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.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PubMed

Yeh, Szu-Tsen; Zambrano, Cristina M; Koch, Walter J; Purcell, Nicole H

2018-05-25

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Leucine-rich repeat kinase 2 inhibitors: a patent review (2014-2016).

PubMed

Galatsis, Paul

2017-06-01

Leucine-rich repeat kinase 2 (LRRK2) is a member of the Tyrosine Kinase-Like (TKL) branch of the kinome tree and is a multi-domain protein that includes GTPase and kinase activity. While genome-wide association studies (GWAS) has linked LRRK2 with Crohn's disease and leprosy, it has received the greatest attention due to it being implicated as one of the genetic loci associated with autosomal dominant inheritance in Parkinson's disease (PD). Areas covered: In this review, the small molecule patent literature from 2014-2016 with a focus on composition of matter and use patents was surveyed. Scifinder was primarily searched using 'LRRK2' as the query to identify all relevant literature and then triaged for small molecule patents. Expert opinion: The patent landscape around LRRK2 continues to develop. The early patents covered using existing kinase inhibitors for use against LRRK2. This evolved to compounds specifically designed for selectivity against LRRK2, but key exemplified compounds lacked sufficient brain exposure to affect sufficient efficacy. More recent compounds have addressed this deficiency and show greater potential for treating PD. While potency will be necessary to generate medicines with low human daily doses, brain penetration and safety will be the key differentiators for ultimately determining the most effective LRRK2 disease-modifying treatment for PD.

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

PubMed

Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

2018-04-27

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed Central

Kwak, Su-Hwan

2008-01-01

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. PMID:19704725

G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization

PubMed Central

Papkovskaia, Tatiana D.; Chau, Kai-Yin; Inesta-Vaquera, Francisco; Papkovsky, Dmitri B.; Healy, Daniel G.; Nishio, Koji; Staddon, James; Duchen, Michael R.; Hardy, John; Schapira, Anthony H.V.; Cooper, J. Mark

2012-01-01

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD. PMID:22736029

Polysaccharide Peptide-Induced Virus Resistance Depends on Ca2+ Influx by Increasing the Salicylic Acid Content and Upregulating the Leucine-Rich Repeat Gene in Arabidopsis thaliana.

PubMed

Zhao, Lei; Chen, Yujia; Yang, Wen; Zhang, Yuanle; Chen, Wenbao; Feng, Chaohong; Wang, Qaochun; Wu, Yunfeng

2018-05-01

Plant viral diseases cause severe economic losses in agricultural production. The development of biosource-derived antiviral agents provides an alternative strategy to efficiently control plant viral diseases. We previously reported that the exogenous application of polysaccharide peptide (PSP) exerts significant inhibitive effects on Tobacco mosaic virus infection in Nicotiana tabacum. In this study, we studied in additional detail the mechanism by which PSP can induce virus resistance in Arabidopsis thaliana. We found that PSP significantly induced Ca 2+ influx and increased the accumulation of hydrogen peroxide and salicylic acid (SA) in the A. thaliana cells. A gene with a toll interleukin 1 receptor-nucleotide binding site-leucine-rich repeat domain (LRR) was obtained by RNA sequencing in combination with the screening of the gene-deletion mutants of A. thaliana. The LRR gene was deleted, and the inductive response of A. thaliana to PSP was significantly attenuated after mutation. After the heterologous overexpression of the LRR gene in N. benthamiana, the SA content and PR1 gene expression in N. benthamiana were significantly increased. Through analyses of the LRR gene expression and the ability of A. thaliana to resist Cucumber mosaic virus following the treatments of PSP and PSP + ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid, it was shown that PSP enhanced the virus resistance of A. thaliana by inducing Ca 2+ influx and subsequently improving expression of the LRR gene, which further increased the SA content.

Leucine-rich repeat kinase 2 inhibitors: a review of recent patents (2011 - 2013).

PubMed

Kethiri, Raghava R; Bakthavatchalam, Rajagopal

2014-07-01

Leucine-rich repeat kinase 2 (LRRK2) is a large (2527 residues) complex multi-domain protein that has GTPase and kinase domains. Autosomal dominant missense mutations in LRRK2 have been found in individuals with Parkinson's disease (PD) and are considered responsible for 1% of all cases of PD. Among the mutations confirmed to contribute to PD pathogenicity, G2019S is the most common cause of PD and it increases the kinase activity of LRRK2 by around threefold. LRRK2 has received considerable attention as a therapeutic target for PD, and LRRK2 inhibitors may help prevent and/or treat the disease. LRRK2 inhibitors are being investigated by various industrial and academic institutions. The present review covers patents literature on small-molecule LRRK2 inhibitors patented between 2011 and 2013. Currently, wild-type and mutant LRRK2 are being examined as therapeutic targets for PD. In testimony to the significance of these novel targets, over 20 patent applications related to LRRK2 have been filed in the last 3 years. Several distinct chemotypes have been reported to be LRRK2 inhibitors with very good potency. These compounds are being used to elucidate the physiological and pathophysiological functions of LRRK2, and some may even emerge as therapeutics for PD.

Leucine-Rich Repeat Kinase 2 in Parkinson's Disease: Updated from Pathogenesis to Potential Therapeutic Target.

PubMed

Chen, Jinhua; Chen, Ying; Pu, Jiali

2018-04-27

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the midbrain. The pathogenesis of PD is not fully understood but is likely caused by a combination of genetic and environmental factors. Several genes are associated with the onset and progression of familial PD. There is increasing evidence that leucine-rich repeat kinase 2 (LRRK2) plays a significant role in PD pathophysiology. Many studies have been conducted to elucidate the functions of LRRK2 and identify effective LRRK2 inhibitors for PD treatment. In this review, we discuss the role of LRRK2 in PD and recent progress in the use of LRRK2 inhibitors as therapeutic agents. Key Messages: LRRK2 plays a significant role in the pathophysiology of PD, and pharmacological inhibition of LRRK2 has become one of the most promising potential therapies for PD. Further research is warranted to determine the functions of LRRK2 and expand the applications of LRRK2 inhibitors in PD treatment. © 2018 S. Karger AG, Basel.

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

Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution*

PubMed Central

Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina

2014-01-01

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733

A MicroRNA Superfamily Regulates Nucleotide Binding Site–Leucine-Rich Repeats and Other mRNAs[W][OA

PubMed Central

Shivaprasad, Padubidri V.; Chen, Ho-Ming; Patel, Kanu; Bond, Donna M.; Santos, Bruno A.C.M.; Baulcombe, David C.

2012-01-01

Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack. PMID:22408077

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

PubMed Central

Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

2009-01-01

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

Distinct Secondary Structures of the Leucine-Rich Repeat Proteoglycans Decorin and Biglycan: Glycosylation-Dependent Conformational Stability

NASA Technical Reports Server (NTRS)

Krishnan, Priya; Hocking, Anne M.; Scholtz, J. Martin; Pace, C. Nick; Holik, Kimberly K.; McQuillan, David J.

1998-01-01

Biglycan and decorin, closely related small leucine-rich repeat proteoglycans, have been overexpressed in eukaryotic cers and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans. A comparative biophysical study of these glycoforms has revealed that the overall secondary structures of biglycan and decorin are different. Far-UV Circular Dichroism (CD) spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly Beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to. the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the fmal form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that at least in this specific domain, the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provided further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1-2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan

A Tomato Nucleotide Binding Sites-Leucine-Rich Repeat Gene Is Positively Involved in Plant Resistance to Phytophthora infestans.

PubMed

Jiang, Ning; Cui, Jun; Meng, Jun; Luan, Yushi

2018-06-14

The nucleotide binding sites-leucine-rich repeat (NBS-LRR) genes are key regulatory components of plant to pathogens. Phytophthora infestans-inducible coding sequence encoding an NBS-LRR (SpNBS-LRR) protein in tomato (Solanum pimpinellifolium L3708) was cloned and characterized based on our RNA-Seq data and tomato genome. After sequence analysis, SpNBS-LRR was identified as a hydrophilic protein with no transmembrane topological structure and no signal peptide. SpNBS-LRR had a close genetic relationship to RPS2 of Arabidopsis thaliana by phylogenetic analysis. In addition, SpNBS-LRR gene was mainly expressed in root, with low expression observed in leaf and stem. To further investigate the role of SpNBS-LRR in tomato-P. infestans interaction, SpNBS-LRR was introduced in susceptible tomatoes and three transgenic lines with higher expression level of SpNBS-LRR were selected. These transgenic tomato plants that overexpressed SpNBS-LRR displayed greater resistance than wild-type tomato plants after infection with P. infestans, as shown by decreased disease index, lesion diameters, number of necrotic cells, P. infestans abundance, and higher expression levels of the defense-related genes. This information provides insight into SpNBS-LRR involved in the resistance of tomato to P. infestans infection and candidate for breeding to enhance biotic stress-resistance in tomato.

Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease

PubMed Central

Beilina, Alexandria; Rudenko, Iakov N.; Kaganovich, Alice; Civiero, Laura; Chau, Hien; Kalia, Suneil K.; Kalia, Lorraine V.; Lobbestael, Evy; Chia, Ruth; Ndukwe, Kelechi; Ding, Jinhui; Nalls, Mike A.; Olszewski, Maciej; Hauser, David N.; Kumaran, Ravindran; Lozano, Andres M.; Baekelandt, Veerle; Greene, Lois E.; Taymans, Jean-Marc; Greggio, Elisa; Cookson, Mark R.; Nalls, Mike A.; Plagnol, Vincent; Martinez, Maria; Hernandez, Dena G; Sharma, Manu; Sheerin, Una-Marie; Saad, Mohamad; Simón-Sánchez, Javier; Schulte, Claudia; Lesage, Suzanne; Sveinbjörnsdóttir, Sigurlaug; Arepalli, Sampath; Barker, Roger; Ben-Shlomo, Yoav; Berendse, Henk W; Berg, Daniela; Bhatia, Kailash; de Bie, Rob M A; Biffi, Alessandro; Bloem, Bas; Bochdanovits, Zoltan; Bonin, Michael; Bras, Jose M; Brockmann, Kathrin; Brooks, Janet; Burn, David J; Charlesworth, Gavin; Chen, Honglei; Chong, Sean; Clarke, Carl E; Cookson, Mark R; Cooper, J Mark; Corvol, Jean Christophe; Counsell, Carl; Damier, Philippe; Dartigues, Jean-François; Deloukas, Panos; Deuschl, Günther; Dexter, David T; van Dijk, Karin D; Dillman, Allissa; Durif, Frank; Dürr, Alexandra; Edkins, Sarah; Evans, Jonathan R; Foltynie, Thomas; Gao, Jianjun; Gardner, Michelle; Gibbs, J Raphael; Goate, Alison; Gray, Emma; Guerreiro, Rita; Gústafsson, Ómar; Harris, Clare; van Hilten, Jacobus J; Hofman, Albert; Hollenbeck, Albert; Holton, Janice; Hu, Michele; Huang, Xuemei; Huber, Heiko; Hudson, Gavin; Hunt, Sarah E; Huttenlocher, Johanna; Illig, Thomas; München, Helmholtz Zentrum; Jónsson, Pálmi V; Lambert, Jean-Charles; Langford, Cordelia; Lees, Andrew; Lichtner, Peter; München, Helmholtz Zentrum; Limousin, Patricia; Lopez, Grisel; Lorenz, Delia; McNeill, Alisdair; Moorby, Catriona; Moore, Matthew; Morris, Huw R; Morrison, Karen E; Mudanohwo, Ese; O’Sullivan, Sean S; Pearson, Justin; Perlmutter, Joel S; Pétursson, Hjörvar; Pollak, Pierre; Post, Bart; Potter, Simon; Ravina, Bernard; Revesz, Tamas; Riess, Olaf; Rivadeneira, Fernando; Rizzu, Patrizia; Ryten, Mina; Sawcer, Stephen; Schapira, Anthony; Scheffer, Hans; Shaw, Karen; Shoulson, Ira; Sidransky, Ellen; Smith, Colin; Spencer, Chris C A; Stefánsson, Hreinn; Steinberg, Stacy; Stockton, Joanna D; Strange, Amy; Talbot, Kevin; Tanner, Carlie M; Tashakkori-Ghanbaria, Avazeh; Tison, François; Trabzuni, Daniah; Traynor, Bryan J; Uitterlinden, André G; Velseboer, Daan; Vidailhet, Marie; Walker, Robert; van de Warrenburg, Bart; Wickremaratchi, Mirdhu; Williams, Nigel; Williams-Gray, Caroline H; Winder-Rhodes, Sophie; Stefánsson, Kári; Hardy, John; Heutink, Peter; Brice, Alexis; Gasser, Thomas; Singleton, Andrew B; Wood, Nicholas W; Chinnery, Patrick F; Arepalli, Sampath; Cookson, Mark R; Dillman, Allissa; Ferrucci, Luigi; Gibbs, J Raphael; Hernandez, Dena G; Johnson, Robert; Longo, Dan L; Majounie, Elisa; Nalls, Michael A; O’Brien, Richard; Singleton, Andrew B; Traynor, Bryan J; Troncoso, Juan; van der Brug, Marcel; Zielke, H Ronald; Zonderman, Alan B

2014-01-01

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms. PMID:24510904

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

PubMed Central

Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

2014-01-01

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Haiying; Cui, Yazhou; Luan, Jing

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit themore » level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.« less

Interplay between Leucine-Rich Repeat Kinase 2 (LRRK2) and p62/SQSTM-1 in Selective Autophagy.

PubMed

Park, Sangwook; Han, Seulki; Choi, Insup; Kim, Beomsue; Park, Seung Pyo; Joe, Eun-Hye; Suh, Young Ho

2016-01-01

The deposit of polyubiquitinated aggregates has been implicated in the pathophysiology of Parkinson's disease (PD), and growing evidence indicates that selective autophagy plays a critical role in the clearance of ubiquitin-positive protein aggregates by autophagosomes. The selective autophagic receptor p62/SQSTM-1, which associates directly with both ubiquitin and LC3, transports ubiquitin conjugates to autophagosomes for degradation. Leucine-rich repeat kinase 2 (LRRK2), a PD-associated protein kinase, is tightly controlled by autophagy-lysosome degradation as well as by the ubiquitin-proteasome pathway. However, little is known about the degradation of ubiquitinated LRRK2 via selective autophagy. In the present study, we found that p62/SQSTM-1 physically interacts with LRRK2 as a selective autophagic receptor. The overexpression of p62 leads to the robust degradation of LRRK2 through the autophagy-lysosome pathway. In addition, LRRK2 indirectly regulates Ser351 and Ser403 phosphorylation of p62. Of particular interest, the interaction between phosphorylated p62 and Keap1 is reduced by LRRK2 overexpression. Therefore, we propose that the interplay between LRRK2 and p62 may contribute to the pathophysiological function and homeostasis of LRRK2 protein.

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

DOE PAGES

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.; ...

2017-04-19

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

A harpin elicitor induces the expression of a coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene and others functioning during defense to parasitic nematodes.

PubMed

Aljaafri, Weasam A R; McNeece, Brant T; Lawaju, Bisho R; Sharma, Keshav; Niruala, Prakash M; Pant, Shankar R; Long, David H; Lawrence, Kathy S; Lawrence, Gary W; Klink, Vincent P

2017-12-01

The bacterial effector harpin induces the transcription of the Arabidopsis thaliana NON-RACE SPECIFIC DISEASE RESISTANCE 1/HARPIN INDUCED1 (NDR1/HIN1) coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene. In Glycine max, Gm-NDR1-1 transcripts have been detected within root cells undergoing a natural resistant reaction to parasitism by the syncytium-forming nematode Heterodera glycines, functioning in the defense response. Expressing Gm-NDR1-1 in Gossypium hirsutum leads to resistance to Meloidogyne incognita parasitism. In experiments presented here, the heterologous expression of Gm-NDR1-1 in G. hirsutum impairs Rotylenchulus reniformis parasitism. These results are consistent with the hypothesis that Gm-NDR1-1 expression functions broadly in generating a defense response. To examine a possible relationship with harpin, G. max plants topically treated with harpin result in induction of the transcription of Gm-NDR1-1. The result indicates the topical treatment of plants with harpin, itself, may lead to impaired nematode parasitism. Topical harpin treatments are shown to impair G. max parasitism by H. glycines, M. incognita and R. reniformis and G. hirsutum parasitism by M. incognita and R. reniformis. How harpin could function in defense has been examined in experiments showing it also induces transcription of G. max homologs of the proven defense genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), TGA2, galactinol synthase, reticuline oxidase, xyloglucan endotransglycosylase/hydrolase, alpha soluble N-ethylmaleimide-sensitive fusion protein (α-SNAP) and serine hydroxymethyltransferase (SHMT). In contrast, other defense genes are not directly transcriptionally activated by harpin. The results indicate harpin induces pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) defense processes in the root, activating defense to parasitic nematodes. Copyright © 2017. Published by Elsevier

Nucleotide-Binding Domain Leucine-Rich Repeat Containing Proteins and Intestinal Microbiota: Pivotal Players in Colitis and Colitis-Associated Cancer Development.

PubMed

Prossomariti, Anna; Sokol, Harry; Ricciardiello, Luigi

2018-01-01

The nucleotide-binding domain leucine-rich repeat containing (NLR) proteins play a fundamental role in innate immunity and intestinal tissue repair. A dysbiotic intestinal microbiota, developed as a consequence of alterations in NLR proteins, has recently emerged as a crucial hit for the development of ulcerative colitis (UC) and colitis-associated cancer (CAC). The concept of the existence of functional axes interconnecting bacteria with NLR proteins in a causal role in intestinal inflammation and CAC aroused a great interest for the potential development of preventive and therapeutic strategies against UC and CAC. However, the most recent scientific evidence, which highlights many confounding factors in studies based on microbiota characterization, underlines the need for an in-depth reconsideration of the data obtained until now. The purpose of this review is to discuss the recent findings concerning the cross talk between the NLR signaling and the intestinal microbiota in UC and CAC development, and to highlight the open issues that should be explored and addressed in future studies.

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

The wheat homolog of putative nucleotide-binding site-leucine-rich repeat resistance gene TaRGA contributes to resistance against powdery mildew.

PubMed

Wang, Defu; Wang, Xiaobing; Mei, Yu; Dong, Hansong

2016-03-01

Powdery mildew, one of the most destructive wheat diseases worldwide, is caused by Blumeria graminis f. sp. tritici (Bgt), a fungal species with a consistently high mutation rate that makes individual resistance (R) genes ineffective. Therefore, effective resistance-related gene cloning is vital for breeding and studying the resistance mechanisms of the disease. In this study, a putative nucleotide-binding site-leucine-rich repeat (NBS-LRR) R gene (TaRGA) was cloned using a homology-based cloning strategy and analyzed for its effect on powdery mildew disease and wheat defense responses. Real-time reverse transcription-PCR (RT-PCR) analyses revealed that a Bgt isolate 15 and salicylic acid stimulation significantly induced TaRGA in the resistant variety. Furthermore, the silencing of TaRGA in powdery mildew-resistant plants increased susceptibility to Bgt15 and prompted conidia propagation at the infection site. However, the expression of TaRGA in leaf segments after single-cell transient expression assay highly increased the defense responses to Bgt15 by enhancing callose deposition and phenolic autofluorogen accumulation at the pathogen invading sites. Meanwhile, the expression of pathogenesis-related genes decreased in the TaRGA-silenced plants and increased in the TaRGA-transient-overexpressing leaf segments. These results implied that the TaRGA gene positively regulates the defense response to powdery mildew disease in wheat.

Crystal structure of LGR4-Rspo1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs).

PubMed

Xu, Jin-Gen; Huang, Chunfeng; Yang, Zhengfeng; Jin, Mengmeng; Fu, Panhan; Zhang, Ni; Luo, Jian; Li, Dali; Liu, Mingyao; Zhou, Yan; Zhu, Yongqun

2015-01-23

Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Two rice GRAS family genes responsive to N -acetylchitooligosaccharide elicitor are induced by phytoactive gibberellins: evidence for cross-talk between elicitor and gibberellin signaling in rice cells.

PubMed

Day, R Bradley; Tanabe, Shigeru; Koshioka, Masaji; Mitsui, Toshiaki; Itoh, Hironori; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto; Kaku, Hanae; Shibuya, Naoto; Minami, Eiichi

2004-01-01

In this study, we present data showing that two members of the GRAS family of genes from rice, CIGR1 and CIGR2 (chitin-inducible gibberellin-responsive), inducible by the potent elicitor N -acetylchitooligosaccharide (GN), are rapidly induced by exogenous gibberellins. The pattern of mRNA accumulation was dependent on the dose and biological activity of the gibberellins, suggesting that the induction of the genes by gibberellin is mediated by a biological receptor capable of specific recognition and signal transduction upon perception of the phytoactive compounds. Further pharmacological analysis revealed that the CIGR1 and CIGR2 mRNA accumulation by treatment with gibberellin is dependent upon protein phosphorylation/dephosphorylation events. In rice calli derived from slender rice 1, a constitutive gibberellin-responsive mutant, or d1, a mutant deficient in the alpha -subunit of the heterotrimeric G-protein, CIGR1 and CIGR2 were induced by a GN elicitor, yet not by gibberellin. Neither gibberellin nor GN showed related activities in defense or development, respectively. These results strongly suggested that the signal transduction cascade from gibberellin is independent of that from GN, and further implied that CIGR1 and CIGR2 have dual, distinct roles in defense and development.

OsLRR-RLK1, an early responsive leucine-rich repeat receptor-like kinase, initiates rice defense responses against a chewing herbivore.

PubMed

Hu, Lingfei; Ye, Meng; Kuai, Peng; Ye, Miaofen; Erb, Matthias; Lou, Yonggen

2018-06-07

Plants are constantly exposed to a variety of environmental stresses, including herbivory. How plants perceive herbivores on a molecular level is poorly understood. Leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest subfamily of RLKs, are essential for plants to detect external stress signals, and may therefore also be involved in herbivore perception. Here, we employed RNA interference silencing, phytohormone profiling and complementation, as well as herbivore resistance assays, to investigate the requirement of an LRR-RLK for the initiation of rice (Oryza sativa) defenses against the chewing herbivore striped stem borer (SSB) Chilo suppressalis. We discovered a plasma membrane-localized LRR-RLK, OsLRR-RLK1, whose transcription is strongly up-regulated by SSB attack and treatment with oral secretions of Spodoptera frugiperda. OsLRR-RLK1 acts upstream of mitogen-activated protein kinase (MPK) cascades, and positively regulates defense-related MPKs and WRKY transcription factors. Moreover, OsLRR-RLK1 is a positive regulator of SSB-elicited, but not wound-elicited, levels of jasmonic acid and ethylene, trypsin protease inhibitor activity and plant resistance towards SSB. OsLRR-RLK1 therefore plays an important role in herbivory-induced defenses of rice. Given the well-documented role of LRR-RLKs in the perception of stress-related molecules, we speculate that OsLRR-RLK1 may be involved in the perception of herbivory-associated molecular patterns. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Oryza sativa BRASSINOSTEROID UPREGULATED1 LIKE1 Induces the Expression of a Gene Encoding a Small Leucine-Rich-Repeat Protein to Positively Regulate Lamina Inclination and Grain Size in Rice

PubMed Central

Jang, Seonghoe; Li, Hsing-Yi

2017-01-01

Oryza sativa BRASSINOSTEROID UPREGULATED1 LIKE1 (OsBUL1) positively affects lamina inclination and grain size. OsBUL1 knock-out (osbul1) plants as well as transgenic rice with reduced level of OsBUL1 expression produce erect leaves and small grains. Here, we identified a putative downstream gene of OsBUL1, OsBUL1 DOWNSTREAM GENE1 (OsBDG1) encoding a small protein with short leucine-rich-repeats by cDNA microarray analyses in the lamina joint and panicles of wild-type and osbul1 plants. Transgenic rice plants with increased OsBDG1 expression exhibit increased leaf angle and grain size, which is similar to an OsBDG1 activation tagging line whereas double stranded RNA interference (dsRNAi) lines for OsBDG1 knock-down generate erect leaves with smaller grains. Moreover, transgenic rice expressing OsBDG1 under the control of OsBUL1 promoter also shows enlarged leaf bending and grain size phenotypes. Two genes, OsAP2 (OsAPETALA2) and OsWRKY24 were identified as being upregulated transcriptional activators in the lamina joint of pOsBUL1:OsBDG1 plants and induced expression of the two genes driven by OsBUL1 promoter caused increased lamina inclination and grain size in rice. Thus, our work demonstrates that a series of genes showing expression cascades are involved in the promotion of cell elongation in lamina joints and functionally cause increased lamina inclination. PMID:28769958

The intracellular nucleotide binding leucine-rich repeat receptor - SlNRC4a enhances immune signaling elicited by extracellular perception.

PubMed

Leibman-Markus, Meirav; Pizarro, Lorena; Schuster, Silvia; Lin, Z J Daniel; Gershony, Ofir; Bar, Maya; Coaker, Gitta; Avni, Adi

2018-05-23

Plant recognition and defense against pathogens employs a two-tiered perception system. Surface localized pattern recognition receptors (PRRs) act to recognize microbial features, while intracellular nucleotide binding leucine-rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signaling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB-LRR Required for HR-associated Cell death-4). Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defense responses while silencing SlNRC4 reduces plant immunity. Moreover, the coiled-coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. Based on these findings, we propose that SlNRC4a acts as a non-canonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perception. This article is protected by copyright. All rights reserved.

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection in the Drosophila neuromuscular system

PubMed Central

Kurusu, Mitsuhiko; Cording, Amy; Taniguchi, Misako; Menon, Kaushiki; Suzuki, Emiko; Zinn, Kai

2008-01-01

Summary In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron. PMID:18817735

PnLRR-RLK27, a novel leucine-rich repeats receptor-like protein kinase from the Antarctic moss Pohlia nutans, positively regulates salinity and oxidation-stress tolerance

PubMed Central

Wang, Jing; Liu, Shenghao; Li, Chengcheng; Wang, Tailin; Chen, Kaoshan

2017-01-01

Leucine-rich repeats receptor-like kinases (LRR-RLKs) play important roles in plant growth and development as well as stress responses. Here, 56 LRR-RLK genes were identified in the Antarctic moss Pohlia nutans transcriptome, which were further classified into 11 subgroups based on their extracellular domain. Of them, PnLRR-RLK27 belongs to the LRR II subgroup and its expression was significantly induced by abiotic stresses. Subcellular localization analysis showed that PnLRR-RLK27 was a plasma membrane protein. The overexpression of PnLRR-RLK27 in Physcomitrella significantly enhanced the salinity and ABA tolerance in their gametophyte growth. Similarly, PnLRR-RLK27 heterologous expression in Arabidopsis increased the salinity and ABA tolerance in their seed germination and early root growth as well as the tolerance to oxidative stress. PnLRR-RLK27 overproduction in these transgenic plants increased the expression of salt stress/ABA-related genes. Furthermore, PnLRR-RLK27 increased the activities of reactive oxygen species (ROS) scavengers and reduced the levels of malondialdehyde (MDA) and ROS. Taken together, these results suggested that PnLRR-RLK27 as a signaling regulator confer abiotic stress response associated with the regulation of the stress- and ABA-mediated signaling network. PMID:28241081

Age-related pathology after adenoviral overexpression of the leucine-rich repeat kinase 2 in the mouse striatum.

PubMed

Kritzinger, Astrid; Ferger, Boris; Gillardon, Frank; Stierstorfer, Birgit; Birk, Gerald; Kochanek, Stefan; Ciossek, Thomas

2018-06-01

Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

A QUICK Screen for Lrrk2 Interaction Partners – Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics*

PubMed Central

Meixner, Andrea; Boldt, Karsten; Van Troys, Marleen; Askenazi, Manor; Gloeckner, Christian J.; Bauer, Matthias; Marto, Jarrod A.; Ampe, Christophe; Kinkl, Norbert; Ueffing, Marius

2011-01-01

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function. PMID:20876399

Origin and diversification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in plants.

