoding a novel leucine zipper motif-containing protein

    SciTech Connect

    Sun, Der-Shan; Chang, Nan-Chi A.; Chang, A.C.

    1996-08-15

    cDNA clones encoding a novel protein (LUZP) with three leucine zipper motifs were first identified from a murine bone marrow cDNA library. After screening two additional cDNA libraries of activated peritoneal exudate cells, 32 positive clones were obtained from 1.3 x 10{sup 7} phage plaques. Four overlapping clones constituting a total of 7399 hp were sequenced on both strands. The complete open reading frame of LUZP is 1067 amino acids. In addition to three leucine zipper motifs located at the NH2 terminus, there are three nuclear localization signals and a large number of putative Ser/Thr phosphorylation sites. Western blot analyses indicate that LUZP is predominantly expressed in brain, whereas immunocytochemistry data clearly reveal its presence in the nucleus of neutrons. Interspecific backcross analyses have mapped Luzp to mouse chromosome 4 in proximity to Gpcr14. Comparative mapping data suggest that the human homolog of Luzp will map to human chromosome 1p36. 32 refs., 4 figs.

  2. Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms1[OPEN

    PubMed Central

    Dufayard, Jean-François; Chantret, Nathalie

    2016-01-01

    Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family. PMID:26773008

  3. Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule.

    PubMed Central

    Krantz, D E; Zipursky, S L

    1990-01-01

    Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. PMID:2189727

  4. 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.

  5. Basic Skills. NIACE Briefing Sheet.

    ERIC Educational Resources Information Center

    National Inst. of Adult Continuing Education, Leicester (England).

    Skills For Life, since 2001 the United Kingdom's national adult basic skills strategy, aims to improve literacy, numeracy, or English for speakers of other languages (ESOL) skills among people over the age of 16. Basic skills programs may be full- or part-time and are delivered in colleges, community venues, neighborhood learning centers, adult…

  6. Basic electronics for clinical neurophysiology.

    PubMed

    Misulis, K E

    1989-01-01

    This article reviews the basic electronics that are important to clinical neurophysiology. It is divided into six sections: basic principles of electronics; filters; transistors and amplifiers; displays; electrodes and the electrode-amplifier interface; and electrical safety. In addition, at the end of the review is a brief electronics glossary (Appendix A) and an annotated bibliography (Appendix B) to guide further reading.

  7. Children and Their Basic Needs.

    ERIC Educational Resources Information Center

    Prince, Debra Lindsey; Howard, Esther M.

    2002-01-01

    Describes obstacles presented by poverty in the fulfillment of the basic needs of children. Individually addresses Maslow's five basic needs with regard to children reared in poverty: (1) physiological needs; (2) safety needs; (3) belonging and love needs; (4) self-esteem needs; and (5) self-actualization needs. (Author/SD)

  8. Basic Skills: Dealing with Deficiencies.

    ERIC Educational Resources Information Center

    New Mexico State Univ., Las Cruces.

    Research findings on college instruction and basic skills deficiencies are discussed in 12 papers from the first Regional Conference on University Teaching. Titles and authors are as follows: "Basic Skills: Dealing with Deficiencies" (Susanne D. Roueche, with responses by Gary B. Donart, Betty Harris, and James Nordyke); "Is Higher Education an…

  9. Chinese-Cantonese Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    This nine-volume basic course in Cantonese Chinese is designed for 47 weeks of intense audiolingual instruction. The first book of the series introduces the pronunciation, with emphasis on the tone system, and the basic aspects of the grammar. Also introduced in this volume is the romanization system used in this series (the U.S. Army Language…

  10. Creating Adult Basic Education Programs.

    ERIC Educational Resources Information Center

    Harris, Dolores M.

    Adult basic education programs must teach the "social living skills" disadvantaged adults need, as well as basic literacy skills. In creating an ABE program, one must first assess the needs of the target population--through surveys, group meetings, an advisory council of members of the target population, demographic studies, and consideration of…

  11. Basic sciences: an alternative career?

    PubMed

    Khatri, R

    2013-01-01

    Career selection is a crucial and a complex process which is also true for the medical profession. In the context of our country, due to the limited opportunity and proper guidance, migration of medical graduates to foreign countries is increasing. Though, clinical subjects have a huge attraction, basic science field has failed to impress our medical graduates. In current scenario, basic science field seems to be a dumping site for the incompetent as the candidates who have failed trying their luck elsewhere stumble upon basic science careers though it is not true for all. Moreover, a very few medical graduates are interested in developing their career as a basic scientist. Therefore, to motivate today's young medical graduates, there is a need of a good mentor along with a proper career guidance which can help them to understand the basic science field as an alternative career. PMID:23774420

  12. Leucine supplementation does not affect protein turnover and impairs the beneficial effects of endurance training on glucose homeostasis in healthy mice.