PubMed

Liu, Ping-Li; Du, Liang; Huang, Yuan; Gao, Shu-Min; Yu, Meng

2017-02-07

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases in plants and play crucial roles in development and stress responses. The evolutionary relationships among LRR-RLK genes have been investigated in flowering plants; however, no comprehensive studies have been performed for these genes in more ancestral groups. The subfamily classification of LRR-RLK genes in plants, the evolutionary history and driving force for the evolution of each LRR-RLK subfamily remain to be understood. We identified 119 LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes. Furthermore, these LRR-RLK sequences, along with previously reported LRR-RLK sequences from Arabidopsis thaliana and Oryza sativa, were subjected to evolutionary analyses. Phylogenetic analyses revealed that plant LRR-RLKs belong to 19 subfamilies, eighteen of which were established in early land plants, and one of which evolved in flowering plants. More importantly, we found that the basic structures of LRR-RLK genes for most subfamilies are established in early land plants and conserved within subfamilies and across different plant lineages, but divergent among subfamilies. In addition, most members of the same subfamily had common protein motif compositions, whereas members of different subfamilies showed variations in protein motif compositions. The unique gene structure and protein motif compositions of each subfamily differentiate the subfamily classifications and, more importantly, provide evidence for functional divergence among LRR-RLK subfamilies. Maximum likelihood analyses showed that some sites within four subfamilies were under positive selection. Much of the diversity of plant LRR-RLK genes was established in early land plants. Positive selection contributed to the evolution of a few LRR-RLK subfamilies.

The extracellular Leucine-Rich Repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns

PubMed Central

Dolan, Jackie; Walshe, Karen; Alsbury, Samantha; Hokamp, Karsten; O'Keeffe, Sean; Okafuji, Tatsuya; Miller, Suzanne FC; Tear, Guy; Mitchell, Kevin J

2007-01-01

Background Leucine-rich repeats (LRRs) are highly versatile and evolvable protein-ligand interaction motifs found in a large number of proteins with diverse functions, including innate immunity and nervous system development. Here we catalogue all of the extracellular LRR (eLRR) proteins in worms, flies, mice and humans. We use convergent evidence from several transmembrane-prediction and motif-detection programs, including a customised algorithm, LRRscan, to identify eLRR proteins, and a hierarchical clustering method based on TribeMCL to establish their evolutionary relationships. Results This yields a total of 369 proteins (29 in worm, 66 in fly, 135 in mouse and 139 in human), many of them of unknown function. We group eLRR proteins into several classes: those with only LRRs, those that cluster with Toll-like receptors (Tlrs), those with immunoglobulin or fibronectin-type 3 (FN3) domains and those with some other domain. These groups show differential patterns of expansion and diversification across species. Our analyses reveal several clusters of novel genes, including two Elfn genes, encoding transmembrane proteins with eLRRs and an FN3 domain, and six genes encoding transmembrane proteins with eLRRs only (the Elron cluster). Many of these are expressed in discrete patterns in the developing mouse brain, notably in the thalamus and cortex. We have also identified a number of novel fly eLRR proteins with discrete expression in the embryonic nervous system. Conclusion This study provides the necessary foundation for a systematic analysis of the functions of this class of genes, which are likely to include prominently innate immunity, inflammation and neural development, especially the specification of neuronal connectivity. PMID:17868438

A direct interaction between leucine-rich repeat kinase 2 and specific β-tubulin isoforms regulates tubulin acetylation.

PubMed

Law, Bernard M H; Spain, Victoria A; Leinster, Veronica H L; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J; Taymans, Jean-Marc; Urban, Mary K; Sancho, Rosa M; Blanca Ramírez, Marian; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R; Berwick, Daniel C; Harvey, Kirsten

2014-01-10

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

PubMed

Glynn, Neil C; Comstock, Jack C; Sood, Sushma G; Dang, Phat M; Chaparro, Jose X

2008-01-01

Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

Fine tuning cellular recognition: The function of the leucine rich repeat (LRR) trans-membrane protein, LRT, in muscle targeting to tendon cells.

PubMed

Gilsohn, Eli; Volk, Talila

2010-01-01

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.

The prognostic role of Leucine-rich repeat-containing G-protein-coupled receptor 5 in gastric cancer: A systematic review with meta-analysis.

PubMed

Huang, Tianchen; Qiu, Xinguang; Xiao, Jianan; Wang, Qingbing; Wang, Yanjun; Zhang, Yong; Bai, Dongxiao

2016-04-01

The prognostic value of Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) in gastric cancer remains controversial. To further investigate this relationship, we performed meta-analyses to systematically review the association between LGR5 expression and various clinical parameters in gastric cancer patients. Eligible studies from PubMed, Embase, Web of Science, CNKI (Chinese National Knowledge Infrastructure), Wangfang (Database of Chinese Ministry of Science & Technology) and CBM (China Biological Medicine) databases were evaluated to investigate the association of LGR5 expression with overall survival (OS) and clinicopathological features of gastric cancer. LGR5 overexpression was significantly associated with poor OS in patients with gastric cancer (HR 1.66, 95% CI 1.02-2.69). LGR5 overexpression was also significantly associated with TNM stage (TIII/TIV vs TI/TII: OR 5.42, 95% CI 1.02-28.72) and lymph node metastasis (positive vs negative: OR 2.30, 95% CI 1.06-5.0). Our meta-analysis indicates that LGR5 may be a predictive factor for invasion and metastasis, and poor prognosis in patients with gastric cancer. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Leucine-Rich Repeat Kinase 2 Controls the Ca2+/Nuclear Factor of Activated T Cells/IL-2 Pathway during Aspergillus Non-Canonical Autophagy in Dendritic Cells.

PubMed

Wong, Alicia Yoke Wei; Oikonomou, Vasilis; Paolicelli, Giuseppe; De Luca, Antonella; Pariano, Marilena; Fric, Jan; Tay, Hock Soon; Ricciardi-Castagnoli, Paola; Zelante, Teresa

2018-01-01

The Parkinson's disease-associated protein, Leucine-rich repeat kinase 2 (LRRK2), a known negative regulator of nuclear factor of activated T cells (NFAT), is expressed in myeloid cells such as macrophages and dendritic cells (DCs) and is involved in the host immune response against pathogens. Since, the Ca 2+ /NFAT/IL-2 axis has been previously found to regulate DC response to the fungus Aspergillus , we have investigated the role played by the kinase LRRK2 during fungal infection. Mechanistically, we found that in the early stages of the non-canonical autophagic response of DCs to the germinated spores of Aspergillus , LRRK2 undergoes progressive degradation and regulates NFAT translocation from the cytoplasm to the nucleus. Our results shed new light on the complexity of the Ca 2+ /NFAT/IL-2 pathway, where LRRK2 plays a role in controlling the immune response of DCs to Aspergillus .

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

PubMed Central

Jacobsen, S E; Binkowski, K A; Olszewski, N E

1996-01-01

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes. Images Fig. 1 Fig. 2 Fig. 3 PMID:8799194

Auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism

NASA Astrophysics Data System (ADS)

Cai, Weiming; Hu, Liwei; Hu, Xiangyang; Cui, Dayong; Cai, Weiming

Gravitropism is the asymmetric growth or curvature of plant organs in response to gravistimulation. There is a complex signal transduction cascade which involved in the differential growth of plants in response to changes in the gravity vector. The role of auxin in gravitropism has been demonstrated by many experiments, but little is known regarding the molecular details of such effects. In our studies before, mediation of the gravitropic bending of soybean roots and rice leaf sheath bases by nitric oxide, cGMP and gibberellins, are induced by auxin. The asymmetrical distribution of nitric oxide, cGMP and gibberellins resulted from the asymmetrical synthesis of them in bending sites. In soybean roots, inhibitions of NO and cGMP synthesis reduced differential NO and cGMP accumulation respectively, which both of these effects can lead to the reduction of gravitropic bending. Gibberellin-induced OsXET, OsEXPA4 and OsRWC3 were also found involved in the gravitropic bending. These data indicated that auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism. More experiments need to prove the more detailed mechanism of them.

Leucine-rich Repeat Neuronal Protein 1 Regulates Differentiation of Embryonic Stem Cells by Posttranslational Modifications of Pluripotency Factors.

PubMed

Liao, Chien Huang; Wang, Ya-Hui; Chang, Wei-Wei; Yang, Bei-Chia; Wu, Tsai-Jung; Liu, Wei-Li; Yu, Alice L; Yu, John

2018-06-11

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional, novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs prior to differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

PubMed

Dinsmore, P K; O'Sullivan, D J; Klaenhammer, T R

1998-05-28

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

Jerking & confused: Leucine-rich glioma inactivated 1 receptor encephalitis.

PubMed

Casault, Colin; Alikhani, Katayoun; Pillay, Neelan; Koch, Marcus

2015-12-15

This is a case of autoimmune encephalitis with features of faciobrachial dystonic seizures (FBDS) pathognomonic for Leucine Rich Glioma inactivated (LGI)1 antibody encephalitis. This voltage-gated potassium channel complex encephalitis is marked by rapid onset dementia, FBDS and hyponatremia, which is sensitive to management with immunotherapy including steroids, IVIG and other agents. In this case report we review the clinical features, imaging and management of this condition. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Anabolic effects of leucine-rich whey protein, carbohydrate, and soy protein with and without β-hydroxy-β-methylbutyrate (HMB) during fasting-induced catabolism: A human randomized crossover trial.

PubMed

Rittig, Nikolaj; Bach, Ermina; Thomsen, Henrik H; Møller, Andreas B; Hansen, Jakob; Johannsen, Mogens; Jensen, Erik; Serena, Anja; Jørgensen, Jens O; Richelsen, Bjørn; Jessen, Niels; Møller, Niels

2017-06-01

Protein-rich beverages are widely used clinically to preserve muscle protein and improve physical performance. Beverages with high contents of leucine or its keto-metabolite β-hydroxy-β-methylbutyrate (HMB) are especially anabolic in muscle, but it is uncertain whether this also applies to catabolic conditions such as fasting and whether common or separate intracellular signaling cascades are involved. To compare a specific leucine-rich whey protein beverage (LWH) with isocaloric carbohydrate- (CHO), soy protein (SOY), and soy protein +3 g HMB (HMB) during fasting-induced catabolic conditions. Eight healthy lean male subjects underwent four interventions (LWH, CHO, SOY, and HMB) using a randomized crossover design. Each trial included a 36 h fast and consisted of a 3 h basal fasting period and a 4 h 'sipping' period. Forearm net balances of phenylalanine (NB phe , measure of net protein loss) improved for all groups (p < 0.05), but more prominently so for LWH and HMB compared with SOY (p < 0.05). Muscle protein phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets eukaryotic translation factor 4E-binding protein 1 (4EBP1) and ribosomal S6 kinase 1 (S6) were distinctly increased during LWH consumption (p < 0.05). The ratio between autophagy protein microtubule-associated protein 1 light chain-3β II and I (LC3II/LC3I, a measure of autophagy activity) was decreased during LWH and SOY intake compared with the fasting period (p < 0.05). LWH and HMB have superior anabolic effects on muscle protein kinetics after 36 h of fasting, and LWH distinctly activates the mTOR pathway. These novel findings suggest that leucine-rich whey protein and/or HMB are specifically beneficial during fasting-induced catabolic conditions. Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Overexpression of rice LRK1 restricts internode elongation by down-regulating OsKO2.

PubMed

Yang, Mengfei; Qi, Weiwei; Sun, Fan; Zha, Xiaojun; Chen, Mingluan; Huang, Yunqing; Feng, Yu-Qi; Yang, Jinshui; Luo, Xiaojin

2013-01-01

Rice (Oryza sativa) has the potential to undergo rapid internodal elongation which determines plant height. Gibberellin is involved in internode elongation. Leucine-rich repeat receptor-like kinases (LRR-RLKs) are the largest subfamily of transmembrane receptor-like kinases in plants. LRR-RLKs play important functions in mediating a variety of cellular processes and regulating responses to environmental signals. LRK1, a PSK receptor homolog, is a member of the LRR-RLK family. In the present study, differences in ectopic expression of LRK1 were consistent with extent of rice internode elongation. Analyses of gene expression demonstrated that LRK1 restricts gibberellin biosynthesis during the internode elongation process by down-regulation of the gibberellin biosynthetic gene coding for ent-kaurene oxidase.

Genomic evidence for genes encoding leucine-rich repeat receptors linked to resistance against the eukaryotic extra- and intracellular Brassica napus pathogens Leptosphaeria maculans and Plasmodiophora brassicae.

PubMed

Stotz, Henrik U; Harvey, Pascoe J; Haddadi, Parham; Mashanova, Alla; Kukol, Andreas; Larkan, Nicholas J; Borhan, M Hossein; Fitt, Bruce D L

2018-01-01

Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

[Unexplicated neuropsychiatric disorders: Do not ignore dysimmune encephalitis. A case report of a dysimmune encephalitis with anti-leucine rich glioma inactivated 1 (LGI-1) antibodies].

PubMed

Le Dault, E; Lagarde, S; Guedj, E; Dufournet, B; Rey, C; Kaphan, E; Tanguy, G; Bregigeon, M; Sagui, E; Brosset, C

2016-02-01

Anti-leucine rich glioma inactivated 1 encephalitis is a common and a treatable etiology of autoimmune encephalitis. Its diagnosis is a challenge because the initial diagnostic work-up is often normal. A 48-year-old man experienced cognitive and behavioral troubles, facio-brachial dystonic seizures and a syndrome of inappropriate antidiuretic hormone secretion. First line tests excluded infectious, neoplastic, systemic inflammatory, endrocrine or toxic etiologies. Cerebral (18)Fluoro-desoxy-glucose (FDG) position emission tomography and research of specific antibodies in cerebro-spinal fluid and serum led to diagnose an anti-leucine rich glioma inactivated 1 encephalitis. Intravenous immunoglobulins and corticosteroids were partially effective. Cyclophosphamid permitted a good recovery. In the presence of acute neuropsychiatric disorders with a negative etiologic research, physician should think about dysimmune encephalitis. Facio-brachial dystonic seizures and syndrome of inappropriate antidiuretic hormone secretion are highly evocative of anti-leucine rich glioma inactivated 1 encephalitis. The diagnosis needs specific diagnostic tests (cerebral (18)FDG position emission tomography and antibodies research in cerebro-spinal fluid and in serum), after the exclusion of alternative diagnoses. Extensive and repeated diagnostic work-up for neoplasia is required. Immunosupressive therapies are effective in most cases. Copyright © 2015 Société nationale française de médecine interne (SNFMI). Published by Elsevier SAS. All rights reserved.

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation.

PubMed

Sritrakul, Tepyuda; Nitipan, Supachai; Wajjwalku, Worawidh; La-Ard, Anchalee; Suphatpahirapol, Chattip; Petkarnjanapong, Wimol; Ongphiphadhanakul, Boonsong; Prapong, Siriwan

2017-11-01

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC 50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should

Leucine-rich diet alters the 1H-NMR based metabolomic profile without changing the Walker-256 tumour mass in rats.

PubMed

Viana, Laís Rosa; Canevarolo, Rafael; Luiz, Anna Caroline Perina; Soares, Raquel Frias; Lubaczeuski, Camila; Zeri, Ana Carolina de Mattos; Gomes-Marcondes, Maria Cristina Cintra

2016-10-03

Cachexia is one of the most important causes of cancer-related death. Supplementation with branched-chain amino acids, particularly leucine, has been used to minimise loss of muscle tissue, although few studies have examined the effect of this type of nutritional supplementation on the metabolism of the tumour-bearing host. Therefore, the present study evaluated whether a leucine-rich diet affects metabolomic derangements in serum and tumour tissues in tumour-bearing Walker-256 rats (providing an experimental model of cachexia). After 21 days feeding Wistar female rats a leucine-rich diet, distributed in L-leucine and LW-leucine Walker-256 tumour-bearing groups, we examined the metabolomic profile of serum and tumour tissue samples and compared them with samples from tumour-bearing rats fed a normal protein diet (C - control; W - tumour-bearing groups). We utilised 1 H-NMR as a means to study the serum and tumour metabolomic profile, tumour proliferation and tumour protein synthesis pathway. Among the 58 serum metabolites examined, we found that 12 were altered in the tumour-bearing group, reflecting an increase in activity of some metabolic pathways related to energy production, which diverted many nutrients toward tumour growth. Despite displaying increased tumour cell activity (i.e., higher Ki-67 and mTOR expression), there were no differences in tumour mass associated with changes in 23 metabolites (resulting from valine, leucine and isoleucine synthesis and degradation, and from the synthesis and degradation of ketone bodies) in the leucine-tumour group. This result suggests that the majority of nutrients were used for host maintenance. A leucine rich-diet, largely used to prevent skeletal muscle loss, did not affect Walker 256 tumour growth and led to metabolomic alterations that may partially explain the positive effects of leucine for the whole tumour-bearing host.

Anti-leucine rich glioma inactivated 1 protein and anti-N-methyl-D-aspartate receptor encephalitis show distinct patterns of brain glucose metabolism in 18F-fluoro-2-deoxy-d-glucose positron emission tomography

PubMed Central

2014-01-01

Background Pathogenic autoantibodies targeting the recently identified leucine rich glioma inactivated 1 protein and the subunit 1 of the N-methyl-D-aspartate receptor induce autoimmune encephalitis. A comparison of brain metabolic patterns in 18F-fluoro-2-deoxy-d-glucose positron emission tomography of anti-leucine rich glioma inactivated 1 protein and anti-N-methyl-D-aspartate receptor encephalitis patients has not been performed yet and shall be helpful in differentiating these two most common forms of autoimmune encephalitis. Methods The brain 18F-fluoro-2-deoxy-d-glucose uptake from whole-body positron emission tomography of six anti-N-methyl-D-aspartate receptor encephalitis patients and four patients with anti-leucine rich glioma inactivated 1 protein encephalitis admitted to Hannover Medical School between 2008 and 2012 was retrospectively analyzed and compared to matched controls. Results Group analysis of anti-N-methyl-D-aspartate encephalitis patients demonstrated regionally limited hypermetabolism in frontotemporal areas contrasting an extensive hypometabolism in parietal lobes, whereas the anti-leucine rich glioma inactivated 1 protein syndrome was characterized by hypermetabolism in cerebellar, basal ganglia, occipital and precentral areas and minor frontomesial hypometabolism. Conclusions This retrospective 18F-fluoro-2-deoxy-d-glucose positron emission tomography study provides novel evidence for distinct brain metabolic patterns in patients with anti-leucine rich glioma inactivated 1 protein and anti-N-methyl-D-aspartate receptor encephalitis. PMID:24950993

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

PubMed

Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

2017-10-06

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Hypothesis: Do miRNAs Targeting the Leucine-Rich Repeat Kinase 2 Gene (LRRK2) Influence Parkinson's Disease Susceptibility?

PubMed

Yılmaz, Şenay Görücü; Geyik, Sırma; Neyal, Ayşe Münife; Soko, Nyarai D; Bozkurt, Hakan; Dandara, Collet

2016-04-01

Parkinson's disease (PD) is a frequently occurring neurodegenerative motor disorder adversely impacting global health. There is a paucity of biomarkers and diagnostics that can forecast susceptibility to PD. A new research frontier for PD pathophysiology is the study of variations in microRNA (miRNA) expression whereby miRNAs serve as "upstream regulators" of gene expression in relation to functioning of the dopamine neuronal pathways. Leucine-Rich Repeat Kinase 2 (LRRK2) is a frequently studied gene in PD. Little is known about the ways in which expression of miRNAs targeting LRKK2 impact PD susceptibility. In a sample of 204 unrelated subjects (102 persons with PD and 102 healthy controls), we report here candidate miRNA expression in whole blood samples as measured by real-time PCR (hsa-miR-4671-3p, hsa-miR-335-3p, hsa-miR-561-3p, hsa-miR-579-3p, and hsa-miR-3143) that target LRRK2. Using step-wise logistic regression, and controlling for covariates such as age, gender, PD disease severity, concomitant medications, and co-morbidity, we found that the combination of has-miR-335-3p, has-miR-561-3p, and has-miR-579-3p account for 50% of the variation in regards to PD susceptibility (p<0.0001). Notably, the hsa-miR-561-3p expression was the most robust predictor of PD in both univariate and multivariate analyses (p<0.001). Moreover, the biological direction (polarity) of the association was plausible in that the candidate miRNAs displayed a diminished expression in patients. This is consistent with the hypothesis that decreased levels of miRNAs targeting LRRK2 might result in a gain of function for LRRK2, and by extension, loss of neuronal viability. To the best of our knowledge, this is the first clinical association study of the above candidate miRNAs' expression in PD using peripheral samples. These observations may guide future clinical diagnostics research on PD.

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice.

PubMed

Liu, Yaju; Xu, Yunyuan; Xiao, Jun; Ma, Qibin; Li, Dan; Xue, Zhen; Chong, Kang

2011-07-01

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice. Copyright © 2011 Elsevier GmbH. All rights reserved.

Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant.

PubMed

Liu, Min; Kang, Stephanie; Ray, Soumya; Jackson, Justin; Zaitsev, Alexandra D; Gerber, Scott A; Cuny, Gregory D; Glicksman, Marcie A

2011-11-01

Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance.

PubMed

Dellett, Margaret; Hu, Wanzhou; Papadaki, Vasiliki; Ohnuma, Shin-ichi

2012-04-01

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury. © 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

Helminthosporic acid functions as an agonist for gibberellin receptor.

PubMed

Miyazaki, Sho; Jiang, Kai; Kobayashi, Masatomo; Asami, Tadao; Nakajima, Masatoshi

2017-11-01

Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure-activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

PubMed Central

Van der Does, Dieuwertje; Boutrot, Freddy; Vernhettes, Samantha; Tintor, Nico; Veerabagu, Manikandan; Miedes, Eva; Segonzac, Cécile; Hardtke, Christian S.; Molina, Antonio; Höfte, Herman; Hamann, Thorsten

2017-01-01

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues. PMID:28604776

Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2.

PubMed

Slootweg, Erik J; Spiridon, Laurentiu N; Roosien, Jan; Butterbach, Patrick; Pomp, Rikus; Westerhof, Lotte; Wilbers, Ruud; Bakker, Erin; Bakker, Jaap; Petrescu, Andrei-José; Smant, Geert; Goverse, Aska

2013-07-01

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.

[Effects of redox state of disulfide bonds on the intrinsic fluorescence and denaturation of Trx-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea].

PubMed

Zhang, Teng; Feng, Juan; Li, Yang; Chen, Rui; Tang, Li-Xia; Pang, Xiao-Feng; Ren, Zheng-Long

2010-02-01

In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni(2+) -NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol x L(-1) GSSG or 5 mmol x L(-1) peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol x L(-1) DTT was absent or present, the fusion protein could not be fully unfolded with lambda(max) < 350 nm following the treatment of 6 mol x L(-1) GdnHCl. (5) Fusion protein Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (deltaG) of 3.7 kJ x mol(-1). However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

Metabolic Mechanism for l-Leucine-Induced Metabolome To Eliminate Streptococcus iniae.

PubMed

Du, Chao-Chao; Yang, Man-Jun; Li, Min-Yi; Yang, Jun; Peng, Bo; Li, Hui; Peng, Xuan-Xian

2017-05-05

Crucial metabolites that modulate hosts' metabolome to eliminate bacterial pathogens have been documented, but the metabolic mechanisms are largely unknown. The present study explores the metabolic mechanism for l-leucine-induced metabolome to eliminate Streptococcus iniae in tilapia. GC-MS-based metabolomics was used to investigate the tilapia liver metabolic profile in the presence of exogenous l-leucine. Thirty-seven metabolites of differential abundance were determined, and 11 metabolic pathways were enriched. Pattern recognition analysis identified serine and proline as crucial metabolites, which are the two metabolites identified in survived tilapias during S. iniae infection, suggesting that the two metabolites play crucial roles in l-leucine-induced elimination of the pathogen by the host. Exogenous l-serine reduces the mortality of tilapias infected by S. iniae, providing a robust proof supporting the conclusion. Furthermore, exogenous l-serine elevates expression of genes IL-1β and IL-8 in tilapia spleen, but not TNFα, CXCR4 and Mx, suggesting that the metabolite promotes a phagocytosis role of macrophages, which is consistent with the finding that l-leucine promotes macrophages to kill both Gram-positive and Gram-negative bacterial pathogens. Therefore, the ability of phagocytosis enhanced by exogenous l-leucine is partly attributed to elevation of l-serine. These results demonstrate a metabolic mechanism by which exogenous l-leucine modulates tilapias' metabolome to enhance innate immunity and eliminate pathogens.

The petunia homologue of tomato gast1: transcript accumulation coincides with gibberellin-induced corolla cell elongation.

PubMed

Ben-Nissan, G; Weiss, D

1996-12-01

Gibberellins (GAs) regulate petunia corolla pigmentation and elongation. To study this hormone's effect at the molecular level, we used the tomato gast1 gene as a probe to isolate a gibberellin-induced gene (gip) from petunia corollas. The deduced sequence of gip exhibited 82% identity with GAST1 protein and contained a short, highly hydrophobic N-terminal region. High levels of gip expression were detected in elongating corollas and young stem intemodes. When detached corollas were grown in vitro in sucrose medium, gip expression was strongly induced by gibberellic acid (GA3). GA3-induced gip expression in corollas was inhibited by abscisic acid (ABA). The expression of the gene was also induced by GA3 in detached young stem segments. Sucrose was not essential for GA-induced gip expression in corollas but enhanced its effect. In stems, on the other hand, sucrose inhibited the effect of the hormone. The results of the present work support the possible role of gip in GA-induced corolla and stem elongation.

Gibberellin Perception by the Gibberellin Receptor and its Effector Recognition

NASA Astrophysics Data System (ADS)

Hakoshima, Toshio; Murase, Kohji; Hirano, Yoshinori; Sun, Tai-Ping

Gibberellins control a diverse range of growth and developmental processes in higher plants and have been widely utilized in the agricultural industry. By binding to a nuclear receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins. The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. We present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the N-terminal DELLA domain of GAI. In this complex, GID1a occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor which is distinct from the hormone-perception mechanism and effector recognition of the known auxin receptors.

Seizures induced by carbachol, morphine, and leucine-enkephalin: a comparison.

PubMed

Snead, O C

1983-04-01

The electrical, behavioral, and pharmacological properties of seizures induced by morphine, leucine-enkephalin, and the muscarinic cholinergic agonist carbachol were examined and compared. Low-dose carbachol given intracerebroventricularly (ICV) produced seizures similar electrically to those produced by ICV morphine and leucine-enkephalin, although there was some difference in site of subcortical origin of onset. Carbachol and morphine were similar in that they had the same anticonvulsant profile, produced similar behavioral changes, caused generalized absence seizures in low doses and generalized convulsive seizures in high doses, and were capable of chemical kindling. However, opiate-induced seizures were not overcome by cholinergic antagonists, nor were carbachol seizures blocked by opiate antagonists. These data suggest that there may be a common noncholinergic, nonopiatergic system involved in mediating carbachol- and morphine-induced seizures but not enkephalin seizures.

Leucine signaling in the pathogenesis of type 2 diabetes and obesity.