    PubMed

    Costa Júnior, José M; Rosa, Morgana R; Protzek, André O; de Paula, Flávia M; Ferreira, Sandra M; Rezende, Luiz F; Vanzela, Emerielle C; Zoppi, Cláudio C; Silveira, Leonardo R; Kettelhut, Isis C; Boschero, Antonio C; de Oliveira, Camila A M; Carneiro, Everardo M

    2015-04-01

    Endurance exercise training as well as leucine supplementation modulates glucose homeostasis and protein turnover in mammals. Here, we analyze whether leucine supplementation alters the effects of endurance exercise on these parameters in healthy mice. Mice were distributed into sedentary (C) and exercise (T) groups. The exercise group performed a 12-week swimming protocol. Half of the C and T mice, designated as the CL and TL groups, were supplemented with leucine (1.5 % dissolved in the drinking water) throughout the experiment. As well known, endurance exercise training reduced body weight and the retroperitoneal fat pad, increased soleus mass, increased VO2max, decreased muscle proteolysis, and ameliorated peripheral insulin sensitivity. Leucine supplementation had no effect on any of these parameters and worsened glucose tolerance in both CL and TL mice. In the soleus muscle of the T group, AS-160(Thr-642) (AKT substrate of 160 kDa) and AMPK(Thr-172) (AMP-Activated Protein Kinase) phosphorylation was increased by exercise in both basal and insulin-stimulated conditions, but it was reduced in TL mice with insulin stimulation compared with the T group. Akt phosphorylation was not affected by exercise but was lower in the CL group compared with the other groups. Leucine supplementation increased mTOR phosphorylation at basal conditions, whereas exercise reduced it in the presence of insulin, despite no alterations in protein synthesis. In trained groups, the total FoxO3a protein content and the mRNA for the specific isoforms E2 and E3 ligases were reduced. In conclusion, leucine supplementation did not potentiate the effects of endurance training on protein turnover, and it also reduced its positive effects on glucose homeostasis.

  13. Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise.

    PubMed

    Moberg, Marcus; Apró, William; Ekblom, Björn; van Hall, Gerrit; Holmberg, Hans-Christer; Blomstrand, Eva

    2016-06-01

    Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (Placebo<Leucineleucine (40%, P < 0.05) and placebo (100%, P < 0.05). In summary, EAA ingestion appears to stimulate translation initiation more effectively than the other supplements, although the results also suggest that this effect is primarily attributable to the BCAA. PMID:27053525

  14. Crystal Engineering of l-Alanine with l-Leucine Additive using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization

    PubMed Central

    2015-01-01

    In this work, we demonstrated that the change in the morphology of l-alanine crystals can be controlled with the addition of l-leucine using the metal-assisted and microwave accelerated evaporative crystallization (MA-MAEC) technique. Crystallization experiments, where an increasing stoichiometric amount of l-leucine is added to initial l-alanine solutions, were carried out on circular poly(methyl methacrylate) (PMMA) disks modified with a 21-well capacity silicon isolator and silver nanoparticle films using microwave heating (MA-MAEC) and at room temperature (control experiments). The use of the MA-MAEC technique afforded for the growth of l-alanine crystals with different morphologies up to ∼10-fold faster than those grown at room temperature. In addition, the length of l-alanine crystals was systematically increased from ∼380 to ∼2000 μm using the MA-MAEC technique. Optical microscope images revealed that the shape of l-alanine crystals was changed from tetragonal shape (without l-leucine additive) to more elongated and wire-like structures with the addition of the l-leucine additive. Further characterization of l-alanine crystals was undertaken by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy and powder X-ray diffraction (PXRD) measurements. In order to elucidate the growth mechanism of l-alanine crystals, theoretical simulations of l-alanine’s morphology with and without l-leucine additive were carried out using Materials Studio software in conjunction with our experimental data. Theoretical simulations revealed that the growth of l-alanine’s {011} and {120} crystal faces were inhibited due to the incorporation of l-leucine into these crystal faces in selected positions. PMID:24839404

  15. Enhanced production of branched-chain amino acids by Gluconacetobacter europaeus with a specific regional deletion in a leucine responsive regulator.

    PubMed

    Akasaka, Naoki; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2014-12-01

    Vinegar with increased amounts of branched-chain amino acids (BCAAs; valine, leucine and isoleucine) is favorable for human health as BCAAs decrease diet-induced obesity and hyperglycemia. To construct Gluconacetobacter europaeus which produces BCAAs, leucine responsive regulator (GeLrp) is focused and two Gelrp mutants were constructed. Wild-type KGMA0119 didn't produce significant amount of valine (0.13 mM) and leucine (0 mM) and strain KGMA7110 which lacks complete Gelrp accumulated valine (0.48 mM) and leucine (0.11 mM) but showed impaired growth, and it was fully restored in the presence of essential amino acids. Strain KGMA7203 was then constructed with a nonsense mutation at codon Trp132 in the Gelrp, which leads a specific deletion at an estimated ligand-sensing region in the C-terminal domain. KGMA7203 produced greater quantities of valine (0.80 mM) and leucine (0.26 mM) and showed the same growth characteristics as KGMA0119. mRNA levels of BCAAs biosynthesis genes (ilvI and ilvC) and probable BCAAs efflux pump (leuE) were determined by quantitative reverse-transcription PCR. Expression rates of ilvI and ilvC in the two Gelrp disruptants were greater than those in KGMA0119. leuE was highly expressed in KGMA7110 only, suggesting that the accumulation in KGMA7110 culture was caused by increased expression of the biosynthesis genes and abnormal enhanced export of amino acids resulting in impaired cell growth. In contrast, KGMA7203 would achieve the high level production through enhanced expression of the biosynthesis genes without enhancing that for the efflux pump. KGMA7203 was considered advantageous for production of vinegar with higher amounts of valine and leucine.

  16. A fifth member of the tomato 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene family harbours a leucine zipper and is anaerobically induced.