PubMed

Melnik, Bodo C

2012-03-15

Epidemiological evidence points to increased dairy and meat consumption, staples of the Western diet, as major risk factors for the development of type 2 diabetes (T2D). This paper presents a new concept and comprehensive review of leucine-mediated cell signaling explaining the pathogenesis of T2D and obesity by leucine-induced over-stimulation of mammalian target of rapamycin complex 1 (mTORC1). mTORC1, a pivotal nutrient-sensitive kinase, promotes growth and cell proliferation in response to glucose, energy, growth factors and amino acids. Dairy proteins and meat stimulate insulin/insulin-like growth factor 1 signaling and provide high amounts of leucine, a primary and independent stimulator for mTORC1 activation. The downstream target of mTORC1, the kinase S6K1, induces insulin resistance by phosphorylation of insulin receptor substrate-1, thereby increasing the metabolic burden of β-cells. Moreover, leucine-mediated mTORC1-S6K1-signaling plays an important role in adipogenesis, thus increasing the risk of obesity-mediated insulin resistance. High consumption of leucine-rich proteins explains exaggerated mTORC1-dependent insulin secretion, increased β-cell growth and β-cell proliferation promoting an early onset of replicative β-cell senescence with subsequent β-cell apoptosis. Disturbances of β-cell mass regulation with increased β-cell proliferation and apoptosis as well as insulin resistance are hallmarks of T2D, which are all associated with hyperactivation of mTORC1. In contrast, the anti-diabetic drug metformin antagonizes leucine-mediated mTORC1 signaling. Plant-derived polyphenols and flavonoids are identified as natural inhibitors of mTORC1 and exert anti-diabetic and anti-obesity effects. Furthermore, bariatric surgery in obesity reduces increased plasma levels of leucine and other branched-chain amino acids. Attenuation of leucine-mediated mTORC1 signaling by defining appropriate upper limits of the daily intake of leucine-rich animal and dairy

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa21[C][W][OA

PubMed Central

Slootweg, Erik J.; Spiridon, Laurentiu N.; Roosien, Jan; Butterbach, Patrick; Pomp, Rikus; Westerhof, Lotte; Wilbers, Ruud; Bakker, Erin; Bakker, Jaap; Petrescu, Andrei-José; Smant, Geert; Goverse, Aska

2013-01-01

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins. PMID:23660837

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.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

PubMed Central

2011-01-01

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

PubMed

Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

2011-05-26

Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

The ontogeny of seizures induced by leucine-enkephalin and beta-endorphin.

PubMed

Snead, O C; Stephens, H

1984-06-01

Rats ranging in postnatal age from 6 hours to 28 days were implanted with cortical and depth electrodes as well as an indwelling cannula in the lateral ventricle. We then administered varying amounts of the opiate peptides leucine-enkephalin and beta-endorphin intracerebroventricularly with continuous electroencephalographic monitoring. Leucine-enkephalin produced electrical seizure activity in rats as young as 2 days. beta-Endorphin administration was associated with seizures at the fifth postnatal day, with a high incidence of apnea resulting in death in animals as young as 6 hours. An adult seizure response to beta-endorphin and leucine-enkephalin was seen at 15 and 28 days of age, respectively. Naloxone blocked the seizure produced by these opiate peptides in all age groups. The data indicate that the opiate peptides are potent epileptogenic compounds in developing brain, that seizures induced by leucine-enkephalin differ from those caused by beta-endorphin, and that petit mal-like seizure activity can be an adult response in the rodent.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

PubMed

Ishibashi, Osamu; Akagi, Ichiro; Ogawa, Yota; Inui, Takashi

2018-05-11

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Aberrant expression of epithelial leucine-rich repeat containing G protein-coupled receptor 5-positive cells in the eutopic endometrium in endometriosis and implications in deep-infiltrating endometriosis.

PubMed

Vallvé-Juanico, Júlia; Suárez-Salvador, Elena; Castellví, Josep; Ballesteros, Agustín; Taylor, Hugh S; Gil-Moreno, Antonio; Santamaria, Xavier

2017-11-01

To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5 + ) cells from the endometrium of women with endometriosis. Prospective experimental study. University hospital/fertility clinic. Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Obtaining of uterine aspirates by using a Cornier Pipelle. Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5 + cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Immunofluorescence showed that LGR5 + cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5 + cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5 + versus LGR5 - cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5 + cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Our results revealed the presence of aberrantly located LGR5 + cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes. Copyright © 2017. Published by Elsevier Inc.

Clustering of motor and nonmotor traits in leucine-rich repeat kinase 2 G2019S Parkinson's disease nonparkinsonian relatives: A multicenter family study.

PubMed

Mestre, Tiago A; Pont-Sunyer, Claustre; Kausar, Farah; Visanji, Naomi P; Ghate, Taneera; Connolly, Barbara S; Gasca-Salas, Carmen; Kern, Drew S; Jain, Jennifer; Slow, Elizabeth J; Faust-Socher, Achinoam; Kasten, Meike; Wadia, Pettarusp M; Zadikoff, Cindy; Kumar, Prakash; de Bie, Ronald M; Thomsen, Teri; Lang, Anthony E; Schüle, Birgitt; Klein, Christine; Tolosa, Eduardo; Marras, Connie

2018-04-17

The objective of this study was to determine phenotypic features that differentiate nonparkinsonian first-degree relatives of PD leucine-rich repeat kinase 2 (LRRK2) G2019S multiplex families, regardless of carrier status, from healthy controls because nonparkinsonian individuals in multiplex families seem to share a propensity to present neurological features. We included nonparkinsonian first-degree relatives of LRRK2 G2019S familial PD cases and unrelated healthy controls participating in established multiplex family LRRK2 cohorts. Study participants underwent neurologic assessment including cognitive screening, olfaction testing, and questionnaires for daytime sleepiness, depression, and anxiety. We used a multiple logistic regression model with backward variable selection, validated with bootstrap resampling, to establish the best combination of motor and nonmotor features that differentiates nonparkinsonian first-degree relatives of LRRK2 G2019S familial PD cases from unrelated healthy controls. We included 142 nonparkinsonian family members and 172 unrelated healthy controls. The combination of past or current symptoms of anxiety (adjusted odds ratio, 4.16; 95% confidence interval, 2.01-8.63), less daytime sleepiness (adjusted odds ratio [1 unit], 0.90; 95% confidence interval, 0.83-0.97], and worse motor UPDRS score (adjusted odds ratio [1 unit], 1.4; 95% confidence interval, 1.20-1.67) distinguished nonparkinsonian family members, regardless of LRRK2 G2019S mutation status, from unrelated healthy controls. The model accuracy was good (area under the curve = 79.3%). A set of motor and nonmotor features distinguishes first-degree relatives of LRRK2 G2019S probands, regardless of mutation status, from unrelated healthy controls. Environmental or non-LRRK2 genetic factors in LRRK2-associated PD may influence penetrance of the LRRK2 G2019S mutation. The relationship of these features to actual PD risk requires longitudinal observation of LRRK2 familial PD

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

USDA-ARS?s Scientific Manuscript database

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...

Potent anti-seizure effects of D-leucine

PubMed Central

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

2015-01-01

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

The heterozygous R1441C mutation of leucine-rich repeat kinase 2 gene in a Chinese patient with Parkinson disease: A five-year follow-up and literatures review.

PubMed

Peng, Fang; Sun, Yi-Min; Chen, Chen; Luo, Su-Shan; Li, Da-Ke; Wang, Yi-Xuan; Yang, Ke; Liu, Feng-Tao; Zuo, Chuan-Tao; Ding, Zheng-Tong; An, Yu; Wu, Jian-Jun; Wang, Jian

2017-02-15

Leucine-rich repeat kinase 2 gene (LRRK2) was recognized associated with both familial and sporadic Parkinson Disease (PD). Seven missense mutations (G2019S, R1441C, R1441G, R1441H, Y1699C, I2020T, N1437H) of it have been confirmed disease- causing. They were common among Caucasian PD patients, but rarely reported in Asian, especially in Chinese Han population. We aimed to identify the frequencies of these seven mutations of LRRK2 in Chinese early-onset PD (EOPD) patients and analyze the phenotypes. One hundred and thirty seven EOPD patients were enrolled for genetic testing. The seven disease-causing mutations of LRRK2 were carried out by target sequencing using Illumina HiSeq 2000 Sequencer. The identified variants were further confirmed by Sanger sequence. The clinical materials were investigated retrospectively. Only one patient (0.73%) was found carrying pathogenetic LRRK2 mutation of R1441C. The age at onset of the female patient was 44. She manifested typical motor symptoms of PD and responded well to levodopa therapy. Longitudinal evaluation showed progression of motor symptoms and depression but no cognitive impairment. The dopamine transporter (DAT) imaging via [11C]-2β-carbomethoxy-3β-(4-fluorophenyl) tropan (CFT) and Positron emission computed tomography (PET) revealed typical dopamine transporter uptake reduction. The LRRK2 R1441C mutation was found in a Chinese EOPD patient for the first time. The manifestations of LRRK2-R1441C carriers were indistinguishable from sporadic PD patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

PubMed Central

Usdin, K; Furano, A V

1988-01-01

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed. Images PMID:2837766

Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats in Bacterial Type III Secretion System Effectors.

PubMed

Batkhishig, Dashdavaa; Bilguun, Khurelbaatar; Enkhbayar, Purevjav; Miyashita, Hiroki; Kretsinger, Robert H; Matsushima, Norio

2018-06-01

Leucine rich repeats (LRRs) are present in over 100,000 proteins from viruses to eukaryotes. The LRRs are 20-30 residues long and occur in tandem. LRRs form parallel stacks of short β-strands and then assume a super helical arrangement called a solenoid structure. Individual LRRs are separated into highly conserved segment (HCS) with the consensus of LxxLxLxxNxL and variable segment (VS). Eight classes have been recognized. Bacterial LRRs are short and characterized by two prolines in the VS; the consensus is xxLPxLPxx with Nine residues (N-subtype) and xxLPxxLPxx with Ten residues (T-subtype). Bacterial LRRs are contained in type III secretion system effectors such as YopM, IpaH3/9.8, SspH1/2, and SlrP from bacteria. Some LRRs in decorin, fribromodulin, TLR8/9, and FLRT2/3 from vertebrate also contain the motifs. In order to understand structural features of bacterial LRRs, we performed both secondary structures assignments using four programs-DSSP-PPII, PROSS, SEGNO, and XTLSSTR-and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness), based on the atomic coordinates of their crystal structures. The N-subtype VS adopts a left handed polyproline II helix (PPII) with four, five or six residues and a type I β-turn at the C-terminal side. Thus, the N-subtype is characterized by a super secondary structure consisting of a PPII and a β-turn. In contrast, the T-subtype VS prefers two separate PPIIs with two or three and two residues. The HELFIT analysis indicates that the type I β-turn is a right handed helix. The HELFIT analysis determines three unit vectors of the helix axes of PPII (P), β-turn (B), and LRR domain (A). Three structural parameters using these three helix axes are suggested to characterize the super secondary structure and the LRR domain.

Gibberellin Promotes Shoot Branching in the Perennial Woody Plant Jatropha curcas

PubMed Central

Ni, Jun; Gao, Congcong; Chen, Mao-Sheng; Pan, Bang-Zhen; Ye, Kaiqin; Xu, Zeng-Fu

2015-01-01

Strigolactone (SL), auxin and cytokinin (CK) interact to regulate shoot branching. CK has long been considered to be the only key phytohormone to promote lateral bud outgrowth. Here we report that gibberellin also acts as a positive regulator in the control of shoot branching in the woody plant Jatropha curcas. We show that gibberellin and CK synergistically promote lateral bud outgrowth, and that both hormones influence the expression of putative branching regulators, J. curcas BRANCHED1 and BRANCHED2, which are key transcription factors maintaining bud dormancy. Moreover, treatment with paclobutrazol, an inhibitor of de novo gibberellin biosynthesis, significantly reduced the promotion of bud outgrowth by CK, suggesting that gibberellin is required for CK-mediated axillary bud outgrowth. In addition, SL, a plant hormone involved in the repression of shoot branching, acted antagonistically to both gibberellin and CK in the control of lateral bud outgrowth. Consistent with this, the expression of JcMAX2, a J. curcas homolog of Arabidopsis MORE AXILLARY GROWTH 2 encoding an F-box protein in the SL signaling pathway, was repressed by gibberellin and CK treatment. We also provide physiological evidence that gibberellin also induces shoot branching in many other trees, such as papaya, indicating that a more complicated regulatory network occurs in the control of shoot branching in some perennial woody plants. PMID:26076970

Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds.

PubMed

Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko

2004-06-01

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1

PubMed Central

Narusaka, Mari; Toyoda, Kazuhiro; Shiraishi, Tomonori; Iuchi, Satoshi; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

2016-01-01

Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex. PMID:26750751

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds.

PubMed

Nakajima, Masatoshi; Nakayama, Akira; Xu, Zheng-Jun; Yamaguchi, Isomaro

2004-03-01

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.

Gibberellin Promotes Shoot Branching in the Perennial Woody Plant Jatropha curcas.

PubMed

Ni, Jun; Gao, Congcong; Chen, Mao-Sheng; Pan, Bang-Zhen; Ye, Kaiqin; Xu, Zeng-Fu

2015-08-01

Strigolactone (SL), auxin and cytokinin (CK) interact to regulate shoot branching. CK has long been considered to be the only key phytohormone to promote lateral bud outgrowth. Here we report that gibberellin also acts as a positive regulator in the control of shoot branching in the woody plant Jatropha curcas. We show that gibberellin and CK synergistically promote lateral bud outgrowth, and that both hormones influence the expression of putative branching regulators, J. curcas BRANCHED1 and BRANCHED2, which are key transcription factors maintaining bud dormancy. Moreover, treatment with paclobutrazol, an inhibitor of de novo gibberellin biosynthesis, significantly reduced the promotion of bud outgrowth by CK, suggesting that gibberellin is required for CK-mediated axillary bud outgrowth. In addition, SL, a plant hormone involved in the repression of shoot branching, acted antagonistically to both gibberellin and CK in the control of lateral bud outgrowth. Consistent with this, the expression of JcMAX2, a J. curcas homolog of Arabidopsis MORE AXILLARY GROWTH 2 encoding an F-box protein in the SL signaling pathway, was repressed by gibberellin and CK treatment. We also provide physiological evidence that gibberellin also induces shoot branching in many other trees, such as papaya, indicating that a more complicated regulatory network occurs in the control of shoot branching in some perennial woody plants. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Plant growth enhancement and associated physiological responses are coregulated by ethylene and gibberellin in response to harpin protein Hpa1.

PubMed

Li, Xiaojie; Han, Bing; Xu, Manyu; Han, Liping; Zhao, Yanying; Liu, Zhilan; Dong, Hansong; Zhang, Chunling

2014-04-01

The harpin protein Hpa1 produced by the bacterial blight pathogen of rice induces several growth-promoting responses in plants, activating the ethylene signaling pathway, increasing photosynthesis rates and EXPANSIN (EXP) gene expression levels, and thereby enhancing the vegetative growth. This study was attempted to analyze any mechanistic connections among the above and the role of gibberellin in these responses. Hpa1-induced growth enhancement was evaluated in Arabidopsis, tomato, and rice. And growth-promoting responses were determined mainly as an increase of chlorophyll a/b ratio, which indicates a potential elevation of photosynthesis rates, and enhancements of photosynthesis and EXP expression in the three plant species. In Arabidopsis, Hpa1-induced growth-promoting responses were partially compromised by a defect in ethylene perception or gibberellin biosynthesis. In tomato and rice, compromises of Hpa1-induced growth-promoting responses were caused by a pharmacological treatment with an ethylene perception inhibitor or a gibberellin biosynthesis inhibitor. In the three plant species, moreover, Hpa1-induced growth-promoting responses were significantly impaired, but not totally eliminated, by abolishing ethylene perception or gibberellin synthesis. However, simultaneous nullifications in both ethylene perception and gibberellin biosynthesis almost canceled the full effects of Hpa1 on plant growth, photosynthesis, and EXP2 expression. Theses results suggest that ethylene and gibberellin coregulate Hpa1-induced plant growth enhancement and associated physiological and molecular responses.

d-Leucine: Evaluation in an epilepsy model.

PubMed

Holden, Kylie; Hartman, Adam L

2018-01-01

Current medicines do not provide sufficient seizure control for nearly one-third of patients with epilepsy. New options are needed to address this treatment gap. We recently found that the atypical amino acid d-leucine protected against acutely-induced seizures in mice, but its effect in chronic seizures has not been explored. We hypothesized that d-leucine would protect against spontaneous recurrent seizures. We also investigated whether mice lacking a previously-described d-leucine receptor (Tas1R2/R3) would be protected against acutely-induced seizures. Male FVB/NJ mice were subjected to kainic acid-induced status epilepticus and monitored by video-electroencephalography (EEG) (surgically implanted electrodes) for 4weeks before, during, and after treatment with d-leucine. Tas1R2/R3 knockout mice and controls underwent the maximal electroshock threshold (MES-T) and 6-Hz tests. There was no difference in number of calendar days with seizures or seizure frequency with d-leucine treatment. In an exploratory analysis, mice treated with d-leucine had a lower number of dark cycles with seizures. Tas1R2/R3 knockout mice had elevated seizure thresholds in the MES-T test but not the 6-Hz test. d-Leucine treatment was ineffective against chronic seizures after kainic acid-induced status epilepticus, but there was some efficacy during the dark cycle. Because d-leucine is highly concentrated in the pineal gland, these data suggest that d-leucine may be useful as a tool for studying circadian patterns in epilepsy. Deletion of the Tas1R2/R3 receptor protected against seizures in the MES-T test and, therefore, may be a novel target for treating seizures. Published by Elsevier Inc.

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

The wheat Sr50 gene reveals rich diversity at a cereal disease resistance locus

USDA-ARS?s Scientific Manuscript database

We identify the wheat stem rust resistance gene Sr50 by physical mapping, mutation and complementation as homologous to barley Mla encoding a Coiled-Coil-Nucleotide-Binding-Leucine-Rich Repeat (CC-NB-LRR) protein. We show that Sr50 confers a unique resistance specificity, different from Sr31 and oth...

Small Leucine-Rich Proteoglycans in Renal Inflammation: Two Sides of the Coin.

PubMed

Nastase, Madalina V; Janicova, Andrea; Roedig, Heiko; Hsieh, Louise Tzung-Harn; Wygrecka, Malgorzata; Schaefer, Liliana

2018-04-01

It is now well-established that members of the small leucine-rich proteoglycan (SLRP) family act in their soluble form, released proteolytically from the extracellular matrix (ECM), as danger-associated molecular patterns (DAMPs). By interacting with Toll-like receptors (TLRs) and the inflammasome, the two SLRPs, biglycan and decorin, autonomously trigger sterile inflammation. Recent data indicate that these SLRPs, besides their conventional role as pro-inflammatory DAMPs, additionally trigger anti-inflammatory signaling pathways to tightly control inflammation. This is brought about by selective employment of TLRs, their co-receptors, various adaptor molecules, and through crosstalk between SLRP-, reactive oxygen species (ROS)-, and sphingolipid-signaling. In this review, the complexity of SLRP signaling in immune and kidney resident cells and its relevance for renal inflammation is discussed. We propose that the dichotomy in SLRP signaling (pro- and anti-inflammatory) allows for fine-tuning the inflammatory response, which is decisive for the outcome of inflammatory kidney diseases.

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

Pediatric Dilated Cardiomyopathy-Associated LRRC10 (Leucine-Rich Repeat-Containing 10) Variant Reveals LRRC10 as an Auxiliary Subunit of Cardiac L-Type Ca2+ Channels.

PubMed

Woon, Marites T; Long, Pamela A; Reilly, Louise; Evans, Jared M; Keefe, Alexis M; Lea, Martin R; Beglinger, Carl J; Balijepalli, Ravi C; Lee, Youngsook; Olson, Timothy M; Kamp, Timothy J

2018-02-03

Genetic causes of dilated cardiomyopathy (DCM) are incompletely understood. LRRC10 (leucine-rich repeat-containing 10) is a cardiac-specific protein of unknown function. Heterozygous mutations in LRRC10 have been suggested to cause DCM, and deletion of Lrrc10 in mice results in DCM. Whole-exome sequencing was carried out on a patient who presented at 6 weeks of age with DCM and her unaffected parents, filtering for rare, deleterious, recessive, and de novo variants. Whole-exome sequencing followed by trio-based filtering identified a homozygous recessive variant in LRRC10 , I195T. Coexpression of I195T LRRC10 with the L-type Ca 2+ channel (Ca v 1.2, β 2CN2 , and α 2 δ subunits) in HEK293 cells resulted in a significant ≈0.5-fold decrease in I Ca,L at 0 mV, in contrast to the ≈1.4-fold increase in I Ca,L by coexpression of LRRC10 (n=9-12, P <0.05). Coexpression of LRRC10 or I195T LRRC10 did not alter the surface membrane expression of Ca v 1.2. LRRC10 coexpression with Ca v 1.2 in the absence of auxiliary β 2CN2 and α 2 δ subunits revealed coassociation of Ca v 1.2 and LRRC10 and a hyperpolarizing shift in the voltage dependence of activation (n=6-9, P <0.05). Ventricular myocytes from Lrrc10 -/- mice had significantly smaller I Ca,L , and coimmunoprecipitation experiments confirmed association between LRRC10 and the Ca v 1.2 subunit in mouse hearts. Examination of a patient with DCM revealed homozygosity for a previously unreported LRRC10 variant: I195T. Wild-type and I195T LRRC10 function as cardiac-specific subunits of L-type Ca 2+ channels and exert dramatically different effects on channel gating, providing a potential link to DCM. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

DOE PAGES

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; ...

2015-03-16

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Oral Leucine Supplementation Is Sensed by the Brain but neither Reduces Food Intake nor Induces an Anorectic Pattern of Gene Expression in the Hypothalamus

PubMed Central

Zampieri, Thais T.; Pedroso, João A. B.; Furigo, Isadora C.; Tirapegui, Julio; Donato, Jose

2013-01-01

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity. PMID:24349566

Leucine deprivation inhibits proliferation and induces apoptosis of human breast cancer cells via fatty acid synthase

PubMed Central

Xiao, Fei; Wang, Chunxia; Yin, Hongkun; Yu, Junjie; Chen, Shanghai; Fang, Jing; Guo, Feifan

2016-01-01

Substantial studies on fatty acid synthase (FASN) have focused on its role in regulating lipid metabolism and researchers have a great interest in treating cancer with dietary manipulation of amino acids. In the current study, we found that leucine deprivation caused the FASN-dependent anticancer effect. Here we showed that leucine deprivation inhibited cell proliferation and induced apoptosis of MDA-MB-231 and MCF-7 breast cancer cells. In an in vivo tumor xenograft model, the leucine-free diet suppressed the growth of human breast cancer tumors and triggered widespread apoptosis of the cancer cells. Further study indicated that leucine deprivation decreased expression of lipogenic gene FASN in vitro and in vivo. Over-expression of FASN or supplementation of palmitic acid (the product of FASN action) blocked the effects of leucine deprivation on cell proliferation and apoptosis in vitro and in vivo. Moreover, leucine deprivation suppressed the FASN expression via regulating general control non-derepressible (GCN)2 and sterol regulatory element-binding protein 1C (SREBP1C). Taken together, our study represents proof of principle that anticancer effects can be obtained with strategies to deprive tumors of leucine via suppressing FASN expression, which provides important insights in prevention of breast cancer via metabolic intervention. PMID:27579768

RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence.

PubMed

Lü, Peitao; Zhang, Changqing; Liu, Jitao; Liu, Xiaowei; Jiang, Guimei; Jiang, Xinqiang; Khan, Muhammad Ali; Wang, Liangsheng; Hong, Bo; Gao, Junping

2014-05-01

Rose (Rosa hybrida) is one of the most important ornamental plants worldwide; however, senescence of its petals terminates the ornamental value of the flower, resulting in major economic loss. It is known that the hormones abscisic acid (ABA) and ethylene promote petal senescence, while gibberellins (GAs) delay the process. However, the molecular mechanisms underlying the antagonistic effects amongst plant hormones during petal senescence are still unclear. Here we isolated RhHB1, a homeodomain-leucine zipper I transcription factor gene, from rose flowers. Quantitative RT-PCR and GUS reporter analyses showed that RhHB1 was strongly expressed in senescing petals, and its expression was induced by ABA or ethylene in petals. ABA or ethylene treatment clearly accelerated rose petal senescence, while application of the gibberellin GA3 delayed the process. However, silencing of RhHB1 delayed the ABA- or ethylene-mediated senescence, and resulted in higher petal anthocyanin levels and lower expression of RhSAG12. Moreover, treatment with paclobutrazol, an inhibitor of GA biosynthesis, repressed these delays. In addition, silencing of RhHB1 blocked the ABA- or ethylene-induced reduction in expression of the GA20 oxidase encoded by RhGA20ox1, a gene in the GA biosynthetic pathway. Furthermore, RhHB1 directly binds to the RhGA20ox1 promoter, and silencing of RhGA20ox1 promoted petal senescence. Eight senescence-related genes showed substantial differences in expression in petals after treatment with GA3 or paclobutrazol. These results suggest that RhHB1 mediates the antagonistic effect of GAs on ABA and ethylene during rose petal senescence, and that the promotion of petal senescence by ABA or ethylene operates through an RhHB1-RhGA20ox1 regulatory checkpoint. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae).

PubMed

Sousa, Juliana C; Berto, Raquel F; Gois, Elicélia A; Fontenele-Cardi, Nauíla C; Honório, José E R; Konno, Katsuhiro; Richardson, Michael; Rocha, Marcos F G; Camargo, Antônio A C M; Pimenta, Daniel C; Cardi, Bruno A; Carvalho, Krishnamurti M

2009-07-01

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Natural Variation of Molecular and Morphological Gibberellin Responses.

PubMed

Nam, Youn-Jeong; Herman, Dorota; Blomme, Jonas; Chae, Eunyoung; Kojima, Mikiko; Coppens, Frederik; Storme, Veronique; Van Daele, Twiggy; Dhondt, Stijn; Sakakibara, Hitoshi; Weigel, Detlef; Inzé, Dirk; Gonzalez, Nathalie

2017-01-01

Although phytohormones such as gibberellins are essential for many conserved aspects of plant physiology and development, plants vary greatly in their responses to these regulatory compounds. Here, we use genetic perturbation of endogenous gibberellin levels to probe the extent of intraspecific variation in gibberellin responses in natural accessions of Arabidopsis (Arabidopsis thaliana). We find that these accessions vary greatly in their ability to buffer the effects of overexpression of GA20ox1, encoding a rate-limiting enzyme for gibberellin biosynthesis, with substantial differences in bioactive gibberellin concentrations as well as transcriptomes and growth trajectories. These findings demonstrate a surprising level of flexibility in the wiring of regulatory networks underlying hormone metabolism and signaling. © 2017 American Society of Plant Biologists. All Rights Reserved.

Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response.

PubMed

Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J

2015-11-01

In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance. © 2015 American Society of Plant Biologists. All Rights Reserved.

Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response1

PubMed Central

Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A.; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J.

2015-01-01

In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance. PMID:26373661

Immunohistochemistry of active gibberellins and gibberellin-inducible alpha-amylase in developing seeds of morning glory.

PubMed

Nakayama, Akira; Park, Seijin; Zheng-Jun, Xu; Nakajima, Masatoshi; Yamaguchi, Isomaro

2002-07-01

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A(1)-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA(1) and/or GA(3) were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible alpha-amylase in this digestion. We isolated an alpha-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA(3). RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA(1/3). An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA(1/3), was also localized around starch grains in the integument of developing young seeds. The localization of GA(1/3) in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.

Mechanism of gibberellin-dependent stem elongation in peas

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.; Sovonick-Dunford, S. A.

1989-01-01

Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process.

Progress and development of analytical methods for gibberellins.