    PubMed

    Sell, Simone; Hehl, Reinhard

    2005-02-01

    Using the leucine zipper domain of a small anaerobically induced bZIP transcription factor in a yeast two hybrid screen, anaerobically induced genes were identified. One peptide corresponds to an anaerobically induced IDS4-like protein that maybe involved in G-protein signaling. Surprisingly, another interacting peptide corresponds to a novel anaerobically induced 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, designated ACO5. ACO5 harbours a leucine zipper and transcription is mainly induced in fruits and to a lesser extend in leaves. The role of ACO5 in the low oxygen response of tomato is discussed. PMID:16040352

  17. L-leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst.

    PubMed

    Hibi, Makoto; Kawashima, Takashi; Sokolov, Pavel M; Smirnov, Sergey V; Kodera, Tomohiro; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2013-03-01

    L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.

  18. The basic aerodynamics of floatation

    SciTech Connect

    Davies, M.J.; Wood, D.H.

    1983-09-01

    The original derivation of the basic theory governing the aerodynamics of both hovercraft and modern floatation ovens, requires the validity of some extremely crude assumptions. However, the basic theory is surprisingly accurate. It is shown that this accuracy occurs because the final expression of the basic theory can be derived by approximating the full Navier-Stokes equations in a manner that clearly shows the limitations of the theory. These limitations are used in discussing the relatively small discrepancies between the theory and experiment, which may not be significant for practical purposes.

  19. Basic nitrogen in hydrocracked resids

    SciTech Connect

    Somogyvari, A.; Zanzotto, L. ); Jeffries, R. )

    1990-07-01

    No direct link has been established between the basic nitrogen components of hydrocracked residues and the increase in anti-stripping properties of distillation residues containing them. Thus, it is the intent of this report to show that a correlation exists between the basic nitrogen components of hydrocracked residues and the anti-stripping properties of asphalts containing them and that it is not only the quantity but also the type of basic nitrogen compounds found in the hydrocracked residues that account for the enhanced anti-stripping properties of these bitumens and blends containing them.

  20. Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7).

    PubMed

    Narasimhamurthy, S; Naganagowda, G A; Janagani, S; Gururaja, T L; Levine, M J

    2000-08-01

    Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in

  1. Leucine does not affect mechanistic target of rapamycin complex 1 assembly but is required for maximal ribosomal protein s6 kinase 1 activity in human skeletal muscle following resistance exercise.

    PubMed

    Apró, William; Moberg, Marcus; Hamilton, D Lee; Ekblom, Björn; Rooyackers, Olav; Holmberg, Hans-Christer; Blomstrand, Eva

    2015-10-01

    We examined how the stimulatory effect of leucine on the mechanistic target of rapamycin complex 1 (mTORC1) pathway is affected by the presence of the remaining essential amino acids (EAAs). Nine male subjects performed resistance exercise on 4 occasions and were randomly supplied EAAs with leucine, EAAs without leucine (EAA-Leu), leucine alone, or flavored water (placebo; control). Muscle biopsies were taken from the vastus lateralis before and 60 and 90 min after exercise. Biopsies were analyzed for protein phosphorylation, kinase activity, protein-protein interactions, amino acid concentrations, and tracer incorporation. Leucine alone stimulated ribosomal protein s6 kinase 1 (S6K1) phosphorylation ∼280% more than placebo and EAA-Leu after exercise. Moreover, this response was enhanced by 60-75% after intake of EAAs compared with that of leucine alone (P < 0.05). Kinase activity of S6K1 reflected that of S6K1 phosphorylation; 60 min after exercise, the activity was elevated 3.3- and 4.2-fold with intake of leucine alone and with EAAs, respectively (P < 0.05). The interaction between mammalian target of rapamycin and regulatory-associated protein of mammalian target of rapamycin was unaltered in response to both resistance exercise and amino acid provision. Leucine alone stimulates mTORC1 signaling, although this response is enhanced by other EAAs and does not appear to be caused by alterations in mTORC1 assembly.

  2. Plants, Animals and Basic Skills.

    ERIC Educational Resources Information Center

    Pheasant, Robert

    2000-01-01

    Countrystart is a class in which students work with plants and animals, providing numerous opportunities to integrate basic skills teaching. The practical subject area becomes the vehicle to develop other skills needed by students. (JOW)

  3. French Basic Course. Grammatical Index.

    ERIC Educational Resources Information Center

    Defense Language Inst., Monterey, CA.

    This index is intended for use with Volumes 1 through 8 of the French Basic Course. It facilitates the finding of grammatical references in those volumes. The items are cross-referenced and arranged in alphabetical order. (Author/AMH)

  4. Brain Basics: Know Your Brain

    MedlinePlus

    ... fact sheet is a basic introduction to the human brain. It may help you understand how the healthy ... largest and most highly developed part of the human brain: it consists primarily of the cerebrum ( 2 ) and ...

  5. Method development for the determination of D- and L-isomers of leucine in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application to animal plasma samples.

    PubMed

    Sugimoto, Hiroshi; Kakehi, Masaaki; Jinno, Fumihiro

    2015-10-01

    We developed a highly sensitive and specific high-performance liquid chromatography tandem mass spectrometry method with an electrospray ionization for the determination of D- and L-isomers of leucine in human plasma. Phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and quality control samples. The extraction of D- and L-leucine in plasma samples (100 μL) was performed using cationic exchange solid-phase extraction. The enantiomer separation of D- and L-leucine was successfully achieved without derivatization using a CHIRALPAK ZWIX(-) with an isocratic mobile phase comprised of methanol/acetonitrile/1 mol/L ammonium formate/formic acid (500:500:25:2, v/v/v/v) at a flow rate of 0.5 mL/min. In addition, the discrimination of DL-leucine from structural isomers DL-isoleucine and DL-allo-isoleucine was performed using the unique precursor and product ion pair transition of DL-leucine (m/z 132.1 > 43.0) and DL-leucine-d 7 (m/z 139.2 > 93.0) in positive electrospray ionization mode. The standard curves were linear throughout the calibration range from 0.001 to 1 μg/mL for D-leucine and from 1 to 1000 μg/mL for L-leucine, respectively, with acceptable intra- and inter-day precision and accuracy. The stability of D- and L-leucine in human plasma and solvents was confirmed. The endogenous level of D- and L-leucine in human plasma was 0.00197~0.00591 and 9.63~24.7 μg/mL, respectively. This method was also successfully applied to investigate the species difference in the ratios of D-leucine to total leucine from individual plasma concentrations in humans and various animals. The plasma D-leucine concentrations or their ratio to total leucine in rodents was much higher than that in humans. PMID:26345443

  6. From growth to basic needs.

    PubMed

    Streeten, P

    1979-09-01

    Despite growing hostility and basic misconceptions, the concept of "basic human needs" has superseded former approaches, including concentration on growth, creation of employment, and redistribution of benefits to the poor, as the approach by which mass deprivation may be reduced. The new approach can be defined briefly as one which is designed to improve, first, the income earning opportunities for the poor; second, the public services that reach the poor; third, the flow of goods and services to meet the needs of all members of the household; and fourth, participation of the poor in the ways in which their needs are met. All four pillars must be built on a sustainable basis. In addition, basic needs must be met in a shorter period and at a lower level of earned income per capita than has generally been true in the past, or than would have been achieved via the income expansion associated with growth alone. The basic needs approach is concerned with particular goods and services directed at particular, identified human beings. Another advantage of the basic needs approach is that it is a more positive concept than the double negatives of eliminating or reducing unemployment, alleviating poverty, or reducing inequality. The basic needs approach spells out in considerable detail human needs in terms of health, food, education, water, shelter, transport, simple household goods, as well as non-material needs like participation, cultural identity, and a sense of purpose in life and work, which interact with the material needs. PMID:12261287

  7. Influence of age of aggregates and prokaryotic abundance on glucose and leucine uptake by heterotrophic marine prokaryotes.

    PubMed

    Azúa, Iñigo; Unanue, Marian; Ayo, Begoña; Artolozaga, Itxaso; Iriberri, Juan

    2007-03-01

    The kinetics of glucose and leucine uptake in attached and free-living prokaryotes in two types of microcosms with different nutrient qualities were compared. Microcosm type M1, derived from unaltered seawater, and microcosm type M2, from phytoplankton cultures, clearly expressed different kinetic parameters (Vmax/cell and K' m). In aggregates with low cell densities (M1 microcosm), the attached prokaryotes benefited from attachment as reflected in the higher potential uptake rates, while in aggregates with high cell densities (M2 microcosm) differences in the potential uptake rates of attached and free-living prokaryotes were not evident. The aging process and the chemical changes in aggregates of M2 microcosms were followed for 15-20 days. The results showed that as the aggregates aged and prokaryotic abundance increased, attached prokaryotes decreased their potential uptake rate and their K' m for substrate. This suggests an adaptive response by attached prokaryotes when aggregates undergo quantitative and qualitative impoverishment.

  8. A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    PubMed Central

    Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S.; Jiang, Cai-Zhong

    2014-01-01

    Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence. PMID:24551088

  9. Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

    PubMed

    Suzuki, Hirohumi; Yamamoto, Toshiharu

    2014-02-01

    In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH.

  10. Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in gastric cancer

    PubMed Central

    Li, Shen; Li, Ziyu; Guo, Ting; Xing, Xiao-Fang; Cheng, Xiaojing; Du, Hong; Wen, Xian-Zi; Ji, Jia-Fu

    2016-01-01

    Maternal embryonic leucine zipper kinase (MELK) is upregulated in a variety of human tumors, and is considered an attractive molecular target for cancer treatment. We characterized the expression of MELK in gastric cancer (GC) and measured the effects of reducing MELK mRNA levels and protein activity on GC growth. MELK was frequently overexpressed in primary GCs, and higher MELK levels correlated with worse clinical outcomes. Reducing MELK expression or inhibiting kinase activity resulted in growth inhibition, G2/M arrest, apoptosis and suppression of invasive capability of GC cells in vitro and in vivo. MELK knockdown led to alteration of epithelial mesenchymal transition (EMT)-associated proteins. Furthermore, targeting treatment with OTSSP167 in GC patient-derived xenograft (PDX) models had anticancer effects. Thus, MELK promotes cell growth and invasiveness by inhibiting apoptosis and promoting G2/M transition and EMT in GC. These results suggest that MELK may be a promising target for GC treatment. PMID:26701722

  11. A leucine-to-proline substitution causes a defective [alpha]-antichymotrypsin allele associated with familial obstructive lung disease

    SciTech Connect

    Poller, W.; Scholz, S.; Fischer, M. ); Faber, J.P.; Tief, K.; Olek, K.; Kirchgesser, M. ); Weidinger, S. ); Heidtmann, H.H. )

    1993-09-01

    Using denaturing gradient gel electrophoresis and direct sequencing of amplified genomic DNA, the authors have identified two defective mutants of the human [alpha][sub 1]-antichymotrypsin (ACT) gene associated with chronic obstructive pulmonary disease (COPD). A leucine 55-to-proline substitution causing a defective ACT allele (Bochum-1) was observed in a family with COPD in three subsequent generations. Another mutation, proline 229-to-alanine (Bonn-1), was associated with ACT serum deficiency in four patients with a positive family history. These mutations were not detected among 100 healthy control subjects, suggesting a possible pathogenetic role of ACT gene defects in a subset of patients with COPD. 14 refs., 1 fig., 1 tab.