PubMed

Pan, Chaozhi; Tan, Swee Ngin; Yong, Jean Wan Hong; Ge, Liya

2017-01-01

Gibberellins, as a group of phytohormones, exhibit a wide variety of bio-functions within plant growth and development, which have been used to increase crop yields. Many analytical procedures, therefore, have been developed for the determination of the types and levels of endogenous and exogenous gibberellins. As plant tissues contain gibberellins in trace amounts (usually at the level of nanogram per gram fresh weight or even lower), the sample pre-treatment steps (extraction, pre-concentration, and purification) for gibberellins are reviewed in details. The primary focus of this comprehensive review is on the various analytical methods designed to meet the requirements for gibberellins analyses in complex matrices with particular emphasis on high-throughput analytical methods, such as gas chromatography, liquid chromatography, and capillary electrophoresis, mostly combined with mass spectrometry. The advantages and drawbacks of the each described analytical method are discussed. The overall aim of this review is to provide a comprehensive and critical view on the different analytical methods nowadays employed to analyze gibberellins in complex sample matrices and their foreseeable trends. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice.

PubMed

Li, Hongliang; Xu, Mingjiang; Lee, Jiyeon; He, Chaoyong; Xie, Zhonglin

2012-11-15

Leucine supplementation has been shown to prevent high-fat diet (HFD)-induced obesity, hyperglycemia, and dyslipidemia in animal models, but the underlying mechanisms are not fully understood. Recent studies suggest that activation of Sirtuin 1 (SIRT1) is an important mechanism to maintain energy and metabolic homeostasis. We therefore examined the involvement of SIRT1 in leucine supplementation-prevented obesity and insulin resistance. To accomplish this goal, male C57BL/6J mice were fed normal diet or HFD, supplemented with or without leucine. After 2 mo of treatment, alterations in SIRT1 expression, insulin signaling, and energy metabolism were analyzed. Eight weeks of HFD induced obesity, fatty liver, mitochondrial dysfunction, hyperglycemia, and insulin resistance in mice. Addition of leucine to HFD correlated with increased expression of SIRT1 and NAMPT (nicotinamide phosphoribosyltransferase) as well as higher intracellular NAD(+) levels, which decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and forkhead box O1 (FoxO1). The deacetylation of PGC1α may contribute to upregulation of genes controlling mitochondrial biogenesis and fatty acid oxidation, thereby improving mitochondrial function and preventing HFD-induced obesity in mice. Moreover, decreased acetylation of FoxO1 was accompanied by decreased expression of pseudokinase tribble 3 (TRB3) and reduced the association between TRB3 and Akt, which enhanced insulin sensitivity and improved glucose metabolism. Finally, transfection of dominant negative AMPK prevented activation of SIRT1 signaling in HFD-Leu mice. These data suggest that increased expression of SIRT1 after leucine supplementation may lead to reduced acetylation of PGC1α and FoxO1, which is associated with attenuation of HFD-induced mitochondrial dysfunction, insulin resistance, and obesity.

Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice

PubMed Central

Li, Hongliang; Xu, Mingjiang; Lee, Jiyeon; He, Chaoyong

2012-01-01

Leucine supplementation has been shown to prevent high-fat diet (HFD)-induced obesity, hyperglycemia, and dyslipidemia in animal models, but the underlying mechanisms are not fully understood. Recent studies suggest that activation of Sirtuin 1 (SIRT1) is an important mechanism to maintain energy and metabolic homeostasis. We therefore examined the involvement of SIRT1 in leucine supplementation-prevented obesity and insulin resistance. To accomplish this goal, male C57BL/6J mice were fed normal diet or HFD, supplemented with or without leucine. After 2 mo of treatment, alterations in SIRT1 expression, insulin signaling, and energy metabolism were analyzed. Eight weeks of HFD induced obesity, fatty liver, mitochondrial dysfunction, hyperglycemia, and insulin resistance in mice. Addition of leucine to HFD correlated with increased expression of SIRT1 and NAMPT (nicotinamide phosphoribosyltransferase) as well as higher intracellular NAD+ levels, which decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and forkhead box O1 (FoxO1). The deacetylation of PGC1α may contribute to upregulation of genes controlling mitochondrial biogenesis and fatty acid oxidation, thereby improving mitochondrial function and preventing HFD-induced obesity in mice. Moreover, decreased acetylation of FoxO1 was accompanied by decreased expression of pseudokinase tribble 3 (TRB3) and reduced the association between TRB3 and Akt, which enhanced insulin sensitivity and improved glucose metabolism. Finally, transfection of dominant negative AMPK prevented activation of SIRT1 signaling in HFD-Leu mice. These data suggest that increased expression of SIRT1 after leucine supplementation may lead to reduced acetylation of PGC1α and FoxO1, which is associated with attenuation of HFD-induced mitochondrial dysfunction, insulin resistance, and obesity. PMID:22967499

Isolation of gibberellin A8-glucoside from shoot apices of Althaea rosea.

PubMed

Harada, H; Yokota, T

1970-03-01

Gibberellin A8-glucoside has been isolated from shoot apices of Althaea rosea. It showed a weak growth-promoting activity on rice seedlings and oat mesocotyl sections but did not induce germination of lettuce seeds in darkness.

Salicylic-Acid-Induced Chilling- and Oxidative-Stress Tolerance in Relation to Gibberellin Homeostasis, C-Repeat/Dehydration-Responsive Element Binding Factor Pathway, and Antioxidant Enzyme Systems in Cold-Stored Tomato Fruit.

PubMed

Ding, Yang; Zhao, Jinhong; Nie, Ying; Fan, Bei; Wu, Shujuan; Zhang, Yu; Sheng, Jiping; Shen, Lin; Zhao, Ruirui; Tang, Xuanming

2016-11-02

Effects of salicylic acid (SA) on gibberellin (GA) homeostasis, C-repeat/dehydration-responsive element binding factor (CBF) pathway, and antioxidant enzyme systems linked to chilling- and oxidative-stress tolerance in tomato fruit were investigated. Mature green tomatoes (Solanum lycopersicum L. cv. Moneymaker) were treated with 0, 0.5, and 1 mM SA solution for 15 min before storage at 4 °C for 28 days. In comparison to 0 or 0.5 mM SA, 1 mM SA significantly decreased the chilling injury (CI) index in tomato fruit. In the SA-treated fruit, the upregulation of GA biosynthetic gene (GA3ox1) expression was followed by gibberellic acid (GA 3 ) surge and DELLA protein degradation. CBF1 participated in the SA-modulated tolerance and stimulated the expression of GA catabolic gene (GA2ox1). Furthermore, 1 mM SA enhanced activities of antioxidant enzymes and, thus, reduced reactive oxygen species accumulation. Our findings suggest that SA might protect tomato fruit from CI and oxidative damage through regulating GA metabolism, CBF1 gene expression, and antioxidant enzyme activities.

Immunohistochemistry of Active Gibberellins and Gibberellin-Inducible α-Amylase in Developing Seeds of Morning Glory1

PubMed Central

Nakayama, Akira; Park, Seijin; Zheng-Jun, Xu; Nakajima, Masatoshi; Yamaguchi, Isomaro

2002-01-01

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A1-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA1 and/or GA3 were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible α-amylase in this digestion. We isolated an α-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA3. RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA1/3. An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA1/3, was also localized around starch grains in the integument of developing young seeds. The localization of GA1/3 in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds. PMID:12114559

Repeated Administration of D-Amphetamine Induces Distinct Alterations in Behavior and Metabolite Levels in 129Sv and Bl6 Mouse Strains.

PubMed

Vanaveski, Taavi; Narvik, Jane; Innos, Jürgen; Philips, Mari-Anne; Ottas, Aigar; Plaas, Mario; Haring, Liina; Zilmer, Mihkel; Vasar, Eero

2018-01-01

The main goal of the study was to characterize the behavioral and metabolomic profiles of repeated administration (for 11 days) of d-amphetamine (AMPH, 3 mg/kg i. p.), indirect agonist of dopamine (DA), in widely used 129S6/SvEvTac (129Sv) and C57BL/6NTac (Bl6) mouse strains. Acute administration of AMPH (acute AMPH) induced significantly stronger motor stimulation in Bl6. However, repeated administration of AMPH (repeated AMPH) caused stronger motor sensitization in 129Sv compared acute AMPH. Body weight of 129Sv was reduced after repeated saline and AMPH, whereas no change occurred in Bl6. In the metabolomic study, acute AMPH induced an elevation of isoleucine and leucine, branched chain amino acids (BCAA), whereas the level of hexoses was reduced in Bl6. Both BCAAs and hexoses remained on level of acute AMPH after repeated AMPH in Bl6. Three biogenic amines [asymmetric dimethylarginine (ADMA), alpha-aminoadipic acid (alpha-AAA), kynurenine] were significantly reduced after repeated AMPH. Acute AMPH caused in 129Sv a significant reduction of valine, lysophosphatidylcholines (lysoPC a C16:0, lysoPC a C18:2, lysoPC a C20:4), phosphatidylcholine (PC) diacyls (PC aa C34:2, PC aa C36:2, PC aa C36:3, PC aa C36:4) and alkyl-acyls (PC ae C38:4, PC ae C40:4). However, repeated AMPH increased the levels of valine and isoleucine, long-chain acylcarnitines (C14, C14:1-OH, C16, C18:1), PC diacyls (PC aa C38:4, PC aa C38:6, PC aa C42:6), PC acyl-alkyls (PC ae C38:4, PC ae C40:4, PC ae C40:5, PC ae C40:6, PC ae C42:1, PC ae C42:3) and sphingolipids [SM(OH)C22:1, SM C24:0] compared to acute AMPH in 129Sv. Hexoses and kynurenine were reduced after repeated AMPH compared to saline in 129Sv. The established changes probably reflect a shift in energy metabolism toward lipid molecules in 129Sv because of reduced level of hexoses. Pooled data from both strains showed that the elevation of isoleucine and leucine was a prominent biomarker of AMPH-induced behavioral sensitization

Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights.

PubMed

Heeley, Nicholas; Kirwan, Peter; Darwish, Tamana; Arnaud, Marion; Evans, Mark L; Merkle, Florian T; Reimann, Frank; Gribble, Fiona M; Blouet, Clemence

2018-04-01

Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (K ATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca 2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca 2+ current. A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca 2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly

Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution

NASA Astrophysics Data System (ADS)

Kato, Hiroaki; Sato, Tomomi; Ueguchi-Tanaka, Miyako

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1 (GID1) from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an α/β-hydrolase fold similar to that of the hormone-sensitive lipases (HSLs) except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds.

PubMed

Sutoh, Keita; Washio, Kenji; Imai, Ryozo; Wada, Masamitsu; Nakai, Tomonori; Yamauchi, Daisuke

2015-01-01

The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat.

PubMed

Wohleb, Eric S; Hanke, Mark L; Corona, Angela W; Powell, Nicole D; Stiner, La'Tonia M; Bailey, Michael T; Nelson, Randy J; Godbout, Jonathan P; Sheridan, John F

2011-04-27

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

CvADH1, a member of short-chain alcohol dehydrogenase family, is inducible by gibberellin and sucrose in developing watermelon seeds.

PubMed

Kim, Joonyul; Kang, Hong-Gyu; Jun, Sung-Hoon; Lee, Jinwon; Yim, Jieun; An, Gynheung

2003-01-01

To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3beta-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-N'-phenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA(3) strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development.

Fungal Diversity and Community Composition of Culturable Fungi in Stanhopea trigrina Cast Gibberellin Producers

PubMed Central

Salazar-Cerezo, Sonia; Martinez-Montiel, Nancy; Cruz-Lopez, Maria del Carmen; Martinez-Contreras, Rebeca D.

2018-01-01

Stanhopea tigrina is a Mexican endemic orchid reported as a threatened species. The naturally occurring microorganisms present in S. tigrina are unknown. In this work, we analyzed the diversity of endophytic and epiphytic culturable fungi in S. tigrina according to morphological and molecular identification. Using this combined approach, in this study we retrieved a total of 634 fungal isolates that presented filamentous growth, which were grouped in 134 morphotypes that were associated to 63 genera, showing that S. tigrina harbors a rich diversity of both endophytic and epiphytic fungi. Among these, the majority of the isolates corresponded to Ascomycetes, with Trichoderma and Penicillium as the most frequent genera followed by Fusarium and Aspergillus. Non-ascomycetes isolated were associated only to the genus Mucor (Mucoromycota) and Schizophyllum (Basidiomycota). Identified genera showed a differential distribution considering their epiphytic or endophytic origin, the tissue from which they were isolated, and the ability of the orchid to grow on different substrates. To our knowledge, this work constitutes the first study of the mycobiome of S. tigrina. Interestingly, 21 fungal isolates showed the ability to produce gibberellins. Almost half of the isolates were related to the gibberellin-producer genus Penicillium based on morphological and molecular identification. However, the rest of the isolates were related to the following genera, which have not been reported as gibberellin producers so far: Bionectria, Macrophoma, Nectria, Neopestalotiopsis, Talaromyces, Trichoderma, and Diplodia. Taken together, we found that S. tigrina possess a significant fungal diversity that could be a rich source of fungal metabolites with the potential to develop biotechnological approaches oriented to revert the threatened state of this orchid in the near future. PMID:29670591

Fungal Diversity and Community Composition of Culturable Fungi in Stanhopea trigrina Cast Gibberellin Producers.

PubMed

Salazar-Cerezo, Sonia; Martinez-Montiel, Nancy; Cruz-Lopez, Maria Del Carmen; Martinez-Contreras, Rebeca D

2018-01-01

Stanhopea tigrina is a Mexican endemic orchid reported as a threatened species. The naturally occurring microorganisms present in S. tigrina are unknown. In this work, we analyzed the diversity of endophytic and epiphytic culturable fungi in S. tigrina according to morphological and molecular identification. Using this combined approach, in this study we retrieved a total of 634 fungal isolates that presented filamentous growth, which were grouped in 134 morphotypes that were associated to 63 genera, showing that S. tigrina harbors a rich diversity of both endophytic and epiphytic fungi. Among these, the majority of the isolates corresponded to Ascomycetes, with Trichoderma and Penicillium as the most frequent genera followed by Fusarium and Aspergillus . Non-ascomycetes isolated were associated only to the genus Mucor (Mucoromycota) and Schizophyllum (Basidiomycota). Identified genera showed a differential distribution considering their epiphytic or endophytic origin, the tissue from which they were isolated, and the ability of the orchid to grow on different substrates. To our knowledge, this work constitutes the first study of the mycobiome of S. tigrina . Interestingly, 21 fungal isolates showed the ability to produce gibberellins. Almost half of the isolates were related to the gibberellin-producer genus Penicillium based on morphological and molecular identification. However, the rest of the isolates were related to the following genera, which have not been reported as gibberellin producers so far: Bionectria, Macrophoma, Nectria, Neopestalotiopsis, Talaromyces, Trichoderma , and Diplodia . Taken together, we found that S. tigrina possess a significant fungal diversity that could be a rich source of fungal metabolites with the potential to develop biotechnological approaches oriented to revert the threatened state of this orchid in the near future.

Plant cytoskeleton: DELLA connects gibberellins to microtubules.

PubMed

Dixit, Ram

2013-06-03

A new study reveals that DELLA proteins directly interact with the prefoldin complex, thus regulating tubulin subunit availability in a gibberellin-dependent manner. This finding provides a mechanistic link between the growth-promoting plant hormone gibberellin and cortical microtubule organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nutritional leucine supplementation attenuates cardiac failure in tumour-bearing cachectic animals.

PubMed

Toneto, Aline Tatiane; Ferreira Ramos, Luiz Alberto; Salomão, Emilianne Miguel; Tomasin, Rebeka; Aereas, Miguel Arcanjo; Gomes-Marcondes, Maria Cristina Cintra

2016-12-01

The condition known as cachexia presents in most patients with malignant tumours, leading to a poor quality of life and premature death. Although the cancer-cachexia state primarily affects skeletal muscle, possible damage in the cardiac muscle remains to be better characterized and elucidated. Leucine, which is a branched chain amino acid, is very useful for preserving lean body mass. Thus, this amino acid has been studied as a coadjuvant therapy in cachectic cancer patients, but whether this treatment attenuates the effects of cachexia and improves cardiac function remains poorly understood. Therefore, using an experimental cancer-cachexia model, we evaluated whether leucine supplementation ameliorates cachexia in the heart. Male Wistar rats were fed either a leucine-rich or a normoprotein diet and implanted or not with subcutaneous Walker-256 carcinoma. During the cachectic stage (approximately 21 days after tumour implantation), when the tumour mass was greater than 10% of body weight, the rats were subjected to an electrocardiogram analysis to evaluate the heart rate, QT-c, and T wave amplitude. The myocardial tissues were assayed for proteolytic enzymes (chymotrypsin, alkaline phosphatase, cathepsin, and calpain), cardiomyopathy biomarkers (myeloperoxidase, tissue inhibitor of metalloproteinases, and total plasminogen activator inhibitor 1), and caspase-8, -9, -3, and -7 activity. Both groups of tumour-bearing rats, especially the untreated group, had electrocardiography alterations that were suggestive of ischemia, dilated cardiomyopathy, and sudden death risk. Additionally, the rats in the untreated tumour-bearing group but not their leucine-supplemented littermates exhibited remarkable increases in chymotrypsin activity and all three heart failure biomarkers analysed, including an increase in caspase-3 and -7 activity. Our data suggest that a leucine-rich diet could modulate heart damage, cardiomyocyte proteolysis, and apoptosis driven by cancer

Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onder, Zeynep; Chang, Vivian; Moroianu, Junona, E-mail: moroianu@bc.edu

2015-01-01

We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7more » interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.« less

Opposing effects of external gibberellin and Daminozide on Stevia growth and metabolites.

PubMed

Karimi, Mojtaba; Hashemi, Javad; Ahmadi, Ali; Abbasi, Alireza; Pompeiano, Antonio; Tavarini, Silvia; Guglielminetti, Lorenzo; Angelini, Luciana G

2015-01-01

Steviol glycosides (SVglys) and gibberellins are originated from the shared biosynthesis pathway in Stevia (Stevia rebaudiana Bertoni). In this research, two experiments were conducted to study the opposing effects of external gibberellin (GA3) and Daminozide (a gibberellin inhibitor) on Stevia growth and metabolites. Results showed that GA3 significantly increased the stem length and stem dry weight in Stevia. Total soluble sugar content increased while the SVglys biosynthesis was decreased by external GA3 applying in Stevia leaves. In another experiment, the stem length was reduced by Daminozide spraying on Stevia shoots. The Daminozide did not affect the total SVglys content, while in 30 ppm concentration, significantly increased the soluble sugar production in Stevia leaves. Although the gibberellins biosynthesis pathway has previously invigorated in Stevia leaf, the Stevia response to external gibberellins implying on high precision regulation of gibberellins biosynthesis in Stevia and announces that Stevia is able to kept endogenous gibberellins in a low quantity away from SVglys production. Moreover, the assumption that the internal gibberellins were destroyed by Daminozide, lack of Daminozide effects on SVglys production suggests that gibberellins biosynthesis could not act as a competitive factor for SVglys production in Stevia leaves.

MicroRNA-137 dysregulation predisposes to osteoporotic fracture by impeding ALP activity and expression via suppression of leucine-rich repeat-containing G-protein-coupled receptor 4 expression.

PubMed

Liu, Xiangjun; Xu, Xiaohui

2018-08-01

Osteoporosis is defined as a loss of bone mass and deterioration of its architecture resulting in bone weakness, which becomes prone to fracture. The objective of this study was to investigate the molecular mechanism by which miR-137 can reduce the risk of fracture in patients with osteoporosis. An online miRNA database and a luciferase reporter assay system were used to confirm that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) was the target of miR-137. Real-time PCR and western blot analysis were used to study miR-137 mRNA, the expression of LGR4 mRNA and protein among different groups or cells transfected with a scrambled miRNA control, miR-137 mimic, LGR4 siRNA and miR-137 inhibitor. Expression of miR-137 was upregulated to higher levels in cells isolated from osteoporosis patients with fracture than in those without fracture. The 'seed sequence' was found to be located within the 3' untranslated region (3'-UTR) of LGR4 mRNA by searching an online miRNA database. Luciferase reporter assay was performed to confirm that LGR4 is a direct target gene of miR-137 with a potential binding site in the 3'UTR of LGR4. Luciferase activity of cells transfected with wild-type LGR4 3'UTR was much lower than that of the cells transfected with mutant LGR4 3'UTR. The results of real-time PCR and immunohistochemistry experiments demonstrated that the expression levels of LGR4 mRNA and protein were much higher in osteoporosis patients with fracture than osteoporosis patients without fracture. We found that the expression levels of LGR4 mRNA and protein were clearly upregulated following transfection with miR-137 inhibitor, while noticeably downregulated following transfection with miR-137 mimic when compared with the scramble control. Furthermore, the expression of ALP mRNA and ALP activity in bone tissue were much higher in osteoporosis patients with fracture than those without fracture. In conclusion, these data prove that the overexpression of

Sepsis-induced alterations in protein-protein interactions within mTOR complex 1 and the modulating effect of leucine on muscle protein synthesis.

PubMed

Kazi, Abid A; Pruznak, Anne M; Frost, Robert A; Lang, Charles H

2011-02-01

Sepsis-induced muscle atrophy is produced in part by decreased protein synthesis mediated by inhibition of mTOR (mammalian target of rapamycin). The present study tests the hypothesis that alteration of specific protein-protein interactions within the mTORC1 (mTOR complex 1) contributes to the decreased mTOR activity observed after cecal ligation and puncture in rats. Sepsis decreased in vivo translational efficiency in gastrocnemius and reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein (BP) 1, S6 kinase (S6K) 1, and mTOR, compared with time-matched pair-fed controls. Sepsis decreased T246-phosphorylated PRAS40 (proline-rich Akt substrate 40) and reciprocally increased S792-phosphorylated raptor (regulatory associated protein of mTOR). Despite these phosphorylation changes, sepsis did not alter PRAS40 binding to raptor. The amount of the mTOR-raptor complex did not differ between groups. In contrast, the binding and retention of both 4E-BP1 and S6K1 to raptor were increased, and, conversely, the binding of raptor with eIF3 was decreased in sepsis. These changes in mTORC1 in the basal state were associated with enhanced 5'-AMP activated kinase activity. Acute in vivo leucine stimulation increased muscle protein synthesis in control, but not septic rats. This muscle leucine resistance was associated with coordinated changes in raptor-eIF3 binding and 4E-BP1 phosphorylation. Overall, our data suggest the sepsis-induced decrease in muscle protein synthesis may be mediated by the inability of 4E-BP1 and S6K1 to be phosphorylated and released from mTORC1 as well as the decreased recruitment of eIF3 necessary for a functional 48S complex. These data provide additional mechanistic insight into the molecular mechanisms by which sepsis impairs both basal protein synthesis and the anabolic response to the nutrient signal leucine in skeletal muscle.

Tumor microenvironment: Modulation by decorin and related molecules harboring leucine-rich tandem motifs.

PubMed

Goldoni, Silvia; Iozzo, Renato V

2008-12-01

Decorin, the prototype member of the small leucine-rich proteoglycans, resides in the tumor microenvironment and affects the biology of various types of cancer by downregulating the activity of several receptors involved in cell growth and survival. Decorin binds to and modulates the signaling of the epidermal growth factor receptor and other members of the ErbB family of receptor tyrosine kinases. It exerts its antitumor activity by a dual mechanism: via inhibition of these key receptors through their physical downregulation coupled with attenuation of their signaling, and by binding to and sequestering TGFbeta. Decorin also modulates the insulin-like growth factor receptor and the low-density lipoprotein receptor-related protein 1, which indirectly affects the TGFbeta receptor pathway. When expressed in tumor xenograft-bearing mice or injected systemically, decorin inhibits both primary tumor growth and metastatic spreading. In this review, we summarize the latest reports on decorin and related molecules that are relevant to cancer and bring forward the idea of decorin as an anticancer therapeutic and possible prognostic marker for patients affected by various types of tumors. We also discuss the role of lumican and LRIG1, a novel cell growth inhibitor homologous to decorin. (c) 2008 Wiley-Liss, Inc.

Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan’s syndrome and acquired neuromyotonia

PubMed Central

Irani, Sarosh R.; Alexander, Sian; Waters, Patrick; Kleopa, Kleopas A.; Pettingill, Philippa; Zuliani, Luigi; Peles, Elior; Buckley, Camilla; Lang, Bethan

2010-01-01

Antibodies that immunoprecipitate 125I-α-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan’s syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with 125I-α-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan’s syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein

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

PubMed

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

2015-12-15

Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. Copyright © 2015 Elsevier B.V. All rights reserved.

Wounding induces changes in cytokinin and auxin content in potato tuber, but does not induce formation of gibberellins

USDA-ARS?s Scientific Manuscript database

Cytokinin, auxin and gibberellin content in resting and wound-responding potato tuber are not clearly defined. Consequently, the coordination and possible networking of these classical hormones in the regulation of wound-healing processes are poorly understood. Using a well-defined tuber wound-hea...

Genome-wide sequencing of longan (Dimocarpus longan Lour.) provides insights into molecular basis of its polyphenol-rich characteristics

PubMed Central

Lin, Yuling; Min, Jiumeng; Lai, Ruilian; Wu, Zhangyan; Chen, Yukun; Yu, Lili; Cheng, Chunzhen; Jin, Yuanchun; Tian, Qilin; Liu, Qingfeng; Liu, Weihua; Zhang, Chengguang; Lin, Lixia; Hu, Yan; Zhang, Dongmin; Thu, Minkyaw; Zhang, Zihao; Liu, Shengcai; Zhong, Chunshui; Fang, Xiaodong; Wang, Jian; Yang, Huanming

2017-01-01

Abstract Longan (Dimocarpus longan Lour.), an important subtropical fruit in the family Sapindaceae, is grown in more than 10 countries. Longan is an edible drupe fruit and a source of traditional medicine with polyphenol-rich traits. Tree size, alternate bearing, and witches' broom disease still pose serious problems. To gain insights into the genomic basis of longan traits, a draft genome sequence was assembled. The draft genome (about 471.88 Mb) of a Chinese longan cultivar, “Honghezi,” was estimated to contain 31 007 genes and 261.88 Mb of repetitive sequences. No recent whole-genome-wide duplication event was detected in the genome. Whole-genome resequencing and analysis of 13 cultivated D. longan accessions revealed the extent of genetic diversity. Comparative transcriptome studies combined with genome-wide analysis revealed polyphenol-rich and pathogen resistance characteristics. Genes involved in secondary metabolism, especially those from significantly expanded (DHS, SDH, F3΄H, ANR, and UFGT) and contracted (PAL, CHS, and F3΄5΄H) gene families with tissue-specific expression, may be important contributors to the high accumulation levels of polyphenolic compounds observed in longan fruit. The high number of genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) and leucine-rich repeat receptor-like kinase proteins, as well as the recent expansion and contraction of the NBS-LRR family, suggested a genomic basis for resistance to insects, fungus, and bacteria in this fruit tree. These data provide insights into the evolution and diversity of the longan genome. The comparative genomic and transcriptome analyses provided information about longan-specific traits, particularly genes involved in its polyphenol-rich and pathogen resistance characteristics. PMID:28368449

Leucine Supplementation Improves Skeletal Muscle Regeneration after Cryolesion in Rats

PubMed Central

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. PMID:24416379

Cold-inducible transcription factor, CaCBF, is associated with a homeodomain leucine zipper protein in hot pepper (Capsicum annuum L.).

PubMed

Kim, Sihyun; An, Chung Sun; Hong, Young-Nam; Lee, Kwang-Woong

2004-12-31

C-Repeat/drought responsive element binding factor (CBF1/DREB1b) is a well known transcriptional activator that is induced at low temperature and in turn induces the CBF regulon (CBF-targeted genes). We have cloned and characterized two CBF1-like cDNAs, CaCBF1A and CaCBF1B, from hot pepper. CaCBF1A and CaCBF1B were not produced in response to mechanical wounding or abscisic acid but were induced by low-temperature stress at 4 degrees . When plants were returned to 25 degrees , their transcript levels of the CBF1-like genes decreased markedly within 40 min. Long-term exposure to chilling resulted in continuous expression of these genes. The critical temperature for induction of CaCBF1A was between 10 and 15 degrees . Low temperature led to its transcription in roots, stems, leaves, fruit without seeds, and apical meristems, and a monoclonal antibody against it revealed a significant increase in CaCBF1A protein by 4 h at 4 degrees . Two-hybrid screening led to the isolation of an homeodomain leucine zipper (HD-Zip) protein that interacts with CaCBF1B. Expression of HD-Zip was elevated by low temperature and drought.

NLRP6 Induces Pyroptosis by Activation of Caspase-1 in Gingival Fibroblasts.

PubMed

Liu, W; Liu, J; Wang, W; Wang, Y; Ouyang, X

2018-05-01

NLRP6, a member of the nucleotide-binding domain, leucine-rich repeat-containing (NLR) innate immune receptor family, has been reported to participate in inflammasome formation. Activation of inflammasome triggers a caspase-1-dependent programming cell death called pyroptosis. However, whether NLRP6 induces pyroptosis has not been investigated. In this study, we showed that NLRP6 overexpression activated caspase-1 and gasdermin-D and then induced pyroptosis of human gingival fibroblasts, resulting in release of proinflammatory mediators interleukin (IL)-1β and IL-18. Moreover, NLRP6 was highly expressed in gingival tissue of periodontitis compared with healthy controls. Porphyromonas gingivalis, which is a commensal bacterium and has periodontopathic potential, induced pyroptosis of gingival fibroblasts by activation of NLRP6. Together, we, for the first time, identified that NLRP6 could induce pyroptosis of gingival fibroblasts by activation of caspase-1 and may play a role in periodontitis.

Phenotypic Suppression of the Gibberellin-Insensitive Mutant (gai) of Arabidopsis.

PubMed Central

Wilson, R. N.; Somerville, C. R.

1995-01-01

The semidominant gibberellin-insensitive (gai) mutant of Arabidopsis thaliana shows impairment in multiple responses to the plant hormone gibberellin A3, which include effects on seed germination, stem elongation, apical dominance, and rapid flowering in short days. Results presented here show that the gai mutation also interferes with development of fertile flowers in continuous light. Mu-tagenesis of the gai mutant resulted in recovery of 17 independent mutants in which the gibberellin-insensitive phenotype is partially or completely suppressed. Sixteen of the suppressor mutations act semidominantly to restore gibberellin responsiveness. One representative of this class, the gar1 mutation, could not be genetically separated from the gai locus and is proposed to cause inactivation of the gai gene. The exceptional gar2 mutation partially suppresses the gai phenotype, is completely dominant, and is not linked to the gai locus. The gar2 mutation may define a new gene involved in gibberellin signaling. A recessive allele of the spindly (SPY) locus, spy-5, was also found to partially suppress the gai mutant phenotype. PMID:12228487

Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC

PubMed Central

Malkani, Niyati; Biggar, Kyle; Shehab, Majida Abu; Li, Shawn; Jansson, Thomas; Gupta, Madhulika B.

2016-01-01

Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth. PMID:26733150

Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis

PubMed Central

Druhan, Lawrence J.; Lance, Amanda; Li, Shimena; Price, Andrea E.; Emerson, Jacob T.; Baxter, Sarah A.; Gerber, Jonathan M.; Avalos, Belinda R.

2017-01-01

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1. PMID:28081565

Endurance Exercise Attenuates Postprandial Whole-Body Leucine Balance in Trained Men.

PubMed

Mazzulla, Michael; Parel, Justin T; Beals, Joseph W; VAN Vliet, Stephan; Abou Sawan, Sidney; West, Daniel W D; Paluska, Scott A; Ulanov, Alexander V; Moore, Daniel R; Burd, Nicholas A

2017-12-01

Endurance exercise increases indices of small intestinal damage and leucine oxidation, which may attenuate dietary amino acid appearance and postprandial leucine balance during postexercise recovery. Therefore, the purpose of this study was to examine the effect of an acute bout of endurance exercise on postprandial leucine kinetics and net leucine balance. In a crossover design, seven trained young men (age = 25.6 ± 2.3 yr; V˙O2peak = 61.4 ± 2.9 mL·kg·min; mean ± SEM) received a primed constant infusion of L-[1-C]leucine before and after ingesting a mixed macronutrient meal containing 18 g whole egg protein intrinsically labeled with L-[5,5,5-H3]leucine, 17 g fat, and 60 g carbohydrate at rest and after 60 min of treadmill running at 70% V˙O2peak. Plasma intestinal fatty acid binding protein concentrations and leucine oxidation both increased (P < 0.01) to peaks that were ~2.5-fold above baseline values during exercise with a concomitant decrease (P < 0.01) in nonoxidative leucine disposal. Meal ingestion attenuated (P < 0.01) endogenous leucine rates of appearance at rest and after exercise. There were no differences (both, P > 0.05) in dietary leucine appearance rates or in the amount of dietary protein-derived leucine that appeared into circulation over the 5-h postprandial period at rest and after exercise (62% ± 2% and 63% ± 2%, respectively). Leucine balance over the 5-h postprandial period was positive (P < 0.01) in both conditions but was negative (P < 0.01) during the exercise trial after accounting for exercise-induced leucine oxidation. We demonstrate that endurance exercise does not modulate dietary leucine availability from a mixed meal but attenuates postprandial whole-body leucine balance in trained young men.

Clinical oversight and the avoidance of repeat induced abortion.

PubMed

Jacovetty, Erica L; Clare, Camille A; Squire, Mary-Beatrice; Kubal, Keshar P; Liou, Sherry; Inchiosa, Mario A

2018-06-03

To evaluate the impact of patient counseling, demographics, and contraceptive methods on repeat induced abortion in women attending family planning clinics. A retrospective chart review of repeat induced abortions was performed. The analysis included patients with an initial induced abortion obtained between January 1, 2001, and March 31, 2014, at New York City Health + Hospitals/Metropolitan. The duration of involvement in the family planning program, the use of contraceptive interventions, and 18 patient factors were analyzed for their correlation with the incidence of repeat induced abortions per year of follow-up. A decreased rate of repeat induced abortions was associated with a longer duration of clinical oversight (r 2 =0.449, P<0.001), a higher contraceptive efficacy score (r=0.280, P=0.025), and a larger number of clinic visits for contraception (r=0.333, P=0.007). A continuum of contact with all of the services of a family planning clinic demonstrated a strong efficacy to limit repeat induced abortions. By determining the patient characteristics that most influence repeat induced abortion rates, providers can best choose the most efficacious method of contraception available. © 2018 International Federation of Gynecology and Obstetrics.

Leucine facilitates insulin signaling through a Gαi protein-dependent signaling pathway in hepatocytes.

PubMed

Yang, Xuefeng; Mei, Shuang; Wang, Xiaolei; Li, Xiang; Liu, Rui; Ma, Yan; Hao, Liping; Yao, Ping; Liu, Liegang; Sun, Xiufa; Gu, Haihua; Liu, Zhenqi; Cao, Wenhong

2013-03-29

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt(473) and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes*

PubMed Central

Yang, Xuefeng; Mei, Shuang; Wang, Xiaolei; Li, Xiang; Liu, Rui; Ma, Yan; Hao, Liping; Yao, Ping; Liu, Liegang; Sun, Xiufa; Gu, Haihua; Liu, Zhenqi; Cao, Wenhong

2013-01-01

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt473 and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly. PMID:23404499

Efficient arachidonic acid-rich oil production by Mortierella alpina through a repeated fed-batch fermentation strategy.

PubMed

Ji, Xiao-Jun; Zhang, Ai-Hui; Nie, Zhi-Kui; Wu, Wen-Jia; Ren, Lu-Jing; Huang, He

2014-10-01

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process. Copyright © 2014 Elsevier Ltd. All rights reserved.

TGFβ1-induced leucine limitation uncovered by differential ribosome codon reading.

PubMed

Loayza-Puch, Fabricio; Rooijers, Koos; Zijlstra, Jelle; Moumbeini, Behzad; Zaal, Esther A; Oude Vrielink, Joachim F; Lopes, Rui; Ugalde, Alejandro P; Berkers, Celia R; Agami, Reuven

2017-04-01

Cancer cells modulate their metabolic networks to support cell proliferation and a higher demand of building blocks. These changes may restrict the availability of certain amino acids for protein synthesis, which can be utilized for cancer therapy. However, little is known about the amino acid demand changes occurring during aggressive and invasive stages of cancer. Recently, we developed diricore, an approach based on ribosome profiling that can uncover amino acid limitations. Here, we applied diricore to a cellular model in which epithelial breast cells respond rapidly to TGFβ1, a cytokine essential for cancer progression and metastasis, and uncovered shortage of leucine. Further analyses indicated that TGFβ1 treatment of human breast epithelial cells reduces the expression of SLC3A2, a subunit of the leucine transporter, which diminishes leucine uptake and inhibits cell proliferation. Thus, we identified a specific amino acid limitation associated with the TGFβ1 response, a vulnerability that might be associated with aggressiveness in cancer. © 2017 The Authors.

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

PubMed Central

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

2017-01-01

Insulin-like growth factor-1 (IGF-I) is the key regulator of fetal growth. IGF-I bioavailability is markedly diminished by IGF binding protein-1 (IGFBP-1) phosphorylation. Leucine deprivation strongly induces IGFBP-1hyperphosphorylation, and plays an important role in fetal growth restriction (FGR). FGR is characterized by decreased amino acid availability, which activates the amino acid response (AAR) and inhibits the mechanistic target of rapamycin (mTOR) pathway. We investigated the role of AAR and mTOR in mediating IGFBP-1 secretion and phosphorylation in HepG2 cells in leucine deprivation. mTOR inhibition (rapamycin or raptor+rictor siRNA), or activation (DEPTOR siRNA) demonstrated a role of mTOR in leucine deprivation-induced IGFBP-1 secretion but not phosphorylation. When the AAR was blocked (U0126, or ERK/GCN2 siRNA), both IGFBP-1 secretion and phosphorylation (Ser101/Ser119/Ser169) due to leucine deprivation were prevented. CK2 inhibition by TBB also attenuated IGFBP-1 phosphorylation in leucine deprivation. These results suggest that the AAR and mTOR independently regulate IGFBP-1 secretion and phosphorylation in leucine deprivation. PMID:25957086

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

USDA-ARS?s Scientific Manuscript database

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

Leucine aminopeptidase - urine

MedlinePlus

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

Plant hormones in arbuscular mycorrhizal symbioses: an emerging role for gibberellins.

PubMed

Foo, Eloise; Ross, John J; Jones, William T; Reid, James B

2013-05-01

Arbuscular mycorrhizal symbioses are important for nutrient acquisition in >80 % of terrestrial plants. Recently there have been major breakthroughs in understanding the signals that regulate colonization by the fungus, but the roles of the known plant hormones are still emerging. Here our understanding of the roles of abscisic acid, ethylene, auxin, strigolactones, salicylic acid and jasmonic acid is discussed, and the roles of gibberellins and brassinosteroids examined. Pea mutants deficient in gibberellins, DELLA proteins and brassinosteroids are used to determine whether fungal colonization is altered by the level of these hormones or signalling compounds. Expression of genes activated during mycorrhizal colonization is also monitored. Arbuscular mycorrhizal colonization of pea roots is substantially increased in gibberellin-deficient na-1 mutants compared with wild-type plants. This is reversed by application of GA3. Mutant la cry-s, which lacks gibberellin signalling DELLA proteins, shows reduced colonization. These changes were parallelled by changes in the expression of genes associated with mycorrhizal colonization. The brassinosteroid-deficient lkb mutant showed no change in colonization. Biologically active gibberellins suppress arbuscule formation in pea roots, and DELLA proteins are essential for this response, indicating that this role occurs within the root cells.

A glycine-leucine-rich peptide structurally related to the plasticins from skin secretions of the frog Leptodactylus laticeps (Leptodactylidae).

PubMed

Conlon, J Michael; Abdel-Wahab, Yasser H A; Flatt, Peter R; Leprince, Jérôme; Vaudry, Hubert; Jouenne, Thierry; Condamine, Eric

2009-05-01

A glycine-leucine-rich peptide was isolated from norepinephrine-stimulated skin secretions of the Sante Fe frog Leptodactylus laticeps (Leptodactylidae) whose primary structure (Gly-Leu-Val-Asn-Gly-Leu-Leu-Ser-Ser-Val-Leu-Gly-Gly-Gly-Gln-Gly-Gly-Gly-Gly-Leu-Leu-Gly-Gly-Ile-Leu) contains the (GXXXG)(3) motif found in the plasticins, previously identified only in phyllomedusid frogs (Hylidae). Circular dichroism studies showed that the secondary structure of the peptide, termed plasticin-L1, was markedly solvent-dependent displaying a random coil conformation in water, a beta-sheet structure in methanol, and an alpha-helical conformation in 50% trifluoroethanol-water. A synthetic replicate of the peptide did not inhibit the growth of Escherichia coli or Staphylococcus aureus or lyse human erythrocytes at concentrations up to 500 microM. At relatively high concentrations (>or=1 microM), the peptide produced a significant (P<0.05), although modest (139% of basal rate at 3 microM), increase in the rate of glucose-induced release of insulin from rat clonal BRIN-BD11 beta cells without increasing the rate of release of lactate dehydrogenase. A peptide, termed ocellatin-L2 was also identified in the skin secretion that was identical to the previously described ocellatin-L1 except for the substitution Asn(23)-->Asp. Ocellatin-L2 was devoid of antimicrobial and hemolytic activity but also showed significant activity in stimulating insulin release from BRIN-BD11 cells (181% of basal rate at 3 microM).

Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing.

PubMed

Magne, Hugues; Savary-Auzeloux, Isabelle; Migné, Carole; Peyron, Marie-Agnès; Combaret, Lydie; Rémond, Didier; Dardevet, Dominique

2012-04-15

During ageing, immobilization periods increase and are partially responsible of sarcopaenia by inducing a muscle atrophy which is hardly recovered from. Immobilization-induced atrophy is due to an increase of muscle apoptotic and proteolytic processes and decreased protein synthesis. Moreover, previous data suggested that the lack of muscle mass recovery might be due to a defect in protein synthesis response during rehabilitation. This study was conducted to explore protein synthesis during reloading and leucine supplementation effect as a nutritional strategy for muscle recovery. Old rats (22–24 months old) were subjected to unilateral hindlimb casting for 8 days (I8) and allowed to recover for 10–40 days (R10–R40). They were fed a casein (±leucine) diet during the recovery. Immobilized gastrocnemius muscles atrophied by 20%, and did not recover even at R40. Amount of polyubiquitinated conjugates and chymotrypsin- and trypsin-like activities of the 26S proteasome increased. These changes paralleled an ‘anabolic resistance' of the protein synthesis at the postprandial state (decrease of protein synthesis, P-S6 and P-4E-BP1). During the recovery, proteasome activities remained elevated until R10 before complete normalization and protein synthesis was slightly increased. With free leucine supplementation during recovery, if proteasome activities were normalized earlier and protein synthesis was higher during the whole recovery, it nevertheless failed in muscle mass gain. This discrepancy could be due to a ‘desynchronization' between the leucine signal and the availability of amino acids coming from casein digestion. Thus, when supplemented with leucine-rich proteins (i.e. whey) and high protein diets, animals partially recovered the muscle mass loss.

Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing

PubMed Central

Magne, Hugues; Savary-Auzeloux, Isabelle; Migné, Carole; Peyron, Marie-Agnès; Combaret, Lydie; Rémond, Didier; Dardevet, Dominique

2012-01-01

During ageing, immobilization periods increase and are partially responsible of sarcopaenia by inducing a muscle atrophy which is hardly recovered from. Immobilization-induced atrophy is due to an increase of muscle apoptotic and proteolytic processes and decreased protein synthesis. Moreover, previous data suggested that the lack of muscle mass recovery might be due to a defect in protein synthesis response during rehabilitation. This study was conducted to explore protein synthesis during reloading and leucine supplementation effect as a nutritional strategy for muscle recovery. Old rats (22–24 months old) were subjected to unilateral hindlimb casting for 8 days (I8) and allowed to recover for 10–40 days (R10–R40). They were fed a casein (±leucine) diet during the recovery. Immobilized gastrocnemius muscles atrophied by 20%, and did not recover even at R40. Amount of polyubiquitinated conjugates and chymotrypsin- and trypsin-like activities of the 26S proteasome increased. These changes paralleled an ‘anabolic resistance’ of the protein synthesis at the postprandial state (decrease of protein synthesis, P-S6 and P-4E-BP1). During the recovery, proteasome activities remained elevated until R10 before complete normalization and protein synthesis was slightly increased. With free leucine supplementation during recovery, if proteasome activities were normalized earlier and protein synthesis was higher during the whole recovery, it nevertheless failed in muscle mass gain. This discrepancy could be due to a ‘desynchronization’ between the leucine signal and the availability of amino acids coming from casein digestion. Thus, when supplemented with leucine-rich proteins (i.e. whey) and high protein diets, animals partially recovered the muscle mass loss. PMID:22351629

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

Plant hormones in arbuscular mycorrhizal symbioses: an emerging role for gibberellins

PubMed Central

Foo, Eloise; Ross, John J.; Jones, William T.; Reid, James B.

2013-01-01

Background and Aims Arbuscular mycorrhizal symbioses are important for nutrient acquisition in >80 % of terrestrial plants. Recently there have been major breakthroughs in understanding the signals that regulate colonization by the fungus, but the roles of the known plant hormones are still emerging. Here our understanding of the roles of abscisic acid, ethylene, auxin, strigolactones, salicylic acid and jasmonic acid is discussed, and the roles of gibberellins and brassinosteroids examined. Methods Pea mutants deficient in gibberellins, DELLA proteins and brassinosteroids are used to determine whether fungal colonization is altered by the level of these hormones or signalling compounds. Expression of genes activated during mycorrhizal colonization is also monitored. Key Results Arbuscular mycorrhizal colonization of pea roots is substantially increased in gibberellin-deficient na-1 mutants compared with wild-type plants. This is reversed by application of GA3. Mutant la cry-s, which lacks gibberellin signalling DELLA proteins, shows reduced colonization. These changes were parallelled by changes in the expression of genes associated with mycorrhizal colonization. The brassinosteroid-deficient lkb mutant showed no change in colonization. Conclusions Biologically active gibberellins suppress arbuscule formation in pea roots, and DELLA proteins are essential for this response, indicating that this role occurs within the root cells. PMID:23508650

Gibberellins in Penicillium strains: Challenges for endophyte-plant host interactions under salinity stress.

PubMed

Leitão, Ana Lúcia; Enguita, Francisco J

2016-02-01

The genus Penicillium is one of the most versatile "mycofactories", comprising some species able to produce gibberellins, bioactive compounds that can modulate plant growth and development. Although plants have the ability to synthesize gibberellins, their levels are lower when plants are under salinity stress. It has been recognized that detrimental abiotic conditions, such as saline stress, have negative effects on plants, being the availability of bioactive gibberellins a critical factor for their growth under this conditions. This review summarizes the interplay existing between endophytic Penicillium strains and plant host interactions, with focus on bioactive gibberellins production as a fungal response that allows plants to overcome salinity stress. Copyright © 2015 Elsevier GmbH. All rights reserved.

Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry

NASA Technical Reports Server (NTRS)

Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko

2004-01-01

Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.

Effect of Rituximab in Patients With Leucine-Rich, Glioma-Inactivated 1 Antibody–Associated Encephalopathy

PubMed Central

Irani, Sarosh R.; Gelfand, Jeffrey M.; Bettcher, Brianne M.; Singhal, Neel S.; Geschwind, Michael D.

2015-01-01

IMPORTANCE This observational study describes the efficacy and safety of rituximab in 5 patients with voltage-gated potassium channel (VGKC)–complex/leucine-rich, glioma-inactivated 1 (LGI1) antibody–associated encephalopathy. Rituximab is a monoclonal antibody that targets CD20 and is used to treat other neurologic and nonneurologic diseases. OBSERVATIONS This case series reports sequential seizure frequencies, modified Rankin Scale scores, and VGKC-complex antibody titers in 5 adult patients (median age, 65 years; range, 48–73 years) treated with rituximab. Median time from symptom onset to rituximab initiation was 414 days (range, 312–851 days). One patient showed a rapid clinical improvement after treatment with rituximab alone and experienced a rituximab-responsive clinical relapse. Another showed possible improvement on neuropsychometric memory indexes after rituximab therapy. In contrast, all patients showed robust responses to treatment with glucocorticoids, intravenous immunoglobulins, and/or plasma exchange at some point in their illness. Treatment with glucocorticoids—less so with intravenous immunoglobulins and plasma exchange—was associated with the most marked reductions in VGKC-complex antibodies. The only patient who did not receive glucocorticoids showed the poorest clinical and serologic responses. CONCLUSIONS AND RELEVANCE Rituximab was well tolerated in this predominantly older adult patient population and may be an effective option for some patients with LGI1 antibody–associated encephalopathy. Glucocorticoid therapy appears particularly efficacious. Earlier rituximab administration and randomized trials are required to formally assess efficacy. PMID:24842754

Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity, and fiber differentiation in tobacco stems.

PubMed

Dayan, Jonathan; Voronin, Nickolay; Gong, Fan; Sun, Tai-ping; Hedden, Peter; Fromm, Hillel; Aloni, Roni

2012-01-01

The gibberellins (GAs) are a group of endogenous compounds that promote the growth of most plant organs, including stem internodes. We show that in tobacco (Nicotiana tabacum) the presence of leaves is essential for the accumulation of bioactive GAs and their immediate precursors in the stem and consequently for normal stem elongation, cambial proliferation, and xylem fiber differentiation. These processes do not occur in the absence of maturing leaves but can be restored by application of C(19)-GAs, identifying the presence of leaves as a requirement for GA signaling in stems and revealing the fundamental role of GAs in secondary growth regulation. The use of reporter genes for GA activity and GA-directed DELLA protein degradation in Arabidopsis thaliana confirms the presence of a mobile signal from leaves to the stem that induces GA signaling.

Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene.

PubMed

Qi, Weiwei; Sun, Fan; Wang, Qianjie; Chen, Mingluan; Huang, Yunqing; Feng, Yu-Qi; Luo, Xiaojin; Yang, Jinshui

2011-09-01

Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops.

Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide.

PubMed

Kaiser, Julienne C; King, Alyssa N; Grigg, Jason C; Sheldon, Jessica R; Edgell, David R; Murphy, Michael E P; Brinsmade, Shaun R; Heinrichs, David E

2018-01-01

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.

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

Downstream change in leucine aminopeptidase activity and leucine assimilation by epilithic microbiota along the River Swale, northern England.

PubMed

Ainsworth, A M; Goulder, R

2000-05-05

Parallel determinations of epilithic extracellular leucine aminopeptidase activity and leucine assimilation were made at five sites along 112 km of the River Swale and also in two tributaries, the River Wiske and Cod Beck. Epilithic leucine aminopeptidase activity along the Swale increased with distance downstream; this increase was gradual, rather than stepwise in response to specific sewage-works outfalls. Epilithic leucine assimilation, in contrast, did not consistently increase along the river. Epilithic leucine aminopeptidase activity and leucine assimilation were both potentially controlled by epilithic microbial variables (bacterial abundance and chlorophyll a) while leucine aminopeptidase activity was also strongly related to water-quality variables, especially temperature, pH and conductivity. Epilithic leucine aminopeptidase activity and leucine assimilation were coupled, but the magnitude of aminopeptidase activity was always substantially greater than that of leucine assimilation. Arguments are presented, however, which suggest that this did not necessarily indicate the constant availability of excess leucine, and by inference amino-acid nitrogen, to epilithic bacteria. Values of epilithic leucine aminopeptidase activity and leucine assimilation, expressed relative to rates in overlying water, suggested that most activity and assimilation was epilithic rather than planktonic, although the planktonic contribution was proportionately greater at the deeper, more downstream, sites. In the tributaries, River Wiske and Cod Beck, values of epilithic leucine aminopeptidase activity and epilithic microbial abundance, as well as those of many water-quality variables, resembled values in the middle and lower Swale. Thus, these tributaries were essentially lowland, enriched watercourses being very different from the headstreams of the main river.

Structure and Functional Characterization of the RNA-Binding Element of the NLRX1 Innate Immune Modulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Minsun; Yoon, Sung-il; Wilson, Ian A.

2012-06-20

Mitochondrial NLRX1 is a member of the family of nucleotide-binding domain and leucine-rich-repeat-containing proteins (NLRs) that mediate host innate immunity as intracellular surveillance sensors against common molecular patterns of invading pathogens. NLRX1 functions in antiviral immunity, but the molecular mechanism of its ligand-induced activation is largely unknown. The crystal structure of the C-terminal fragment (residues 629975) of human NLRX1 (cNLRX1) at 2.65 {angstrom} resolution reveals that cNLRX1 consists of an N-terminal helical (LRRNT) domain, central leucine-rich repeat modules (LRRM), and a C-terminal three-helix bundle (LRRCT). cNLRX1 assembles into a compact hexameric architecture that is stabilized by intersubunit and interdomain interactionsmore » of LRRNT and LRRCT in the trimer and dimer components of the hexamer, respectively. Furthermore, we find that cNLRX1 interacts directly with RNA and supports a role for NLRX1 in recognition of intracellular viral RNA in antiviral immunity.« less

Amelogenin exons 8 and 9 encoded peptide enhances leucine rich amelogenin peptide mediated dental pulp repair.

PubMed

Huang, Yulei; Goldberg, Michel; Le, Thuan; Qiang, Ran; Warner, Douglas; Witkowska, Halina Ewa; Liu, Haichuan; Zhu, Li; Denbesten, Pamela; Li, Wu

2012-01-01

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair. Copyright © 2012 S. Karger AG, Basel.

Cryptochrome and Phytochrome Cooperatively but Independently Reduce Active Gibberellin Content in Rice Seedlings under Light Irradiation

PubMed Central

Hirose, Fumiaki; Inagaki, Noritoshi; Hanada, Atsushi; Yamaguchi, Shinjiro; Kamiya, Yuji; Miyao, Akio; Hirochika, Hirohiko; Takano, Makoto

2012-01-01

In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4–OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants. PMID:22764280

Cryptochrome and phytochrome cooperatively but independently reduce active gibberellin content in rice seedlings under light irradiation.

PubMed

Hirose, Fumiaki; Inagaki, Noritoshi; Hanada, Atsushi; Yamaguchi, Shinjiro; Kamiya, Yuji; Miyao, Akio; Hirochika, Hirohiko; Takano, Makoto

2012-09-01

In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4-OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants.