  12. Posterior amorphous corneal dystrophy is associated with a deletion of small leucine-rich proteoglycans on chromosome 12.

    PubMed

    Kim, Michelle J; Frausto, Ricardo F; Rosenwasser, George O D; Bui, Tina; Le, Derek J; Stone, Edwin M; Aldave, Anthony J

    2014-01-01

    Posterior amorphous corneal dystrophy (PACD) is a rare, autosomal dominant disorder affecting the cornea and iris. Next-generation sequencing of the previously identified PACD linkage interval on chromosome 12q21.33 failed to yield a pathogenic mutation. However, array-based copy number analysis and qPCR were used to detect a hemizygous deletion in the PACD linkage interval containing 4 genes encoding small leucine-rich proteoglycans (SLRPs): KERA, LUM, DCN, and EPYC. Two other unrelated families with PACD also demonstrated deletion of these SLRPs, which play important roles in collagen fibrillogenesis and matrix assembly. Given that these genes are essential to the maintenance of corneal clarity and the observation that knockout murine models display corneal phenotypic similarities to PACD, we provide convincing evidence that PACD is associated with haploinsufficiency of these SLRPs.

  13. VUV action spectroscopy of protonated leucine-enkephalin peptide in the 6-14 eV range

    SciTech Connect

    Ranković, M. Lj.; Canon, F.; Nahon, L.; Giuliani, A.; Milosavljević, A. R.

    2015-12-28

    We have studied the Vacuum Ultraviolet (VUV) photodissociation of gas-phase protonated leucine-enkephalin peptide ion in the 5.7 to 14 eV photon energy range by coupling a linear quadrupole ion trap with a synchrotron radiation source. We report VUV activation tandem mass spectra at 6.7, 8.4, and 12.8 eV photon energies and photodissociation yields for a number of selected fragments. The obtained results provide insight into both near VUV radiation damage and electronic properties of a model peptide. We could distinguish several absorption bands and assign them to particular electronic transitions, according to previous theoretical studies. The photodissociation yields appear to be very different for the various observed fragmentation channels, depending on both the types of fragments and their position along the peptide backbone. The present results are discussed in light of recent gas-phase spectroscopic data on peptides.

  14. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

    PubMed Central

    Kalunke, Raviraj M.; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

    2015-01-01

    Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens. PMID:25852708

  15. [Proteolysis of simple glyprolines by leucine aminopeptidase and enzymes from nasal slime, brain membranes, and rat blood].

    PubMed

    Shevchenko, K V; V'iunova, T V; Nagaev, I Iu; Andreeva, L A; Miasoedov, N F

    2013-01-01

    Proteolysis of Pro-Gly-Pro-Leu, Pro-Gly-Pro-Gly and Pro-Gly-Pro were studied comparatively to Met-Glu-His-Phe-Pro-Gly-Pro (semax). It is shown that all three peptides are considerably more stable to proteolysis by N-leucine-aminopeptidase (EC 3.4.11.1, Sigma, type VI, 9.2 units/mg), and by enzymes of nasal slime, brain microsomal fractions, and rat blood. Metabolites of the proteolysis showed that semax derives His-Phe-Pro-Gly-Pro only, Pro-Gly-Pro-Leu forms Gly-Pro-Leu, Pro-Gly-Pro and Gly-Pro, Pro-Gly-Pro-Gly gives Pro-Gly-Pro and Gly-Pro, and Pro-Gly-Pro forms Gly-Pro.

  16. Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

    PubMed

    Suzuki, Hirohumi; Yamamoto, Toshiharu

    2014-02-01

    In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH. PMID:24034715

  17. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens.

    PubMed

    Kalunke, Raviraj M; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

    2015-01-01

    Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens.

  18. Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit

    SciTech Connect

    Badouel, Caroline; Chartrain, Isabelle; Blot, Joelle; Tassan, Jean-Pierre

    2010-08-01

    MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

  19. VUV action spectroscopy of protonated leucine-enkephalin peptide in the 6-14 eV range

    SciTech Connect

    Ranković, M. Lj.; Canon, F.; Nahon, L.; Giuliani, A.; Milosavljević, A. R.

    2015-12-29

    We have studied the VUV photodissociation of gas-phase protonated leucine-enkephalin peptide ion in the 5.7 to 14 eV photon energy range by coupling a linear quadrupole ion trap with a synchrotron radiation source. We report VUV activation tandem mass spectra at 6.7, 8.4 and 12.8 eV photon energies and photodissociation yields for a number of selected fragments. The obtained results provide insights into both near VUV radiation damage and electronic properties of a model peptide. We could distinguish several absorption bands and assign them to particular electronic transitions, according to previous theoretical studies. Furthermore, the photodissociation yields appear to be very different for the various observed fragmentation channels, depending both on the type of fragments and their position along the peptide backbone. The present results are discussed in light of recent gas-phase spectroscopic data on peptides.

  20. VUV action spectroscopy of protonated leucine-enkephalin peptide in the 6-14 eV range

    DOE PAGES

    Ranković, M. Lj.; Canon, F.; Nahon, L.; Giuliani, A.; Milosavljević, A. R.