A Novel Gibberellin-Induced Gene from Rice and Its Potential Regulatory Role in Stem Growth1

PubMed Central

van der Knaap, Esther; Kim, Jeong Hoe; Kende, Hans

2000-01-01

Os-GRF1 (Oryza sativa-GROWTH-REGULATING FACTOR1) was identified in a search for genes that are differentially expressed in the intercalary meristem of deepwater rice (Oryza sativa L.) internodes in response to gibberellin (GA). Os-GRF1 displays general features of transcription factors, contains a functional nuclear localization signal, and has three regions with similarities to sequences in the database. One of these regions is similar to a protein interaction domain of SWI2/SNF2, which is a subunit of a chromatin-remodeling complex in yeast. The two other domains are novel and found only in plant proteins of unknown function. To study its role in plant growth, Os-GRF1 was expressed in Arabidopsis. Stem elongation of transformed plants was severely inhibited, and normal growth could not be recovered by the application of GA. Our results indicate that Os-GRF1 belongs to a novel class of plant proteins and may play a regulatory role in GA-induced stem elongation. PMID:10712532

Leucine aminopeptidase blood test

MedlinePlus

Serum leucine aminopeptidase; LAP - serum ... Chernecky CC, Berger BJ. Leucine aminopeptidase (LAP) - blood. In: Chernecky CC, Berger BJ, eds. Laboratory Tests and Diagnostic Procedures . 6th ed. St Louis, MO: Elsevier ...

Differential Effects of Leucine Supplementation in Young and Aged Mice at the Onset of Skeletal Muscle Regeneration

PubMed Central

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

2016-01-01

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

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori

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-tailmore » 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.« less

Leucine reduces reactive oxygen species levels via an energy metabolism switch by activation of the mTOR-HIF-1α pathway in porcine intestinal epithelial cells.

PubMed

Hu, Jun; Nie, Yangfan; Chen, Shifeng; Xie, Chunlin; Fan, Qiwen; Wang, Zhichang; Long, Baisheng; Yan, Guokai; Zhong, Qing; Yan, Xianghua

2017-08-01

Leucine serves not only as a substrate for protein synthesis, but also as a signal molecule involved in protein metabolism. However, whether the levels of cellular reactive oxygen species (ROS), which have damaging effects on cellular DNA, proteins, and lipids, are regulated by leucine is still unclear. Here, we report that leucine supplementation reduces ROS levels in intestinal epithelial cells of weaned piglets. A proteomics analysis revealed that leucine supplementation induces an energy metabolism switch from oxidative phosphorylation (OXPHOS) towards glycolysis. The leucine-induced ROS reduction and the energy metabolism switch were further validated in cultured cells. Mechanistically, our data revealed that leucine-induced ROS reduction actually depends on the energy metabolism switch from OXPHOS towards glycolysis through the mechanistic target of rapamycin (mTOR)- hypoxia-inducible factor-1alpha (HIF-1α) pathway. These findings reveal a vital regulatory role of leucine as the signal molecule involved in an energy metabolism switch in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements.

PubMed

Smith, C D; Baglia, L A; Curristin, S M; Ruddell, A

1994-10-01

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

PubMed Central

Martin, Neil R.W.; Turner, Mark C.; Farrington, Robert; Player, Darren J.

2017-01-01

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology. PMID:28409828

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro.

PubMed

Martin, Neil R W; Turner, Mark C; Farrington, Robert; Player, Darren J; Lewis, Mark P

2017-10-01

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP-1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co-incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly-controlled investigations into nutritional regulation of muscle physiology. © 2017 The Authors. Journal of Cellular Physiology Published by wiley periodicals, Inc.

Protein phosphatase PHLPP1 controls the light-induced resetting of the circadian clock

PubMed Central

Masubuchi, Satoru; Gao, Tianyan; O'Neill, Audrey; Eckel-Mahan, Kristin; Newton, Alexandra C.; Sassone-Corsi, Paolo

2010-01-01

The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) differentially attenuates Akt, PKC, and ERK1/2 signaling, thereby controlling the duration and amplitude of responses evoked by these kinases. PHLPP1 is expressed in the mammalian central clock, the suprachiasmatic nucleus, where it oscillates in a circadian fashion. To explore the role of PHLPP1 in vivo, we have generated mice with a targeted deletion of the PHLPP1 gene. Here we show that PHLPP1-null mice, although displaying normal circadian rhythmicity, have a drastically impaired capacity to stabilize the circadian period after light-induced resetting, producing a large phase shift after light resetting. Our findings reveal that PHLPP1 exerts a previously unappreciated role in circadian control, governing the consolidation of circadian periodicity after resetting. PMID:20080691

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed Central

Vulpe, Chris D.

2013-01-01

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

Induction of Glucocorticoid-induced Leucine Zipper (GILZ) Contributes to Anti-inflammatory Effects of the Natural Product Curcumin in Macrophages*

PubMed Central

Hoppstädter, Jessica; Hachenthal, Nina; Valbuena-Perez, Jenny Vanessa; Lampe, Sebastian; Astanina, Ksenia; Kunze, Michael M.; Bruscoli, Stefano; Riccardi, Carlo; Schmid, Tobias; Diesel, Britta; Kiemer, Alexandra K.

2016-01-01

GILZ (glucocorticoid-induced leucine zipper) is inducible by glucocorticoids and plays a key role in their mode of action. GILZ attenuates inflammation mainly by inhibition of NF-κB and mitogen-activated protein kinase activation but does not seem to be involved in the severe side effects observed after glucocorticoid treatment. Therefore, GILZ might be a promising target for new therapeutic approaches. The present work focuses on the natural product curcumin, which has previously been reported to inhibit NF-κB. GILZ was inducible by curcumin in macrophage cell lines, primary human monocyte-derived macrophages, and murine bone marrow-derived macrophages. The up-regulation of GILZ was neither associated with glucocorticoid receptor activation nor with transcriptional induction or mRNA or protein stabilization but was a result of enhanced translation. Because the GILZ 3′-UTR contains AU-rich elements (AREs), we analyzed the role of the mRNA-binding protein HuR, which has been shown to promote the translation of ARE-containing mRNAs. Our results suggest that curcumin treatment induces HuR expression. An RNA immunoprecipitation assay confirmed that HuR can bind GILZ mRNA. In accordance, HuR overexpression led to increased GILZ protein levels but had no effect on GILZ mRNA expression. Our data employing siRNA in LPS-activated RAW264.7 macrophages show that curcumin facilitates its anti-inflammatory action by induction of GILZ in macrophages. Experiments with LPS-activated bone marrow-derived macrophages from wild-type and GILZ knock-out mice demonstrated that curcumin inhibits the activity of inflammatory regulators, such as NF-κB or ERK, and subsequent TNF-α production via GILZ. In summary, our data indicate that HuR-dependent GILZ induction contributes to the anti-inflammatory properties of curcumin. PMID:27629417

Morphological characteristics, anatomical structure, and gene expression: novel insights into gibberellin biosynthesis and perception during carrot growth and development.

PubMed

Wang, Guang-Long; Xiong, Fei; Que, Feng; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

2015-01-01

Gibberellins (GAs) are considered potentially important regulators of cell elongation and expansion in plants. Carrot undergoes significant alteration in organ size during its growth and development. However, the molecular mechanisms underlying gibberellin accumulation and perception during carrot growth and development remain unclear. In this study, five stages of carrot growth and development were investigated using morphological and anatomical structural techniques. Gibberellin levels in leaf, petiole, and taproot tissues were also investigated for all five stages. Gibberellin levels in the roots initially increased and then decreased, but these levels were lower than those in the petioles and leaves. Genes involved in gibberellin biosynthesis and signaling were identified from the carrotDB, and their expression was analyzed. All of the genes were evidently responsive to carrot growth and development, and some of them showed tissue-specific expression. The results suggested that gibberellin level may play a vital role in carrot elongation and expansion. The relative transcription levels of gibberellin pathway-related genes may be the main cause of the different bioactive GAs levels, thus exerting influences on gibberellin perception and signals. Carrot growth and development may be regulated by modification of the genes involved in gibberellin biosynthesis, catabolism, and perception.

Repeated Solid-state Dewetting of Thin Gold Films for Nanogap-rich Plasmonic Nanoislands.

PubMed

Kang, Minhee; Park, Sang-Gil; Jeong, Ki-Hun

2015-10-15

This work reports a facile wafer-level fabrication for nanogap-rich gold nanoislands for highly sensitive surface enhanced Raman scattering (SERS) by repeating solid-state thermal dewetting of thin gold film. The method provides enlarged gold nanoislands with small gap spacing, which increase the number of electromagnetic hotspots and thus enhance the extinction intensity as well as the tunability for plasmon resonance wavelength. The plasmonic nanoislands from repeated dewetting substantially increase SERS enhancement factor over one order-of-magnitude higher than those from a single-step dewetting process and they allow ultrasensitive SERS detection of a neurotransmitter with extremely low Raman activity. This simple method provides many opportunities for engineering plasmonics for ultrasensitive detection and highly efficient photon collection.

Quantitative role of splanchnic region in leucine metabolism: L-(1-13C,15N)leucine and substrate balance studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Y.M.; Wagner, D.A.; Tredget, E.E.

1990-07-01

The role of the splanchnic region (Sp) in whole body leucine metabolism was assessed in six chronically catheterized fasting mongrel dogs and in eight dogs during constant enteral feeding of a complete amino acid solution (0.24 g.kg-1.h-1). We used primed continuous intravenous infusions of L-(1-13C,15N)leucine and L-(1-14C)leucine and measurements of arteriovenous isotope and leucine balance across the gut, liver, and Sp. In the fasted condition, 3.5% of arterial leucine supply was oxidized in the Sp, accounting for 13% of total body leucine oxidation, with 10% by liver. With amino acid feeding (1) leucine carbon and nitrogen fluxes and oxidation weremore » increased (P less than 0.01) at the whole body level; (2) the percent of whole body leucine oxidation occurring in the Sp and liver increased (P less than 0.01) to 41 and 27%, respectively; (3) fractional metabolic utilization of leucine delivered to the Sp was reduced (P less than 0.01) from 47 to 35%; (4) the deamination rate of leucine in the gut was increased (P less than 0.05), along with an increased reamination rate of alpha-ketoisocaproic acid in the Sp (P less than 0.05). These findings reveal that the Sp accounts for a small fraction of whole body leucine oxidation during the fasting condition, but it plays a quantitatively important role in total body leucine oxidation during amino acid feeding; the gut and liver play cooperative roles in controlling leucine supply to peripheral tissues.« less

Facio-brachio-crural dystonic episodes and drop attacks due to leucine rich glioma inactivated 1 encephalitis in two elderly Indian women.

PubMed

Maramattom, Boby Varkey; Jeevanagi, Sachin Rajashekar; George, Celinamma

2013-10-01

Two women in their 60's are presented to us with sudden falls of acute onset. Prolonged observation revealed a gradually evolving syndrome of paroxysmal right sided faciobrachial dystonic (FBD) posturing lasting seconds. Both patients went on to develop hyponatremia, following which the episodes worsened and appeared on both sides. In both cases, prolonged electroencephalography monitoring and magnetic resonance imaging brain were normal and the response to conventional anticonvulsants was poor. One patient improved spontaneously over 6 months. The 2(nd) patient developed an amnestic syndrome and was started on intravenous methylprednisolone with which her movement disorder abated. Her amnestic syndrome improved and she was discharged on oral steroids. Both patients tested positive for leucine-rich glioma inactivated 1 (LGi1) antibodies. We present the first case reports of FBD episodes and drop attacks owing to LGi1 encephalitis from India and review the relevant literature pertinent to the subject.

Environmental stress induces trinucleotide repeat mutagenesis in human cells.

PubMed

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

2015-03-24

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.

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

USDA-ARS?s Scientific Manuscript database

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

Morphological characteristics, anatomical structure, and gene expression: novel insights into gibberellin biosynthesis and perception during carrot growth and development

PubMed Central

Wang, Guang-Long; Xiong, Fei; Que, Feng; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

2015-01-01

Gibberellins (GAs) are considered potentially important regulators of cell elongation and expansion in plants. Carrot undergoes significant alteration in organ size during its growth and development. However, the molecular mechanisms underlying gibberellin accumulation and perception during carrot growth and development remain unclear. In this study, five stages of carrot growth and development were investigated using morphological and anatomical structural techniques. Gibberellin levels in leaf, petiole, and taproot tissues were also investigated for all five stages. Gibberellin levels in the roots initially increased and then decreased, but these levels were lower than those in the petioles and leaves. Genes involved in gibberellin biosynthesis and signaling were identified from the carrotDB, and their expression was analyzed. All of the genes were evidently responsive to carrot growth and development, and some of them showed tissue-specific expression. The results suggested that gibberellin level may play a vital role in carrot elongation and expansion. The relative transcription levels of gibberellin pathway-related genes may be the main cause of the different bioactive GAs levels, thus exerting influences on gibberellin perception and signals. Carrot growth and development may be regulated by modification of the genes involved in gibberellin biosynthesis, catabolism, and perception. PMID:26504574

The PPARgamma agonist FMOC-L-leucine protects both mature and immature brain.

PubMed

Maurois, Pierre; Rocchi, Stéphane; Pages, Nicole; Bac, Pierre; Stables, James P; Gressens, Pierre; Vamecq, Joseph

2008-01-01

(N-[9-fluorenylmethoxycarbonyl]-)-L-leucine (FMOC-L-leucine) and rosiglitazone, two ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), were evaluated in mature (adult mice) and immature (pups) brain injury models. In adult magnesium-deficient mice, a model responsive to both neuroprotective and anti-seizure compounds, FMOC-L-leucine, but not rosiglitazone, protected against audiogenic seizures. The protection afforded by FMOC-L-leucine was alleviated by the PPARgamma antagonist GW9662 (1-2 mg/kg) and was induced in 50% animals by 4.8+/-1.2 mg/kg. At this dose, FMOC-L-leucine modified audiogenic seizure phase durations in convulsing mice differently than prototype antiepileptic drugs did. FMOC-L-leucine (up to 100 mg/kg) was inactive in the 6 Hz seizure test, an adult animal model largely responsive to anti-seizure drugs. In a model of neonatal brain injury, FMOC-L-leucine (4 microg/kg) was neuroprotective against cerebral ibotenate toxicity. It reduced significantly the size of lesions in grey but not in white matter, while rosiglitazone (10 microg/kg) was inactive. Taken as a whole, the present data support neuroprotective potentialities of FMOC-L-leucine towards both mature and immature brain. The PPAR-based protection of immature brain is more important as it is known that classic adult brain protectants (GABA(A) activators, N-methyl-D-aspartate and sodium channel blockers) may be toxic for immature brain. The PPARgamma agonist FMOC-L-leucine is likely to be devoid of these classic protective mechanisms because of its inactivity in the 6 Hz seizure test, its activity in the audiogenic test being explained by neuroprotective rather than intrinsic anti-seizure mechanisms. Targeting PPARs might be thus a promising way to protect immature brain.

Environmental stress induces trinucleotide repeat mutagenesis in human cells

PubMed Central

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

2015-01-01

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

Repeated Solid-state Dewetting of Thin Gold Films for Nanogap-rich Plasmonic Nanoislands

PubMed Central

Kang, Minhee; Park, Sang-Gil; Jeong, Ki-Hun

2015-01-01

This work reports a facile wafer-level fabrication for nanogap-rich gold nanoislands for highly sensitive surface enhanced Raman scattering (SERS) by repeating solid-state thermal dewetting of thin gold film. The method provides enlarged gold nanoislands with small gap spacing, which increase the number of electromagnetic hotspots and thus enhance the extinction intensity as well as the tunability for plasmon resonance wavelength. The plasmonic nanoislands from repeated dewetting substantially increase SERS enhancement factor over one order-of-magnitude higher than those from a single-step dewetting process and they allow ultrasensitive SERS detection of a neurotransmitter with extremely low Raman activity. This simple method provides many opportunities for engineering plasmonics for ultrasensitive detection and highly efficient photon collection. PMID:26469768

Suppression of LRRC19 promotes cutaneous wound healing in pressure ulcers in mice.

PubMed

Sun, Jie; Wang, Zhijing; Wang, Xirui

2018-02-20

The ischemia-reperfusion (I/R) induced skin lesion has been identified as primary cause of pressure ulcer. Better understanding of the mechanism is required for new therapy development. Leucine rich repeat containing protein 19 (LRRC19) is a recently discovered transmembrane protein containing leucine-rich repeats and plays a role in immune response. To investigate the role of LRRC19 in pressure ulcers, mouse ulcer model was established with two cycles of I/R. The expression of LRRC19 was assessed during injury. siRNA mediated LRRC19 downregulation was applied to investigate the disease severity, immune cell infiltration and pro-inflammatory cytokines production. The primary skin fibroblasts were stimulated with IL-1β to dissect the molecular mechanism. LRRC19 was readily induced in I/R induced lesion site in a pattern mimicking the disease progress as measured by wound area. Knockdown of LRRC19 by siRNA significantly alleviated the disease severity and attenuated immune cell infiltration and pro-inflammatory cytokines production. In primary skin fibroblast model, siRNA knockdown of LRRC19 suppressed IL-1β mediated NFκB activation and its downstream cytokines production. LRRC19 was a novel factor for I/R-induced tissue damage by promoting NFκB dependent pro-inflammatory response. Our results supported that LRRC19 could be a potential therapeutic target for pressure ulcers.

Rice Ethylene-Response AP2/ERF Factor OsEATB Restricts Internode Elongation by Down-Regulating a Gibberellin Biosynthetic Gene1[W][OA

PubMed Central

Qi, Weiwei; Sun, Fan; Wang, Qianjie; Chen, Mingluan; Huang, Yunqing; Feng, Yu-Qi; Luo, Xiaojin; Yang, Jinshui

2011-01-01

Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops. PMID:21753115

Repeated seizures induce long-term increase in hippocampal benzodiazepine receptors.

PubMed Central

McNamara, J O; Peper, A M; Patrone, V

1980-01-01

Repeated seizures, whether induced by kindling or electroshock, caused a long-lasting (at least 24 hr) increase of [3H]diazepam binding in hippocampal membranes of Sprague-Dawley rats. Scatchard analyses demonstrated that increased numbers of binding sites accounted for the increase. Neither repeated hypoxia nor repeated administration of electrical current without inducing seizures caused an increase of [3H]diazepam binding. Regardless of the method used for seizure induction, the response was graded in that large numbers of seizures were required to induce significant increases, whereas fewer seizures induced only slight increases. We suggest that the receptor increases imply a heightened response to benzodiazepines and more powerful hippocampal recurrent inhibition. PMID:6930682

[Effects of fluridone, gibberellin acid and germination temperature on dormancy-breaking for Epimedium wushanense].

PubMed

Su, He; Wang, Yue; Yang, Yang; Dong, Xue-Hui

2016-07-01

We introduced Epimedium wushanense seed which has been stratified for 90 days at 10/20 ℃ as experimental materials, with which we studied the effects of fluridone, gibberellin acid and temperature on E. wushanense germination. The results were suggested as shown below. ①Temperature, fluridone and gibberellin acid can both solely or jointly affect germination energy, germination rate significantly. Among those factors, fluridone affect germination rate and germination energy the most, followed by gibberellin acid and temperature. The highest germination rate under 4 ℃ and 10/20 ℃ stratification are 79.3%, 72.0% respectively, which resulted from treatment of F10GA300 and F20GA200 respectively. The highest germination energy under 4 ℃ and 10/20 ℃ stratification are 52.7%, 52.0%, respectively, which both resulted from F20GA200. ②Compared with 4 ℃ germination, seed could not germinate at 10/20 ℃ germination. Nontheless, application of fluridone can lead E. wushanense seeds to germinating.③The effects of gibberellin acid and interaction between gibberellin acid and fluridone significantly affect seed rotten rate during germination. In addition, soaking is another remarkable factor which increased seed rotten rate. As a result, it is feasible to promote E. wushanense dormancy releasing with gibberellin acid and fluridone associating with a proper germination temperature. Further, it is necessary taking actions to avoid seed rotten rate for saving E. wushanense nurseries'cost. Copyright© by the Chinese Pharmaceutical Association.

EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice.

PubMed

Luo, Anding; Qian, Qian; Yin, Hengfu; Liu, Xiaoqiang; Yin, Changxi; Lan, Ying; Tang, Jiuyou; Tang, Zuoshun; Cao, Shouyun; Wang, Xiujie; Xia, Kai; Fu, Xiangdong; Luo, Da; Chu, Chengcai

2006-02-01

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.

Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice.

PubMed

Zhang, Yingying; Zhu, Yongyou; Peng, Yu; Yan, Dawei; Li, Qun; Wang, Jianjun; Wang, Linyou; He, Zuhua

2008-03-01

The rice Eui (ELONGATED UPPERMOST INTERNODE) gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins (GAs). In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmy1A and GAmyb were highly upregulated in eui aleurone cells in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 (GIBBERELLIN INSENSITIVE DWARF 1), GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA20ox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.

Leaf-Induced Gibberellin Signaling Is Essential for Internode Elongation, Cambial Activity, and Fiber Differentiation in Tobacco Stems[C][W

PubMed Central

Dayan, Jonathan; Voronin, Nickolay; Gong, Fan; Sun, Tai-ping; Hedden, Peter; Fromm, Hillel; Aloni, Roni

2012-01-01

The gibberellins (GAs) are a group of endogenous compounds that promote the growth of most plant organs, including stem internodes. We show that in tobacco (Nicotiana tabacum) the presence of leaves is essential for the accumulation of bioactive GAs and their immediate precursors in the stem and consequently for normal stem elongation, cambial proliferation, and xylem fiber differentiation. These processes do not occur in the absence of maturing leaves but can be restored by application of C19-GAs, identifying the presence of leaves as a requirement for GA signaling in stems and revealing the fundamental role of GAs in secondary growth regulation. The use of reporter genes for GA activity and GA-directed DELLA protein degradation in Arabidopsis thaliana confirms the presence of a mobile signal from leaves to the stem that induces GA signaling. PMID:22253226

Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

PubMed Central

Brown, Sharon J.; Fuller, Heidi R.; Jones, Philip; Caterson, Bruce; Shirran, Sally L.; Botting, Catherine H.

2016-01-01

The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time. PMID:27994047

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

PubMed

Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A

2008-10-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

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

PubMed Central

Suryawan, Agus; Jeyapalan, Asumthia S.; Orellana, Renan A.; Wilson, Fiona A.; Nguyen, Hanh V.; Davis, Teresa A.

2008-01-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E·eIF4G complex and increased eIF4E·4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein β-subunit-like protein (GβL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors. PMID:18682538

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

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.

FRB 121102: A Starquake-induced Repeater?

NASA Astrophysics Data System (ADS)

Wang, Weiyang; Luo, Rui; Yue, Han; Chen, Xuelei; Lee, Kejia; Xu, Renxin

2018-01-01

Since its initial discovery, the fast radio burst (FRB) FRB 121102 has been found to be repeating with millisecond-duration pulses. Very recently, 14 new bursts were detected by the Green Bank Telescope during its continuous monitoring observations. In this paper, we show that the burst energy distribution has a power-law form which is very similar to the Gutenberg–Richter law of earthquakes. In addition, the distribution of burst waiting time can be described as a Poissonian or Gaussian distribution, which is consistent with earthquakes, while the aftershock sequence exhibits some local correlations. These findings suggest that the repeating FRB pulses may originate from the starquakes of a pulsar. Noting that the soft gamma-ray repeaters (SGRs) also exhibit such distributions, the FRB could be powered by some starquake mechanisms associated with the SGRs, including the crustal activity of a magnetar or solidification-induced stress of a newborn strangeon star. These conjectures could be tested with more repeating samples.

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression

PubMed Central

Moreno, J.C.; Cerda, A.; Simpson, K.; Lopez-Diaz, I.; Carrera, E; Handford, M.; Stange, C.

2016-01-01

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. PMID:26893492

Chromosomal translocations and palindromic AT-rich repeats

PubMed Central

Kato, Takema; Kurahashi, Hiroki; Emanuel1, Beverly S.

2012-01-01

Repetitive DNA sequences constitute 30% of the human genome, and are often sites of genomic rearrangement. Recently, it has been found that several constitutional translocations, especially those that involve chromosome 22, take place utilizing palindromic sequences on 22q11 and on the partner chromosome. Analysis of translocation junction fragments shows that the breakpoints of such palindrome-mediated translocations are localized at the center of palindromic AT-rich repeats (PATRRs). The presence of PATRRs at the breakpoints, indicates a palindrome-mediated mechanism involved in the generation of these constitutional translocations. Identification of these PATRR-mediated translocations suggests a universal pathway for gross chromosomal rearrangement in the human genome. De novo occurrences of PATRR-mediated translocations can be detected by PCR in normal sperm samples but not somatic cells. Polymorphisms of various PATRRs influence their propensity for adopting a secondary structure, which in turn affects de novo translocation frequency. We propose that the PATRRs form an unstable secondary structure, which leads to double-strand breaks at the center of the PATRR. The double-strand breaks appear to be followed by a non-homologous end-joining repair pathway, ultimately leading to the translocations. This review considers recent findings concerning the mechanism of meiosis-specific, PATRR-mediated translocations. PMID:22402448

Hollow fiber-based liquid-liquid-liquid micro-extraction with osmosis: II. Application to quantification of endogenous gibberellins in rice plant.

PubMed

Wu, Qian; Wu, Dapeng; Duan, Chunfeng; Shen, Zheng; Guan, Yafeng

2012-11-23

The phenomenon and benefits of osmosis in hollow fiber-based liquid-liquid-liquid micro-extraction (HF-LLLME) were theoretically discussed in part I of this study. In this work, HF-LLLME with osmosis was coupled with high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-triple quadrupole MS/MS) to analyze eight gibberellins (gibberellin A(1), gibberellin A(3), gibberellin A(4), gibberellin A(7), gibberellin A(8), gibberellin A(9), gibberellin A(19) and gibberellin A(20)) in rice plant samples. According to the theory of HF-LLLME with osmosis, single factor experiments, orthogonal design experiments and mass transfer simulation of extraction process were carried out to select the optimal conditions. Cyclohexanol - n-octanol (1:3, v/v) was selected as organic membrane. Donor phase of 12 mL was adjusted to pH 2 and 20% NaCl (w/v) was added. Acceptor phase with an initial volume of 20 μL was the solution of 0.12 mol L(-1) Na(2)CO(3)-NaHCO(3) buffer (pH 9). Temperature was chosen to be 30 °C and extraction time was selected to be 90 min. Under optimized conditions, this method provided good linearity (r, 0.99552-0.99991) and low limits of detection (0.0016-0.061 ng mL(-1)). Finally, this method was applied to the analysis of endogenous gibberellins from plant extract which was obtained with traditional solvent extraction of rice plant tissues, and the relative recoveries were from 62% to 166%. Copyright © 2012 Elsevier B.V. All rights reserved.

Excessive Leucine-mTORC1-Signalling of Cow Milk-Based Infant Formula: The Missing Link to Understand Early Childhood Obesity.

PubMed

Melnik, Bodo C

2012-01-01

Increased protein supply by feeding cow-milk-based infant formula in comparison to lower protein content of human milk is a well-recognized major risk factor of childhood obesity. However, there is yet no conclusive biochemical concept explaining the mechanisms of formula-induced childhood obesity. It is the intention of this article to provide the biochemical link between leucine-mediated signalling of mammalian milk proteins and adipogenesis as well as early adipogenic programming. Leucine has been identified as the predominant signal transducer of mammalian milk, which stimulates the nutrient-sensitive kinase mammalian target of rapamycin complex 1 (mTORC1). Leucine thus functions as a maternal-neonatal relay for mTORC1-dependent neonatal β-cell proliferation and insulin secretion. The mTORC1 target S6K1 plays a pivotal role in stimulation of mesenchymal stem cells to differentiate into adipocytes and to induce insulin resistance. It is of most critical concern that infant formulas provide higher amounts of leucine in comparison to human milk. Exaggerated leucine-mediated mTORC1-S6K1 signalling induced by infant formulas may thus explain increased adipogenesis and generation of lifelong elevated adipocyte numbers. Attenuation of mTORC1 signalling of infant formula by leucine restriction to physiologic lower levels of human milk offers a great chance for the prevention of childhood obesity and obesity-related metabolic diseases.

Hypothalamic Leucine Metabolism Regulates Liver Glucose Production

PubMed Central

Su, Ya; Lam, Tony K.T.; He, Wu; Pocai, Alessandro; Bryan, Joseph; Aguilar-Bryan, Lydia; Gutiérrez-Juárez, Roger

2012-01-01

Amino acids profoundly affect insulin action and glucose metabolism in mammals. Here, we investigated the role of the mediobasal hypothalamus (MBH), a key center involved in nutrient-dependent metabolic regulation. Specifically, we tested the novel hypothesis that the metabolism of leucine within the MBH couples the central sensing of leucine with the control of glucose production by the liver. We performed either central (MBH) or systemic infusions of leucine in Sprague-Dawley male rats during basal pancreatic insulin clamps in combination with various pharmacological and molecular interventions designed to modulate leucine metabolism in the MBH. We also examined the role of hypothalamic ATP-sensitive K+ channels (KATP channels) in the effects of leucine. Enhancing the metabolism of leucine acutely in the MBH lowered blood glucose through a biochemical network that was insensitive to rapamycin but strictly dependent on the hypothalamic metabolism of leucine to α-ketoisocaproic acid and, further, insensitive to acetyl- and malonyl-CoA. Functional KATP channels were also required. Importantly, molecular attenuation of this central sensing mechanism in rats conferred susceptibility to developing hyperglycemia. We postulate that the metabolic sensing of leucine in the MBH is a previously unrecognized mechanism for the regulation of hepatic glucose production required to maintain glucose homeostasis. PMID:22187376

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland.

PubMed

Syaidah, Rahimi; Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

2013-11-01

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

Rare sugar D-allose suppresses gibberellin signaling through hexokinase-dependent pathway in Oryza sativa L.

PubMed

Fukumoto, Takeshi; Kano, Akihito; Ohtani, Kouhei; Yamasaki-Kokudo, Yumiko; Kim, Bong-Gyu; Hosotani, Kouji; Saito, Miu; Shirakawa, Chikage; Tajima, Shigeyuki; Izumori, Ken; Ohara, Toshiaki; Shigematsu, Yoshio; Tanaka, Keiji; Ishida, Yutaka; Nishizawa, Yoko; Tada, Yasuomi; Ichimura, Kazuya; Gomi, Kenji; Akimitsu, Kazuya

2011-12-01

One of the rare sugars, D-allose, which is the epimer of D-glucose at C3, has an inhibitory effect on rice growth, but the molecular mechanisms of the growth inhibition by D-allose were unknown. The growth inhibition caused by D-allose was prevented by treatment with hexokinase inhibitors, D-mannoheptulose and N-acetyl-D-glucosamine. Furthermore, the Arabidopsis glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1, showed a D-allose-insensitive phenotype. D-Allose strongly inhibited the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half rice seeds. The growth of the slender rice1 (slr1) mutant, which exhibits a constitutive gibberellin-responsive phenotype, was also inhibited by D-allose, and the growth inhibition of the slr1 mutant by D-allose was also prevented by D-mannoheptulose treatment. The expressions of gibberellin-responsive genes were down-regulated by D-allose treatment, and the down-regulations of gibberellin-responsive genes were also prevented by D-mannoheptulose treatment. These findings reveal that D-allose inhibits the gibberellin-signaling through a hexokinase-dependent pathway.

Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage*

PubMed Central

Scranton, Melissa A.; Yee, Ashley; Park, Sang-Youl; Walling, Linda L.

2012-01-01

Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress. PMID:22493451

Leucine facilitates the insulin-stimulated glucose uptake and insulin signaling in skeletal muscle cells: involving mTORC1 and mTORC2.

PubMed

Liu, Hui; Liu, Rui; Xiong, Yufang; Li, Xiang; Wang, Xiaolei; Ma, Yan; Guo, Huailan; Hao, Liping; Yao, Ping; Liu, Liegang; Wang, Di; Yang, Xuefeng

2014-08-01

Leucine, a branched-chain amino acid, has been shown to promote glucose uptake and increase insulin sensitivity in skeletal muscle, but the exact mechanism remains unestablished. We addressed this issue in cultured skeletal muscle cells in this study. Our results showed that leucine alone did not have an effect on glucose uptake or phosphorylation of protein kinase B (AKT), but facilitated the insulin-induced glucose uptake and AKT phosphorylation. The insulin-stimulated glucose uptake and AKT phosphorylation were inhibited by the phosphatidylinositol 3-kinase inhibitor, wortmannin, but the inhibition was partially reversed by leucine. The inhibitor of mammalian target of rapamycin complex 1 (mTORC1), rapamycin, had no effect on the insulin-stimulated glucose uptake, but eliminated the facilitating effect of leucine in the insulin-stimulated glucose uptake and AKT phosphorylation. In addition, leucine facilitation of the insulin-induced AKT phosphorylation was neutralized by knocking down the core component of the mammalian target of rapamycin complex 2 (mTORC2) with specific siRNA. Together, these findings show that leucine can facilitate the insulin-induced insulin signaling and glucose uptake in skeletal muscle cells through both mTORC1 and mTORC2, implicating the potential importance of this amino acid in glucose homeostasis and providing new mechanistic insights.

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation.

PubMed

Hahn, Rebecca T; Hoppstädter, Jessica; Hirschfelder, Kerstin; Hachenthal, Nina; Diesel, Britta; Kessler, Sonja M; Huwer, Hanno; Kiemer, Alexandra K

2014-06-01

Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

PubMed Central

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

2014-01-01

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. PMID:24898843

Glucocorticoid-Induced Leucine Zipper Protects the Retina From Light-Induced Retinal Degeneration by Inducing Bcl-xL in Rats.

PubMed

Gu, Ruiping; Tang, Wenyi; Lei, Boya; Ding, Xinyi; Jiang, Cheng; Xu, Gezhi

2017-07-01

The aim of the present study was to investigate the neuroprotective effects of glucocorticoid-induced leucine zipper (GILZ) in a light-induced retinal degeneration model and to explore the underlying mechanisms. Intravitreal injection of recombinant GILZ-overexpressing lentivirus (OE-GILZ-rLV) and short hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV) was performed to up- and downregulate retinal GILZ, respectively. Three days after stable transduction, rats were exposed to continuous bright light (5000 lux) for 2 days. Retinal function was assessed by full-field electroretinography (ERG), and the retinal structure was examined for photoreceptor survival and death in rats kept under a 12-hour light:2-hour dark cycle following light exposure. The expression levels of retinal Bcl-xL, caspase-9, and caspase-3 were examined by Western blotting or real-time PCR at 1, 3, 5, and 7 days after light exposure. Exposure to bright light downregulated retinal GILZ in parallel with the downregulation of Bcl-xL and the upregulation of active caspase-3. Overexpression of retinal GILZ attenuated the decrease of Bcl-xL and the activation of caspase-9 and caspase-3 at 1, 3, 5, and 7 days after bright light exposure, respectively. GILZ silencing aggravated the downregulation of Bcl-xL induced by bright light exposure. Bright light exposure reduced the amplitude of ERG, increased the number of apoptotic photoreceptor cells, and decreased retinal thickness; and GILZ overexpression could attenuate all these effects. Overexpression of GILZ by OE-GILZ-rLV transduction protected the retina from light-induced cellular damage by activating antiapoptotic pathways.

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression.

PubMed

Moreno, J C; Cerda, A; Simpson, K; Lopez-Diaz, I; Carrera, E; Handford, M; Stange, C

2016-04-01

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Flavanol-rich cocoa ameliorates lipemia-induced endothelial dysfunction.

PubMed

Westphal, Sabine; Luley, Claus

2011-09-01

Consumption of flavanols improves chronic endothelial dysfunction. We investigated whether it can also improve acute lipemia-induced endothelial dysfunction. In this randomized, placebo-controlled, double-blind, crossover trial, 18 healthy subjects received a fatty meal with cocoa either rich in flavanols (918 mg) or flavanol-poor. Flow-mediated dilation (FMD), triglycerides, and free fatty acids were then determined over 6 h. After the flavanol-poor fat loading, the FMD deteriorated over 4 h. The consumption of flavanol-rich cocoa, in contrast, improved this deterioration in hours 2, 3, and 4 without abolishing it completely. Flavanols did not have any influence on triglycerides or on free fatty acids. Flavanol-rich cocoa can alleviate the lipemia-induced endothelial dysfunction, probably through an improvement in endothelial NO synthase.

Effects of mechanical stretching on the morphology of extracellular polymers and the mRNA expression of collagens and small leucine-rich repeat proteoglycans in vaginal fibroblasts from women with pelvic organ prolapse.

PubMed

Wang, Sumei; Lü, Dongyuan; Zhang, Zhenyu; Jia, Xingyuan; Yang, Lei

2018-01-01

To determine the effect of mechanical stretching load and the efficacy of postmenopausal estrogen therapy (ET) on pelvic organ prolapse (POP), vaginal fibroblasts isolated from postmenopausal women with or without POP were subjected to 0.1-Hz uniaxial cyclic mechanical stretching (CS) with 10% elongation and 10-8 M 17-β-estradiol (E2) treatment. We investigated the morphological characteristics of extracellular polymers using scanning electron microscopy (SEM) and monitored the mRNA expression of type I collagen (COL I) and type III collagen (COL III) as well as the small leucine-rich proteoglycan (SLRP) family members decorin (DCN), biglycan (BGN), fibromodulin (FMO), and lumican (LUM), using real-time quantitative polymerase chain reaction (RT-PCR). Using SEM, certain viscoelastic polymers were found to be randomly distributed among fibroblasts, which for normal fibroblasts formed clusters of plum flower-like patterns under static-culture conditions and resembled stretched strips when stretched in culture, whereas polymers among POP fibroblasts resembled stretched strips under static-cultured conditions and presented broken networks when stretched in culture. RT-PCR revealed that COL I, DCN, BGN, FMO, and LUM mRNA expression was significantly higher in POP than in normal fibroblasts under static-culture condition. Following CS, COL I and BGN mRNA expression was significantly up-regulated in normal fibroblasts, and DCN and FMO mRNA expression was down-regulated in POP fibroblasts. Following concomitant CS and E2 treatment, significantly elevated COL I and DCN mRNA expression was observed in normal fibroblasts, and significantly elevated COL I and BGN mRNA expression was observed in POP fibroblasts. COL III mRNA expression was not significantly different between the POP and normal group, and CS did not significantly affect expression in either group, though COL III was down-regulated in normal fibroblasts concomitantly treated with E2 and CS. We conclude that the

Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway.

PubMed

Onder, Zeynep; Chang, Vivian; Moroianu, Junona

2015-01-01

We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP-8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), 76IRTFQELLF84, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. Copyright © 2014 Elsevier Inc. All rights reserved.

[2.2.2]- to [3.2.1]-Bicycle Skeletal Rearrangement Approach to the Gibberellin Family of Natural Products.

PubMed

Smith, Brandon R; Njardarson, Jon T

2018-05-03

Synthetic studies toward the gibberellin family of natural products are reported. An oxidative dearomatization/Diels-Alder cascade assembles the carbon skeleton as a [2.2.2]-bicycle, which is then transformed to the [3.2.1]-bicyclic gibberellin core via a novel Lewis acid catalyzed rearrangement. Strategic synthetic handles allow for late-stage modification of the gibberellin skeleton and provides efficient access to this important family of natural compounds.

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

PubMed

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA.

PubMed

Nonin-Lecomte, Sylvie; Dardel, Frédéric; Lestienne, Patrick

2005-08-01

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.

Repeated folding stress-induced morphological changes in the dermal equivalent.

PubMed

Arai, Koji Y; Sugimoto, Mami; Ito, Kanako; Ogura, Yuki; Akutsu, Nobuko; Amano, Satoshi; Adachi, Eijiro; Nishiyama, Toshio

2014-11-01

Repeated mechanical stresses applied to the same region of the skin are thought to induce morphological changes known as wrinkle. However, the underlying mechanisms are not fully understood. To study the mechanisms, we examined effects of repeated mechanical stress on the dermal equivalent. We developed a novel device to apply repeated folding stress to the dermal equivalent. After applying the mechanical stress, morphological changes of the dermal equivalent and expression of several genes related to extracellular matrix turn over and cell contraction were examined. The repeated folding stress induced a noticeable decrease in the width of the dermal equivalent. The mechanical stress altered orientations of collagen fibrils. Hydroxyproline contents, dry weights and cell viability of the dermal equivalents were not affected by the mechanical stress. On the other hand, Rho-associated coiled-coil-containing kinase (ROCK) specific inhibitor Y27632 completely suppressed the decrease in the width of the dermal equivalent. The present results revealed that either degradation of collagen or changes in the number of cells were not responsible for the decrease in the width of the dermal equivalent and indicate that the repeated mechanical stress induces unidirectional contraction in the dermal equivalent through the RhoA-ROCK signaling pathway. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Acute depletion of plasma glutamine increases leucine oxidation in prednisone-treated humans

PubMed Central

Le Bacquer, Olivier; Mauras, Nelly; Welch, Susan; Haymond, Morey; Darmaun, Dominique

2007-01-01

Background, aims & methods To determine whether depletion in plasma glutamine worsens the catabolic response to corticosteroids, 7 healthy volunteers received oral prednisone for 6 days on 2 separate occasions, at least 2 weeks apart, and in random order. On the 6th day of each treatment course, they received 5h intravenous infusions of L-[1-14C]-leucine and L-[1-13C]-glutamine in the postabsorptive state 1) under baseline conditions (prednisone only day), and 2) after 24h of treatment with phenylbutyrate (prednisone+phenylbutyrate day), a glutamine chelating agent. Results Phenylbutyrate treatment was associated with 1) an ≈15% decline in plasma glutamine concentration (627±39 vs. 530±31 μmol.L-1; P<0.05), 2) no change in leucine appearance rate, an index of protein breakdown (124±9 vs. 128±9 μmol.kg-1.h-1; NS) nor in non oxidative leucine disposal, an index of whole body protein synthesis (94±9 vs. 91±7 μmol.kg -1.h-1; NS); and 3) a ≈25% rise in leucine oxidation (30±1 vs. 38±2 μmol.kg-1.h-1, P<0.05), despite an ≈25% decline (p<0.05) in leucine concentration. Conclusions In a model of mild, stress-induced protein catabolism, depletion of plasma glutamine per se may worsen branched chain amino acid and protein wasting. PMID:17097772

21 CFR 862.1460 - Leucine aminopeptidase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Systems § 862.1460 Leucine aminopeptidase test system. (a) Identification. A leucine aminopeptidase test system is a device intended to measure the activity of the enzyme leucine amino-peptidase in serum... diseases such as viral hepatitis and obstructive jaundice. (b) Classification. Class I (general controls...

21 CFR 862.1460 - Leucine aminopeptidase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Systems § 862.1460 Leucine aminopeptidase test system. (a) Identification. A leucine aminopeptidase test system is a device intended to measure the activity of the enzyme leucine amino-peptidase in serum... diseases such as viral hepatitis and obstructive jaundice. (b) Classification. Class I (general controls...

R-loops: targets for nuclease cleavage and repeat instability.

PubMed

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Interactive Effects of Jasmonic Acid, Salicylic Acid, and Gibberellin on Induction of Trichomes in Arabidopsis1

PubMed Central

Traw, M. Brian; Bergelson, Joy

2003-01-01

Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds. PMID:14551332

Repeat sample intraocular pressure variance in induced and naturally ocular hypertensive monkeys.

PubMed

Dawson, William W; Dawson, Judyth C; Hope, George M; Brooks, Dennis E; Percicot, Christine L

2005-12-01

To compare repeat-sample means variance of laser induced ocular hypertension (OH) in rhesus monkeys with the repeat-sample mean variance of natural OH in age-range matched monkeys of similar and dissimilar pedigrees. Multiple monocular, retrospective, intraocular pressure (IOP) measures were recorded repeatedly during a short sampling interval (SSI, 1-5 months) and a long sampling interval (LSI, 6-36 months). There were 5-13 eyes in each SSI and LSI subgroup. Each interval contained subgroups from the Florida with natural hypertension (NHT), induced hypertension (IHT1) Florida monkeys, unrelated (Strasbourg, France) induced hypertensives (IHT2), and Florida age-range matched controls (C). Repeat-sample individual variance means and related IOPs were analyzed by a parametric analysis of variance (ANOV) and results compared to non-parametric Kruskal-Wallis ANOV. As designed, all group intraocular pressure distributions were significantly different (P < or = 0.009) except for the two (Florida/Strasbourg) induced OH groups. A parametric 2 x 4 design ANOV for mean variance showed large significant effects due to treatment group and sampling interval. Similar results were produced by the nonparametric ANOV. Induced OH sample variance (LSI) was 43x the natural OH sample variance-mean. The same relationship for the SSI was 12x. Laser induced ocular hypertension in rhesus monkeys produces large IOP repeat-sample variance mean results compared to controls and natural OH.

Histochemistry of leucine aminoaphthylamidase (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.

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.

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.

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; ...

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

Incorporation of leucine into phospholipids of Bacteroides thetaiotaomicron.

PubMed Central

Smith, R D; Salyers, A A

1981-01-01

L-[4,5-3H]- or L-[U-14C]leucine was incorporated by Bacteroides thetaiotaomicron into acid-precipitable material even when the bacteria were treated with concentrations of tetracycline high enough to prevent growth. Similar results were obtained when L-[2,3,4-3H]valine or L-[4,5-3H]isoleucine was used instead of leucine. In bacteria which had been treated with tetracycline, the acid-precipitable label was not solubilized by treatment with protease, lysozyme, or deoxyribonuclease. However, virtually all of the label was extractable with chloroform-methanol, indicating that the label had been incorporated into membrane lipids. Since L-[1-14C]leucine was not incorporated into lipids, leucine was probably decarboxylated before incorporation. When a chloroform extract from bacteria which had been labeled with both [32P]phosphate and [3H]leucine was resolved into component phospholipids by two-dimensional thin-layer chromatography, 3H was incorporated into all of the phospholipids. When these phospholipids were deacylated, the 3H from leucine was associated with released fatty acids rather than with the head groups. Thus, it appears that B. thetaiotaomicron can utilize leucine and similar amino acids not only by incorporating them into protein but also by incorporating portions of these amino acids into membrane phospholipids. PMID:7462155

Hepatocyte growth factor limits autoimmune neuroinflammation via glucocorticoid-induced leucine zipper expression in dendritic cells.

PubMed

Benkhoucha, Mahdia; Molnarfi, Nicolas; Dunand-Sauthier, Isabelle; Merkler, Doron; Schneiter, Gregory; Bruscoli, Stefano; Riccardi, Carlo; Tabata, Yasuhiko; Funakoshi, Hiroshi; Nakamura, Toshikazu; Reith, Walter; Santiago-Raber, Marie-Laure; Lalive, Patrice H

2014-09-15

Autoimmune neuroinflammation, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity, is believed to result from immune tolerance dysfunction leading to demyelination and substantial neurodegeneration. We previously showed that CNS-restricted expression of hepatocyte growth factor (HGF), a potent neuroprotective factor, reduced CNS inflammation and clinical deficits associated with EAE. In this study, we demonstrate that systemic HGF treatment ameliorates EAE through the development of tolerogenic dendritic cells (DCs) with high expression levels of glucocorticoid-induced leucine zipper (GILZ), a transcriptional repressor of gene expression and a key endogenous regulator of the inflammatory response. RNA interference-directed neutralization of GILZ expression by DCs suppressed the induction of tolerance caused by HGF. Finally, adoptive transfer of HGF-treated DCs from wild-type but not GILZ gene-deficient mice potently mediated functional recovery in recipient mice with established EAE through effective modulation of autoaggressive T cell responses. Altogether, these results show that by inducing GILZ in DCs, HGF reproduces the mechanism of immune regulation induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids and therefore that HGF therapy may have potential in the treatment of autoimmune dysfunctions. Copyright © 2014 by The American Association of Immunologists, Inc.

Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoidinduced leucine zipper

PubMed Central

Lim, Wonchung; Park, Choa; Shim, Myeong Kuk; Lee, Yong Hee; Lee, You Mie; Lee, YoungJoo

2014-01-01

Background and Purpose The COX-2/PGE2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumourigenesis. However, the mechanism by which glucocorticoid receptors (GRs) inhibit COX-2 during hypoxia has not been elucidated. Hence, we explored the mechanisms underlying glucocorticoid-mediated inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells. Experimental Approach The expressions of COX-2 and glucocorticoid-induced leucine zipper (GILZ) in A549 cells were determined by Western blot and/or quantitative real time-PCR respectively. The anti-invasive effect of GILZ on A549 cells was evaluated using the matrigel invasion assay. Key Results The hypoxia-induced increase in COX-2 protein and mRNA levels and promoter activity were suppressed by dexamethasone, and this effect of dexamethasone was antagonized by the GR antagonist RU486. Overexpression of GILZ in A549 cells also inhibited hypoxia-induced COX-2 expression levels and knockdown of GILZ reduced the glucocorticoid-mediated inhibition of hypoxia-induced COX-2 expression, indicating that the inhibitory effects of dexamethasone on hypoxia-induced COX-2 are mediated by GILZ. GILZ suppressed the expression of hypoxia inducible factor (HIF)-1α at the protein level and affected its signalling pathway. Hypoxia-induced cell invasion was also dramatically reduced by GILZ expression. Conclusion and Implications Dexamethasone-induced upregulation of GILZ not only inhibits the hypoxic-evoked induction of COX-2 expression and cell invasion but further blocks the HIF-1 pathway by destabilizing HIF-1α expression. Taken together, these findings suggest that the suppression of hypoxia-induced COX-2 by glucocorticoids is mediated by GILZ. Hence, GILZ is a potential key therapeutic target for suppression of inflammation under hypoxia. PMID:24172143

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

Factors associated with repeat induced abortion in Kenya.

PubMed

Maina, Beatrice W; Mutua, Michael M; Sidze, Estelle M

2015-10-12

Over six million induced abortions were reported in Africa in 2008 with over two million induced abortions occurring in Eastern Africa. Although a significant proportion of women in the region procure more than one abortion during their reproductive period, there is a dearth of research on factors associated with repeat abortion. Data for this study come from the Magnitude and Incidence of Unsafe Abortion Study conducted by the African Population and Health Research Center in Kenya in 2012. The study used a nationally-representative sample of 350 facilities (level II to level VI) that offer post-abortion services for complications following induced and spontaneous abortions. A prospective morbidity survey tool was used by health providers in 328 facilities to collect information on socio-demographic charateristics, reproductive health history and contraceptive use at conception for all patients presenting for post-abortion services. Our analysis is based on data recorded on 769 women who were classified as having had an induced abortion. About 16 % of women seeking post abortion services for an induced abortion reported to have had a previous induced abortion. Being separated or divorced or widowed, having no education, having unwanted pregnancy, having 1-2 prior births and using traditional methods of contraception were associated with a higher likelihood of a repeat induced abortion. The findings point to the need to address the reasons why women with first time induced abortion do not have the necessary information to prevent unintended pregnancies and further induced abortions. Possible explanations linked to the quality of post-abortion family planning and coverage of long-acting methods should be explored.

The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell-Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features

PubMed Central

Kamke, Janine; Rinke, Christian; Schwientek, Patrick; Mavromatis, Kostas; Ivanova, Natalia; Sczyrba, Alexander; Woyke, Tanja; Hentschel, Ute

2014-01-01

The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake. PMID:24498082

Overexpression and characterization of an extracellular leucine aminopeptidase from Aspergillus oryzae.

PubMed

Matsushita-Morita, Mayumi; Tada, Sawaki; Suzuki, Satoshi; Hattori, Ryota; Marui, Junichiro; Furukawa, Ikuyo; Yamagata, Youhei; Amano, Hitoshi; Ishida, Hiroki; Takeuchi, Michio; Kashiwagi, Yutaka; Kusumoto, Ken-Ichi

2011-02-01

Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5-11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.

The Gene pat-2, Which Induces Natural Parthenocarpy, Alters the Gibberellin Content in Unpollinated Tomato Ovaries1

PubMed Central

Fos, Mariano; Nuez, Fernando; García-Martínez, José L.

2000-01-01

We investigated the role of gibberellins (GAs) in the effect of pat-2, a recessive mutation that induces facultative parthenocarpic fruit development in tomato (Lycopersicon esculentum Mill.) using near-isogenic lines with two different genetic backgrounds. Unpollinated wild-type Madrigal (MA/wt) and Cuarenteno (CU/wt) ovaries degenerated, but GA3 application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of MA/pat-2 and CU/pat-2 fruits, which occurs in the absence of pollination and hormone application, was not affected by GA3. Pollinated MA/wt and parthenocarpic MA/pat-2 ovary development was negated by paclobutrazol, and this inhibitory effect was counteracted by GA3. The main GAs of the early-13-hydroxylation pathway (GA1, GA3, GA8, GA19, GA20, GA29, GA44, GA53, and, tentatively, GA81) and two GAs of the non-13-hydroxylation pathway (GA9 and GA34) were identified in MA/wt ovaries by gas chromatography-selected ion monitoring. GAs were quantified in unpollinated ovaries at flower bud, pre-anthesis, and anthesis. In unpollinated MA/pat-2 and CU/pat-2 ovaries, the GA20 content was much higher (up to 160 times higher) and the GA19 content was lower than in the corresponding non-parthenocarpic ovaries. The application of an inhibitor of 2-oxoglutarate-dependent dioxygenases suggested that GA20 is not active per se. The pat-2 mutation may increase GA 20-oxidase activity in unpollinated ovaries, leading to a higher synthesis of GA20, the precursor of an active GA. PMID:10677440

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

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

The Tyrosine Sulfate Domain of Fibromodulin Binds Collagen and Enhances Fibril Formation.

PubMed

Tillgren, Viveka; Mörgelin, Matthias; Önnerfjord, Patrik; Kalamajski, Sebastian; Aspberg, Anders

2016-11-04

Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine tyrosine residues in the fibromodulin N-terminal domain are O-sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether tyrosine sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal tyrosine-sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The tyrosine-sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its tyrosine sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for tyrosine sulfation in collagen interaction and control of fibril formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Overexpression of SlGRAS40 in Tomato Enhances Tolerance to Abiotic Stresses and Influences Auxin and Gibberellin Signaling

PubMed Central

Liu, Yudong; Huang, Wei; Xian, Zhiqiang; Hu, Nan; Lin, Dongbo; Ren, Hua; Chen, Jingxuan; Su, Deding; Li, Zhengguo

2017-01-01

Abiotic stresses are major environmental factors that inhibit plant growth and development impacting crop productivity. GRAS transcription factors play critical and diverse roles in plant development and abiotic stress. In this study, SlGRAS40, a member of the tomato (Solanum lycopersicum) GRAS family, was functionally characterized. In wild-type (WT) tomato, SlGRAS40 was upregulated by abiotic stress induced by treatment with D-mannitol, NaCl, or H2O2. Transgenic tomato plants overexpressing SlGRAS40 (SlGRAS40-OE) were more tolerant of drought and salt stress than WT. SlGRAS40-OE plants displayed pleiotropic phenotypes reminiscent of those resulting from altered auxin and/or gibberellin signaling. A comparison of WT and SlGRAS40-OE transcriptomes showed that the expression of a large number of genes involved in hormone signaling and stress responses were modified. Our study of SlGRAS40 protein provides evidence of how another GRAS plays roles in resisting abiotic stress and regulating auxin and gibberellin signaling during vegetative and reproductive growth in tomato. PMID:29018467

Effect of a Herbal-Leucine mix on the IL-1β-induced cartilage degradation and inflammatory gene expression in human chondrocytes

PubMed Central

2011-01-01

Background Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO), glycosaminoglycan (GAG), matrix metalloproteinases (MMPs), aggrecan (ACAN) and type II collagen (COL2A1) in human OA chondrocytes and OA cartilage explants. Methods Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml) and then stimulated with IL-1β (5 ng/ml). Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits. Results HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p < 0.05). Supporting these gene expression results, IL-1β-induced cartilage matrix breakdown, as evidenced by GAG release from cartilage explants, was also significantly blocked (p < 0.05). Moreover, in the presence of herbal-Leucine mixture (HLM) up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p < 0.05). The inhibitory effects of HLM were mediated by inhibiting the activation of nuclear factor (NF)-kB in human OA chondrocytes in presence of IL-1β. Conclusion Our data

[Recent advances in the analysis of gibberellins plant hormones].