    2015-12-29

    We have studied the VUV photodissociation of gas-phase protonated leucine-enkephalin peptide ion in the 5.7 to 14 eV photon energy range by coupling a linear quadrupole ion trap with a synchrotron radiation source. We report VUV activation tandem mass spectra at 6.7, 8.4 and 12.8 eV photon energies and photodissociation yields for a number of selected fragments. The obtained results provide insights into both near VUV radiation damage and electronic properties of a model peptide. We could distinguish several absorption bands and assign them to particular electronic transitions, according to previous theoretical studies. Furthermore, the photodissociation yields appear to bemore » very different for the various observed fragmentation channels, depending both on the type of fragments and their position along the peptide backbone. The present results are discussed in light of recent gas-phase spectroscopic data on peptides.« less

  1. Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit.

    PubMed

    Badouel, Caroline; Chartrain, Isabelle; Blot, Joëlle; Tassan, Jean-Pierre

    2010-08-01

    MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

  2. Kinetics of D-glucose and L-leucine transport into sheep and pig intestinal brush border membrane vesicles.

    PubMed

    Wolffram, S; Eggenberger, E; Scharrer, E

    1986-01-01

    The kinetic parameters (Vmax, Kt) of Na+-dependent D-glucose transport into brush border membrane vesicles (BBMV) from sheep and pig jejunum were determined. Due to the fermentation of ingested carbohydrates in the rumen the small intestine of ruminants (sheep) has to absorb much less glucose than the small intestine of monogastric omnivores (pigs) or herbivores. Kinetic analysis of the concentration dependence of D-glucose transport revealed a ten-fold smaller Vmax value combined with a five times lower Kt value in sheep BBMV compared with pig BBMV. The Vmax value for L-leucine transport did not differ between the two species investigated, whereas the Kt value in the sheep exceeded that in the pig. It is concluded from these results that the mechanism for Na+-dependent D-glucose transport in ruminants is adapted to the small amounts of carbohydrates reaching the small intestine.

  3. Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity

    PubMed Central

    Merelo, Paz; Ram, Hathi; Pia Caggiano, Monica; Ohno, Carolyn; Ott, Felix; Straub, Daniel; Graeff, Moritz; Cho, Seok Keun; Yang, Seong Wook; Wenkel, Stephan; Heisler, Marcus G.

    2016-01-01

    A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial–abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets. PMID:27698117

  4. Low Microbial Respiration of Leucine at Ambient Oceanic Concentration in the Mixed Layer of the Central Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Hill, P.; Warwick, P.; Zubkov, M.

    2012-12-01

    Bacterioplankton are the primary consumers of dissolved organic matter in the ocean, thus the quantification of bacterioplankton production (BP) is essential to our understanding of carbon cycling in the largest ecosystems on Earth. We compared BP, measured as the rate of 14C-leucine (14C-Leu) or 3H-leucine (3H-Leu) uptake at close to saturating concentration (20 nM), with ambient Leu uptake estimated from dilution bioassays. The latter uses 3H-Leu additions at a range of concentrations close to ambient to estimate ambient Leu uptake rates, in addition to bioavailability and turnover rates. We hypothesised that saturation with Leu would lead to its respiration as a carbon source, thereby not truly representing ambient BP. Seawater samples were collected from the photic zone (22-170 m) in mesotrophic and oligotrophic regions along a transect through the central Atlantic Ocean. Respiration as a proportion of total consumption (uptake + respiration) of close to ambient (0.4 nM) and close to saturating (20 nM) 14C-Leu additions were compared. Leu uptake rates measured using saturating 3H-Leu additions were generally comparable with ambient rates estimated by dilution bioassays; however, saturating additions may overestimate uptake at low rates and underestimate uptake at high rates. The proportion of total Leu uptake that was respired was 3-fold higher for 20 nM 14C-Leu additions than 0.4 nM 14C-Leu additions (15±8% and 5±4%, respectively). Consequently, microbial efficiency of Leu assimilation - an indicator of bacterioplankton growth efficiency - was significantly higher at close to ambient 14C-Leu additions than at close to saturating concentrations (95±4% and 85±8%, respectively). Thus, saturation of open Atlantic Ocean bacterioplankton with Leu, or other molecules indicative of microbial metabolism, leads to the measurement of a response to nutrient addition, rather than an ambient measurement.

  5. The hypersensitive induced reaction and leucine-rich repeat proteins regulate plant cell death associated with disease and plant immunity.

    PubMed

    Choi, Hyong Woo; Kim, Young Jin; Hwang, Byung Kook

    2011-01-01

    Pathogen-induced programmed cell death (PCD) is intimately linked with disease resistance and susceptibility. However, the molecular components regulating PCD, including hypersensitive and susceptible cell death, are largely unknown in plants. In this study, we show that pathogen-induced Capsicum annuum hypersensitive induced reaction 1 (CaHIR1) and leucine-rich repeat 1 (CaLRR1) function as distinct plant PCD regulators in pepper plants during Xanthomonas campestris pv. vesicatoria infection. Confocal microscopy and protein gel blot analyses revealed that CaLRR1 and CaHIR1 localize to the extracellular matrix and plasma membrane (PM), respectively. Bimolecular fluorescent complementation and coimmunoprecipitation assays showed that the extracellular CaLRR1 specifically binds to the PM-located CaHIR1 in pepper leaves. Overexpression of CaHIR1 triggered pathogen-independent cell death in pepper and Nicotiana benthamiana plants but not in yeast cells. Virus-induced gene silencing (VIGS) of CaLRR1 and CaHIR1 distinctly strengthened and compromised hypersensitive and susceptible cell death in pepper plants, respectively. Endogenous salicylic acid levels and pathogenesis-related gene transcripts were elevated in CaHIR1-silenced plants. VIGS of NbLRR1 and NbHIR1, the N. benthamiana orthologs of CaLRR1 and CaHIR1, regulated Bax- and avrPto-/Pto-induced PCD. Taken together, these results suggest that leucine-rich repeat and hypersensitive induced reaction proteins may act as cell-death regulators associated with plant immunity and disease.

  6. A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

    NASA Astrophysics Data System (ADS)

    Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

    2014-07-01

    De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

  7. 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

  8. Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

    PubMed

    Graveline, Richard; Garneau, Philippe; Martin, Christine; Mourez, Michaël; Hancock, Mark A; Lavoie, Rémi; Harel, Josée

    2014-08-15

    CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell. PMID:24914179

  9. Leucine-enriched essential amino acids attenuate muscle soreness and improve muscle protein synthesis after eccentric contractions in rats.

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Mimura, Masako; Inoue, Yoshiko; Sugita, Mayu; Suzuki, Katsuya; Kobayashi, Hisamine

    2015-06-01

    Eccentric exercise results in prolonged muscle weakness and muscle soreness, which are typical symptoms of muscle damage. Recovery from muscle damage is related to mammalian target of rapamycin (mTOR) activity. Leucine-enriched essential amino acids (LEAAs) stimulate muscle protein synthesis via activation of the mTOR pathway. Therefore, we investigated the effect of LEAAs on muscle protein synthesis and muscle soreness after eccentric contractions (EC). Male Sprague-Dawley rats (9-11 weeks old) were administered an LEAA solution (AminoL40; containing 40 % leucine and 60 % other essential amino acids) at 1 g/kg body weight or distilled water (control) 30 min before and 10 min after EC. Tibialis anterior (TA) muscle was exposed to 500 EC by electrical stimulation under anesthesia. The fractional synthesis rate (FSR; %/h) in the TA muscle was measured by incorporating L-[ring-(2)H5] phenylalanine into skeletal muscle protein. Muscle soreness was evaluated by the paw withdrawal threshold using the Randal-Selitto test with some modifications from 1 to 3 days after EC. The FSR in the EC-control group (0.147 ± 0.016 %/h) was significantly lower than in the sedentary group (0.188 ± 0.016 %/h, p < 0.05). AminoL40 administration significantly mitigated the EC-induced impairment of the FSR (0.172 ± 0.018 %/h). EC decreased the paw withdrawal threshold at 1 and 2 days after EC, which indicated that EC induced muscle soreness. Furthermore, AminoL40 administration alleviated the decreased paw withdrawal threshold. These findings suggest that LEAA supplementation improves the rate of muscle protein synthesis and ameliorates muscle soreness after eccentric exercise. PMID:25772815

  10. Effect of Walker 256 tumor growth on intestinal absorption of leucine, methionine and glucose in newly weaned and mature rats.

    PubMed

    Gomes-Marcondes, M C; Honma, H N; Areas, M A; Cury, L

    1998-10-01

    In tumor-bearing rats, most of the serum amino acids are used for synthesis and oxidation processes by the neoplastic tissue. In the present study, the effect of Walker 256 carcinoma growth on the intestinal absorption of leucine, methionine and glucose was investigated in newly weaned and mature rats. Food intake and carcass weight were decreased in newly weaned (NT) and mature (MT) rats bearing Walker 256 tumor in comparison with control animals (NC and MC). The tumor/carcass weight ratio was higher in NT than in MT rats, whereas nitrogen balance was significantly decreased in both as compared to control animals. Glucose absorption was significantly reduced in MT rats (MT = 47.3 +/- 4.9 vs MC = 99.8 +/- 5.3 nmol min-1 cm-1, Kruskal-Wallis test, P < 0.05) but this fact did not hamper the evolution of cancer. There was a significant increase in methionine absorption in both groups (NT = 4.2 +/- 0.3 and MT = 2.0 +/- 0.1 vs NC = 3.7 +/- 0.1 and MC = 1.2 +/- 0.2 nmol min-1 cm-1, Kruskal-Wallis test, P < 0.05), whereas leucine absorption was increased only in young tumor-bearing rats (NT = 8.6 +/- 0.2 vs NC = 7.7 +/- 0.4 nmol min-1 cm-1, Kruskal-Wallis test, P < 0.05), suggesting that these metabolites are being used for synthesis and oxidation processes by the neoplastic cells, which might ensure their rapid proliferation especially in NT rats.

  11. The Pseudomonas aeruginosa liuE gene encodes the 3-hydroxy-3-methylglutaryl coenzyme A lyase, involved in leucine and acyclic terpene catabolism.

    PubMed

    Chávez-Avilés, Mauricio; Díaz-Pérez, Alma Laura; Reyes-de la Cruz, Homero; Campos-García, Jesús

    2009-07-01

    The enzymes involved in the catabolism of leucine are encoded by the liu gene cluster in Pseudomonas aeruginosa PAO1. A mutant in the liuE gene (ORF PA2011) of P. aeruginosa was unable to utilize both leucine/isovalerate and acyclic terpenes as the carbon source. The liuE mutant grown in culture medium with citronellol accumulated metabolites of the acyclic terpene pathway, suggesting an involvement of liuE in both leucine/isovalerate and acyclic terpene catabolic pathways. The LiuE protein was expressed as a His-tagged recombinant polypeptide purified by affinity chromatography in Escherichia coli. LiuE showed a mass of 33 kDa under denaturing and 79 kDa under nondenaturing conditions. Protein sequence alignment and fingerprint sequencing suggested that liuE encodes 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HMG-CoA lyase), which catalyzes the cleavage of HMG-CoA to acetyl-CoA and acetoacetate. LiuE showed HMG-CoA lyase optimal activity at a pH of 7.0 and 37 degrees C, an apparent K(m) of 100 microM for HMG-CoA and a V(max) of 21 micromol min(-1) mg(-1). These results demonstrate that the liuE gene of P. aeruginosa encodes for the HMG-CoA lyase, an essential enzyme for growth in both leucine and acyclic terpenes.

  12. Differential effects of L-tryptophan and L-leucine administration on brain resting state functional networks and plasma hormone levels

    PubMed Central

    Zanchi, Davide; Meyer-Gerspach, Anne Christin; Suenderhauf, Claudia; Janach, Katharina; le Roux, Carel W.; Haller, Sven; Drewe, Jürgen; Beglinger, Christoph; Wölnerhanssen, Bettina K.; Borgwardt, Stefan

    2016-01-01

    Depending on their protein content, single meals can rapidly influence the uptake of amino acids into the brain and thereby modify brain functions. The current study investigates the effects of two different amino acids on the human gut-brain system, using a multimodal approach, integrating physiological and neuroimaging data. In a randomized, placebo-controlled trial, L-tryptophan, L-leucine, glucose and water were administered directly into the gut of 20 healthy subjects. Functional MRI (fMRI) in a resting state paradigm (RS), combined with the assessment of insulin and glucose blood concentration, was performed before and after treatment. Independent component analysis with dual regression technique was applied to RS-fMRI data. Results were corrected for multiple comparisons. In comparison to glucose and water, L-tryptophan consistently modifies the connectivity of the cingulate cortex in the default mode network, of the insula in the saliency network and of the sensory cortex in the somatosensory network. L-leucine has lesser effects on these functional networks. L-tryptophan and L-leucine also modified plasma insulin concentration. Finally, significant correlations were found between brain modifications after L-tryptophan administration and insulin plasma levels. This study shows that acute L-tryptophan and L-leucine intake directly influence the brain networks underpinning the food-reward system and appetite regulation. PMID:27760995

  13. L-Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway.

    PubMed

    Payne, Elspeth M; Virgilio, Maria; Narla, Anupama; Sun, Hong; Levine, Michelle; Paw, Barry H; Berliner, Nancy; Look, A Thomas; Ebert, Benjamin L; Khanna-Gupta, Arati

    2012-09-13

    Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34⁺ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

  14. Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and pathways that regulate ubiquitin ligase expression in rainbow trout primary myocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of four day old rainbow trout myocytes. Supplementing media with 100 nM IGF-I inc...

  15. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upreg...

  16. Enteral leucine supplementation increases protein synthesis in skeletal and cardiac muscles and visceral tissues of neonatal pigs through mTORC1-dependent pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine activates mammalian target of rapamycin (mTOR) to upregulate protein synthesis (PS). To examine enteral Leu effects on PS and signaling activation, 5-d-old piglets were fed for 24 h diets containing: (i) LP, (ii) LP+L, or (iii) HP. PS in skeletal muscles, heart, liver, pancreas, and jejunum...

  17. Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

  18. Branched-chain amino acid interactions with reference to amino acid requirements in adult men: Valine metabolism at different leucine intakes

    SciTech Connect

    Pelletier, V.; Marks, L.; Wagner, D.A.; Hoerr, R.A.; Young, V.R. )

    1991-08-01

    The authors explored whether the oxidation of valine and by implication the physiological requirement for this amino acid are affected by changes in leucine intake over a physiological range. Six young adult men received, in random order, four L-amino acid-based diets for 5 d supplying either 20 or 10 mg valine.kg body wt-1.d-1, each in combination with 80 or 40 mg leucine.kg-1.d-1. On day 6 subjects were studied with an 8-h continuous intravenous infusion of (1-13C)valine (and (2H3)leucine) to determine valine oxidation in the fasted state (first 3 h) and fed state (last 5 h). Valine oxidation in the fasted state was similar among all diets but was lower (P less than 0.05) in the fed state for the 10 vs 20 mg valine.kg-1.d-1 intake. Leucine intake did not affect valine oxidation. Mean daily valine balance approximated +1.3 mg.kg-1.d-1 for the 20-mg intake and -1.6 mg.kg-1.d-1 for the 10-mg intake. These findings support our previously suggested mean valine requirement estimate of approximately 20 mg.kg-1.d-1.

  19. Role of leucine zipper motifs in association of the Escherichia coli cell division proteins FtsL and FtsB.

    PubMed

    Robichon, Carine; Karimova, Gouzel; Beckwith, Jon; Ladant, Daniel

    2011-09-01

    FtsL and FtsB are two inner-membrane proteins that are essential constituents of the cell division apparatus of Escherichia coli. In this study, we demonstrate that the leucine zipper-like (LZ) motifs, located in the periplasmic domain of FtsL and FtsB, are required for an optimal interaction between these two essential proteins.

  20. Basics.

    ERIC Educational Resources Information Center

    Instructor, 1982

    1982-01-01

    Recommended activities include: (1) etymology exercises for elementary school students; (2) a search for information about Alexander the Great; (3) monthly inspections of the school yard to observe environmental changes; and (4) an art history unit on Cro-Magnon cave drawings. An interdisciplinary unit on transportation is included. (PP)