PubMed

Zhang, Xiaona; Lu, Minghua; Xu, Linfang; Xiao, Rui; Cai, Zongwei

2015-08-01

Gibberellins (GAs) are a class of phytohormones that exert profound and diverse effects on plant growth and development, such as seed germination and leaf expansion. Up to now, 136 members of GAs have been identified and recognized. All known GAs are diterpenoid acids with similar chemical structures, only double bonds, hydroxyl numbers and locations on gibberellin alkane skeleton are different. However, the content of GAs in plants is of ultra trace levels (usually at ng/g and even pg/g levels) with little ultraviolet (UV) absorption, no fluorescence and no distinguishing chemical characteristics. Moreover, the matrix of plant samples is complicated. Thus, quantification of GAs is always extremely difficult. Nowadays, the bottle necks for the study of GAs in plants are due to the lack of efficient sample preparation and sensitive detection techniques. This article reviews the analytical methods for determination of GAs in recent years, hoping to provide some references to develop new methods and techniques.

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Efficacy and Safety of Leucine Supplementation in the Elderly.

PubMed

Borack, Michael S; Volpi, Elena

2016-12-01

Leucine supplementation has grown in popularity due to the discovery of its anabolic effects on cell signaling and protein synthesis in muscle. The current recommendation is a minimum intake of 55 mg ⋅ kg -1 . d -1 Leucine acutely stimulates skeletal muscle anabolism and can overcome the anabolic resistance of aging. The value of chronic leucine ingestion for muscle growth is still unclear. Most of the research into leucine consumption has focused on efficacy. To our knowledge, very few studies have sought to determine the maximum safe level of intake. Limited evidence suggests that intakes of ≤1250 mg ⋅ kg -1 . d -1 do not appear to have any health consequences other than short-term elevated plasma ammonia concentrations. Similarly, no adverse events have been reported for the leucine metabolite β-hydroxy-β-methylbutyrate (HMB), although no studies have tested HMB toxicity in humans. Therefore, future research is needed to evaluate leucine and HMB toxicity in the elderly and in specific health conditions. © 2016 American Society for Nutrition.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake.

PubMed

Renguet, Edith; Ginion, Audrey; Gélinas, Roselle; Bultot, Laurent; Auquier, Julien; Robillard Frayne, Isabelle; Daneault, Caroline; Vanoverschelde, Jean-Louis; Des Rosiers, Christine; Hue, Louis; Horman, Sandrine; Beauloye, Christophe; Bertrand, Luc

2017-08-01

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [ 13 C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart. NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation

The evolution and function of protein tandem repeats in plants.

PubMed

Schaper, Elke; Anisimova, Maria

2015-04-01

Sequence tandem repeats (TRs) are abundant in proteomes across all domains of life. For plants, little is known about their distribution or contribution to protein function. We exhaustively annotated TRs and studied the evolution of TR unit variations for all Ensembl plants. Using phylogenetic patterns of TR units, we detected conserved TRs with unit number and order preserved during evolution, and those TRs that have diverged via recent TR unit gains/losses. We correlated the mode of evolution of TRs to protein function. TR number was strongly correlated with proteome size, with about one-half of all TRs recognized as common protein domains. The majority of TRs have been highly conserved over long evolutionary distances, some since the separation of red algae and green plants c. 1.6 billion yr ago. Conversely, recurrent recent TR unit mutations were rare. Our results suggest that the first TRs by far predate the first plants, and that TR appearance is an ongoing process with similar rates across the plant kingdom. Interestingly, the few detected highly mutable TRs might provide a source of variation for rapid adaptation. In particular, such TRs are enriched in leucine-rich repeats (LRRs) commonly found in R genes, where TR unit gain/loss may facilitate resistance to emerging pathogens. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Members of the gibberellin receptor gene family GID1 (GIBBERELLIN INSENSITIVE DWARF1) play distinct roles during Lepidium sativum and Arabidopsis thaliana seed germination

PubMed Central

Voegele, Antje; Linkies, Ada; Müller, Kerstin; Leubner-Metzger, Gerhard

2011-01-01

Germination of endospermic seeds is partly regulated by the micropylar endosperm, which acts as constraint to radicle protrusion. Gibberellin (GA) signalling pathways control coat-dormancy release, endosperm weakening, and organ expansion during seed germination. Three GIBBERELLIN INSENSITIVE DWARF1 (GID1) GA receptors are known in Arabidopsis thaliana: GID1a, GID1b, and GID1c. Molecular phylogenetic analysis of angiosperm GID1s reveals that they cluster into two eudicot (GID1ac, GID1b) groups and one monocot group. Eudicots have at least one gene from each of the two groups, indicating that the different GID1 receptors fulfil distinct roles during plant development. A comparative Brassicaceae approach was used, in which gid1 mutant and whole-seed transcript analyses in Arabidopsis were combined with seed-tissue-specific analyses of its close relative Lepidium sativum (garden cress), for which three GID1 orthologues were cloned. GA signalling via the GID1ac receptors is required for Arabidopsis seed germination, GID1b cannot compensate for the impaired germination of the gid1agid1c mutant. Transcript expression patterns differed temporarily, spatially, and hormonally, with GID1b being distinct from GID1ac in both species. Endosperm weakening is mediated, at least in part, through GA-induced genes encoding cell-wall-modifying proteins. A suppression subtraction hybridization (SSH) cDNA library enriched for sequences that are highly expressed during early germination in the micropylar endosperm contained expansins and xyloglucan endo-transglycosylases/hydrolases (XTHs). Their transcript expression patterns in both species strongly suggest that they are regulated by distinct GID1-mediated GA signalling pathways. The GID1ac and GID1b pathways seem to fulfil distinct regulatory roles during Brassicaceae seed germination and seem to control their downstream targets distinctly. PMID:21778177

Effect of Starvation on the Turnover and Metabolic Response to Leucine

PubMed Central

Sherwin, Robert S.

1978-01-01

l-Leucine was administered as a primed continuous 3-4-h infusion in nonobese and obese subjects in the postabsorptive state and for 12 h in obese subjects after a 3-day and 4-wk fast. In nonobese and obese subjects studied in the post-absorptive state, the leucine infusion resulted in a 150-200% rise in plasma leucine above preinfusion levels, a small decrease in plasma glucose, and unchanged levels of plasma insulin and glucagon and blood ketones. Plasma isoleucine (60-70%) and valine (35-40%) declined to a greater extent than other amino acids (P < 0.001). After 3 days and 4 wk of fasting, equimolar infusions of leucine resulted in two- to threefold greater increments in plasma leucine as compared to post-absorptive subjects, a 30-40% decline in other plasma amino acids, and a 25-30% decrease in negative nitrogen balance. Urinary excretion of 3-methylhistidine was however, unchanged. Plasma glucose which declined in 3-day fasted subjects after leucine administration, surprisingly rose by 20 mg/100 ml after 4 wk of fasting. The rise in blood glucose occurred in the absence of changes in plasma glucagon and insulin and in the face of a 15% decline in endogenous glucose production (as measured by infusion of [3-3H]glucose). On the other hand, fractional glucose utilization fell by 30% (P < 0.001), thereby accounting for hyperglycemia. The estimated metabolic clearance rate of leucine fell by 48% after 3 days of fasting whereas the plasma delivery rate of leucine was unchanged, thereby accounting for a 40% rise in plasma leucine during early starvation. After a 4-wk fast, the estimated metabolic clearance rate of leucine declined further to 59% below base line. Plasma leucine nevertheless fell to postabsorptive levels as the plasma delivery rate of leucine decreased 65% below postabsorptive values. Conclusions: (a) Infusion of exogenous leucine in prolonged fasting results in a decline in plasma levels of other amino acids, improvement in nitrogen balance and

Glucocorticoid-induced Leucine Zipper (GILZ) and Long GILZ Inhibit Myogenic Differentiation and Mediate Anti-myogenic Effects of Glucocorticoids*

PubMed Central

Bruscoli, Stefano; Donato, Valerio; Velardi, Enrico; Di Sante, Moises; Migliorati, Graziella; Donato, Rosario; Riccardi, Carlo

2010-01-01

Myogenesis is a process whereby myoblasts differentiate and fuse into multinucleated myotubes, the precursors of myofibers. Various signals and factors modulate this process, and glucocorticoids (GCs) are important regulators of skeletal muscle metabolism. We show that glucocorticoid-induced leucine zipper (GILZ), a GC-induced gene, and the newly identified isoform long GILZ (L-GILZ) are expressed in skeletal muscle tissue and in C2C12 myoblasts where GILZ/L-GILZ maximum expression occurs during the first few days in differentiation medium. Moreover, we observed that GC treatment of myoblasts, which increased GILZ/L-GILZ expression, resulted in reduced myotube formation, whereas GILZ and L-GILZ silencing dampened GC effects. Inhibition of differentiation caused by GILZ/L-GILZ overexpression correlated with inhibition of MyoD function and reduced expression of myogenin. Notably, results indicate that GILZ and L-GILZ bind and regulate MyoD/HDAC1 transcriptional activity, thus mediating the anti-myogenic effect of GCs. PMID:20124407

Cheliensisin A (Chel A) induces apoptosis in human bladder cancer cells by promoting PHLPP2 protein degradation.

PubMed

Zhang, Ruowen; Che, Xun; Zhang, Jingjie; Li, Yang; Li, Jingxia; Deng, Xu; Zhu, Junlan; Jin, Honglei; Zhao, Qinshi; Huang, Chuanshu

2016-10-11

Cheliensisin A (Chel A), a styryl-lactone compound extracted from Goniothalamus cheliensis, is reported to have significant anti-cancer effects in various cancer cells. Here we demonstrated that Chel A treatment resulted in apoptosis and an inhibition of anchorage-independent growth in human bladder cancer T24, T24T and U5637 cells. Mechanistic studies showed that such effect is mediated by PH domain and Leucine rich repeat Protein Phosphatases (PHLPP2) protein. Chel A treatment led to PHLPP2 degradation and subsequently increased in c-Jun phosphorylation. Moreover PHLPP2 degradation could be attenuated by inhibition of autophagy, which was mediated by Beclin 1. Collectively, we discover that Chel A treatment induces Beclin-dependent autophagy, consequently mediates PHLPP2 degradation and JNK/C-Jun phosphorylation and activation, further in turn contributing to apoptosis in human bladder cancer cells. Current studies provide a significant insight into understanding of anticancer effect of Chel A in treatment of human bladder cancer.

Cheliensisin A (Chel A) induces apoptosis in human bladder cancer cells by promoting PHLPP2 protein degradation

PubMed Central

Li, Yang; Li, Jingxia; Deng, Xu; Zhu, Junlan; Jin, Honglei; Zhao, Qinshi; Huang, Chuanshu

2016-01-01

Cheliensisin A (Chel A), a styryl-lactone compound extracted from Goniothalamus cheliensis, is reported to have significant anti-cancer effects in various cancer cells. Here we demonstrated that Chel A treatment resulted in apoptosis and an inhibition of anchorage-independent growth in human bladder cancer T24, T24T and U5637 cells. Mechanistic studies showed that such effect is mediated by PH domain and Leucine rich repeat Protein Phosphatases (PHLPP2) protein. Chel A treatment led to PHLPP2 degradation and subsequently increased in c-Jun phosphorylation. Moreover PHLPP2 degradation could be attenuated by inhibition of autophagy, which was mediated by Beclin 1. Collectively, we discover that Chel A treatment induces Beclin-dependent autophagy, consequently mediates PHLPP2 degradation and JNK/C-Jun phosphorylation and activation, further in turn contributing to apoptosis in human bladder cancer cells. Current studies provide a significant insight into understanding of anticancer effect of Chel A in treatment of human bladder cancer. PMID:27556506

Comparison of aerobic and anaerobic [3H]leucine incorporation assays for determining pollution-induced bacterial community tolerance in copper-polluted, irrigated soils.

PubMed

Aaen, Karoline Nolsø; Holm, Peter E; Priemé, Anders; Hung, Ngoc Ngo; Brandt, Kristian Koefoed

2011-03-01

Pollution-induced community tolerance (PICT) constitutes a sensitive and ecologically relevant impact parameter in ecotoxicology. We report the development and application of a novel anaerobic [(3) H]leucine incorporation assay and its comparison with the conventional aerobic [(3) H]leucine incorporation assay for PICT detection in soil bacterial communities. Selection of bacterial communities was performed over 42 d in bulk soil microcosms (no plants) and in rice (Oryza sativa) rhizosphere soil mesocosms. The following experimental treatments were imposed using a full factorial design: two soil types, two soil water regimes, and four Cu application rates (0, 30, 120, or 280 µg g(-1)). Bacterial communities in bulk soil microcosms exhibited similar Cu tolerance patterns when assessed by aerobic and anaerobic PICT assays, whereas aerobic microorganisms tended to be more strongly selected for Cu tolerance than anaerobic microorganisms in rhizosphere soil. Despite similar levels of water-extractable Cu, bacterial Cu tolerance was significantly higher in acid sulfate soil than in alluvial soil. Copper amendment selected for significant PICT development in soils subjected to alternate wetting and drying, but not in continuously flooded soils. Our results demonstrate that soil bacterial communities subjected to alternate wetting and drying may be more affected by Cu than bacterial communities subjected to continuous flooding. We conclude that the parallel use of anaerobic and aerobic [(3) H]leucine PICT assays constitutes a valuable improvement over existing procedures for PICT detection in irrigated soils and other redox gradient environments such as sediments and wetlands. Copyright © 2010 SETAC.

Repeatability and reliability of muscle relaxation properties induced by motor cortical stimulation.

PubMed

Molenaar, Joery P; Voermans, Nicol C; de Jong, Lysanne A; Stegeman, Dick F; Doorduin, Jonne; van Engelen, Baziel G

2018-03-15

Impaired muscle relaxation is a feature of many neuromuscular disorders. However, there are few tests available to quantify muscle relaxation. Transcranial magnetic stimulation (TMS) of the motor cortex can induce muscle relaxation by abruptly inhibiting corticospinal drive. The aim of our study is to investigate if repeatability and reliability of TMS-induced relaxation is greater than voluntary relaxation. Furthermore, effects of sex, cooling and fatigue on muscle relaxation properties were studied. Muscle relaxation of deep finger flexors was assessed in twenty-five healthy subjects (14 M and 11 F, aged 39.1{plus minus}12.7 and 45.3{plus minus}8.7 years old, respectively) using handgrip dynamometry. All outcome measures showed greater repeatability and reliability in TMS-induced relaxation compared to voluntary relaxation. The within-subject coefficient of variability of normalized peak relaxation rate was lower in TMS-induced relaxation than in voluntary relaxation (3.0 vs 19.7% in men, and 6.1 vs 14.3% in women). The repeatability coefficient was lower (1.3 vs 6.1 s -1 in men and 2.3 vs 3.1 s -1 in women), and the intraclass correlation coefficient was higher (0.95 vs 0.53 in men and 0.78 vs 0.69 in women), for TMS-induced relaxation compared to voluntary relaxation. TMS enabled to demonstrate slowing effects of sex, muscle cooling, and muscle fatigue on relaxation properties that voluntary relaxation could not. In conclusion, repeatability and reliability of TMS-induced muscle relaxation was greater compared to voluntary muscle relaxation. TMS-induced muscle relaxation has the potential to be used in clinical practice for diagnostic purposes and therapy effect monitoring in patients with impaired muscle relaxation.

Crystallization of Ca-Al-Rich Inclusions: Experimental Studies on the Effects of Repeated Heating Events

NASA Technical Reports Server (NTRS)

Paque, Julie M.; Lofgren, Gary E.; Le, Loan

2000-01-01

The observed textures and chemistry of Ca-Al-rich inclusions (CAIs) are presumed to be the culmination of a series of repeated heating and cooling events in the early history of the solar nebula. We have examined the effects of these heating/cooling cycles experimentally on a bulk composition representing an average Type B Ca-Al-rich inclusion composition. We have tested the effect of the nature of the starting material. Although the most recent and/or highest temperature event prior to incorporation into the parent body dominates the texture and chemistry of the CAI, prior events also affect the phase compositions and textures. We have determined that heating precursor grains to about 1275 C prior to the final melting event increases the likelihood of anorthite crystallization in subsequent higher temperature events and a prior high temperature even that produced dendritic melilite results in melilite that shows evidence of rapid crystallization in subsequent lower temperature events. Prior low temperature pre-crystallization events produce final ran products with pyroxene compositions similar to Type B Ca-Al-rich inclusions, and the glass (residual liquid) composition is more anorthitic than any other experiments to date. The addition of Pt powder to the starting material appears to enhance the ability of anorthite to nucleate from this composition.

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

PubMed Central

Lange, T; Hedden, P; Graebe, J E

1994-01-01

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases. Images PMID:8078921

Characterization of the molecular mechanism underlying gibberellin perception complex formation in rice.

PubMed

Hirano, Ko; Asano, Kenji; Tsuji, Hiroyuki; Kawamura, Mayuko; Mori, Hitoshi; Kitano, Hidemi; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

2010-08-01

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1(G576V)). The GA-dependent degradation of SLR1(G576V) was reduced in Slr1-d4, and compared with SLR1, SLR1(G576V) showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1(G576V) interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.

Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway

PubMed Central

Yang, Jun; Dolinger, Michael; Ritaccio, Gabrielle; Mazurkiewicz, Joseph; Conti, David; Zhu, Xinjun; Huang, Yunfei

2012-01-01

The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients. PMID:22645144

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

PubMed

Sakai, Miho; Sakamoto, Tomoaki; Saito, Tamio; Matsuoka, Makoto; Tanaka, Hiroshi; Kobayashi, Masatomo

2003-04-01

We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

Lifting DELLA Repression of Arabidopsis Seed Germination by Nonproteolytic Gibberellin Signaling1[C][W][OPEN

PubMed Central

Ariizumi, Tohru; Hauvermale, Amber L.; Nelson, Sven K.; Hanada, Atsushi; Yamaguchi, Shinjiro; Steber, Camille M.

2013-01-01

DELLA repression of Arabidopsis (Arabidopsis thaliana) seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent gibberellin (GA) hormone signaling. GA binding to the GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptors stimulates GID1-GA-DELLA complex formation, which in turn triggers DELLA protein ubiquitination and proteolysis via the SCFSLY1 E3 ubiquitin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sleepy1-2 (sly1-2) F-box mutant, long dry after-ripening and GID1 overexpression can relieve the strong sly1-2 seed dormancy phenotype. It appears that sly1-2 seed dormancy results from abscisic acid (ABA) signaling downstream of DELLA, since dormant sly1-2 seeds accumulate high levels of ABA hormone and loss of ABA sensitivity rescues sly1-2 seed germination. DELLA positively regulates the expression of XERICO, an inducer of ABA biosynthesis. GID1b overexpression rescues sly1-2 germination through proteolysis-independent DELLA down-regulation associated with increased expression of GA-inducible genes and decreased ABA accumulation, apparently as a result of decreased XERICO messenger RNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 down-regulates DELLA through protein binding. After-ripening results in increased GA accumulation and GID1a-dependent GA signaling, suggesting that after-ripening triggers GA-stimulated GID1-GA-DELLA protein complex formation, which in turn blocks DELLA transcriptional activation of the XERICO inhibitor of seed germination. PMID:23818171

Evidence for a Gibberellin Biosynthetic Origin of Ceratopteris Antheridiogen 1

PubMed Central

Warne, Thomas R.; Hickok, Leslie G.

1989-01-01

The species-specific chemical messenger, antheridiogen ACe, mediates the differentiation of male gametophytes in the fern Ceratopteris. In order to investigate the biochemical origin of antheridiogen, the effect of the inhibitors, 2′-isopropyl-4′-(trimethylammoniumchloride)-5′ -methylphenylpiperidine-1-carboxylate (AMO-1618), 2-chloroethyl trimethylammonium chloride (CCC), and α-cyclopropyl-α-(4-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) on gametophytic sex expression was determined in C. richardii. Both AMO-1618 and ancymidol blocked the production of male gametophytes in three genetically defined strains of C. richardii that exhibit different sensitivities to antheridiogen. Antheridiogen supplementation overcame inhibition by AMO-1618 and ancymidol, except in one strain (HaC18) that is insensitive to antheridiogen supplementation. These data suggest that the synthesis of Ceratopteris antheridiogen, a taxon that is insensitive to exogenously supplied gibberellins, occurs via a pathway that may include steps in common with gibberellin biosynthesis or involves similar reactions. PMID:16666578

Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings

PubMed Central

Nugroho, Widyanto Dwi; Yamagishi, Yusuke; Nakaba, Satoshi; Fukuhara, Shiori; Begum, Shahanara; Marsoem, Sri Nugroho; Ko, Jae-Heung; Jin, Hyun-O; Funada, Ryo

2012-01-01

Background and Aims Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings. Methods Gibberellic acid (GA3), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 45° from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems. Key Results It was found that GA3 stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA3 stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting. Conclusions The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus. PMID:22843341

Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings.

PubMed

Nugroho, Widyanto Dwi; Yamagishi, Yusuke; Nakaba, Satoshi; Fukuhara, Shiori; Begum, Shahanara; Marsoem, Sri Nugroho; Ko, Jae-Heung; Jin, Hyun-O; Funada, Ryo

2012-09-01

Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings. Gibberellic acid (GA(3)), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 45° from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems. It was found that GA(3) stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA(3) stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting. The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus.

Confirmation and refinement of the heterozygous deletion of the small leucine-rich proteoglycans associated with posterior amorphous corneal dystrophy.

PubMed

Cervantes, Aleck E; Gee, Katherine M; Whiting, Martha F; Frausto, Ricardo F; Aldave, Anthony J

2018-04-19

To present the clinical and cytogenetic features of a previously unreported family with posterior amorphous corneal dystrophy (PACD) associated with a heterozygous deletion of the small leucine-rich proteoglycan (SRLP) genes on chromosome 12. Clinical characterization was performed using slit lamp biomicroscopic and optical coherence tomography (OCT) imaging. Genomic DNA was collected from affected and unaffected family members, and a cytogenomic array was used to identify copy number variations (CNV) present in the PACD locus. Three members of a Guatemalan family presented with clinical characteristics consistent with PACD: bilateral posterior stromal lamellar opacification, decreased corneal curvature, and iridocorneal adhesions. OCT imaging demonstrated decreased corneal thickness and hyperreflectivity of the posterior third of the corneal stroma. CNV analysis confirmed the presumed clinical diagnosis of PACD by revealing a 0.304 Mb heterozygous deletion in the PACD locus on chromosome 12 that included the four SLRP genes (KERA, LUM, DCN, and EPYC) deleted in each of the PACD families in which CNV analysis has been reported. This is the first report of the OCT appearance of PACD and the second confirmation of a heterozygous deletion of chromosome 12q21.33 as the cause of PACD, highlighting the utility of array-based cytogenomics to confirm the suspected clinical diagnosis of PACD. As the smallest previously reported pathogenic deletion was 0.701 Mb, the 0.304-Mb deletion we report is the smallest identified to date and reduces the size of the PACD locus to 0.275 Mb.

A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice.

PubMed

Lo, Shuen-Fang; Yang, Show-Ya; Chen, Ku-Ting; Hsing, Yue-Ie; Zeevaart, Jan A D; Chen, Liang-Jwu; Yu, Su-May

2008-10-01

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2beta-hydroxylation: a larger class of C(19) GA2oxs and a smaller class of C(20) GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C(20) GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C(20) GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C(20) GA2oxs were found to cause less severe GA-defective phenotypes than C(19) GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C(20) GA2oxs.

A Solution NMR Investigation into the Murine Amelogenin Splice-Variant LRAP (Leucine-Rich Amelogenin Protein).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Tarasevich, Barbara J.; Roberts, Jacky

2010-09-01

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the 1H-15N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like themore » full-length protein, is intrinsically disordered under these solution conditions. The major difference between the 1H-15N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 at the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggest that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region and is likely due to a slow exchange process. As observed with rp(H)M180, residue specific changes in molecular dynamics, manifested by the reduction in intensity and disappearance of 1H-15N HSQC cross peaks, were observed with the addition of NaCl to rp(H)LRAP. These perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different pattern of 1H-15N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences account for the cell signaling properties attributable to LRAP but not the full-length protein.« less

Identification of a negative regulator of gibberellin action, HvSPY, in barley.

PubMed Central

Robertson, M; Swain, S M; Chandler, P M; Olszewski, N E

1998-01-01

To broaden our understanding of the molecular mechanisms of gibberellin (GA) action, we isolated a spindly clone (HvSPY) from barley cultivar Himalaya and tested whether the HvSPY protein would modulate GA action in barley aleurone. The HvSPY cDNA showed high sequence identity to Arabidopsis SPY along its entire length, and the barley protein functionally complemented the spy-3 mutation. HvSPY and SPY proteins showed sequence relatedness with animal O-linked N-acetylglucosamine transferases (OGTs), suggesting that they may also have OGT activity. HvSPY has a locus distinct from that of Sln, a mutation that causes the constitutive GA responses of slender barley, which phenotypically resembles Arabidopsis spy mutants. The possibility that the HvSPY gene encodes a negative regulator of GA action was tested by expressing HvSPY in a barley aleurone transient assay system. HvSPY coexpression largely abolished GA3-induced activity of an alpha-amylase promoter. Surprisingly, HvSPY coexpression increased reporter gene activity from an abscisic acid (ABA)-inducible gene promoter (dehydrin), even in the absence of exogenous ABA. These results show that HvSPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter. PMID:9634587

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).

Further Experiments on Gibberellin-Stimulated Amylase Production in Cereal Grains

ERIC Educational Resources Information Center

Coppage, Jo; Hill, T. A.

1973-01-01

Experiments conducted on wheat and barley grains to analyze activities of alpha- and beta-amylase enzymes. Gibberellins were used exogenously. Techniques are described in detail. Results on different cultivars revealed that beta-amylase was not an invariable result of imbibition. Techniques employed can be used by school students. (PS)

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

PubMed Central

Helliwell, Chris A.; Chandler, Peter M.; Poole, Andrew; Dennis, Elizabeth S.; Peacock, W. James

2001-01-01

We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA12. A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA12. Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase. PMID:11172076

Interplay of sugar, light and gibberellins in expression of Rosa hybrida vacuolar invertase 1 regulation.

PubMed

Rabot, Amélie; Portemer, Virginie; Péron, Thomas; Mortreau, Eric; Leduc, Nathalie; Hamama, Latifa; Coutos-Thévenot, Pierre; Atanassova, Rossitza; Sakr, Soulaiman; Le Gourrierec, José

2014-10-01

Our previous findings showed that the expression of the Rosa hybrida vacuolar invertase 1 gene (RhVI1) was tightly correlated with the ability of buds to grow out and was under sugar, gibberellin and light control. Here, we aimed to provide an insight into the mechanistic basis of this regulation. In situ hybridization showed that RhVI1 expression was localized in epidermal cells of young leaves of bursting buds. We then isolated a 895 bp fragment of the promoter of RhVI1. In silico analysis identified putative cis-elements involved in the response to sugars, light and gibberellins on its proximal part (595 bp). To carry out functional analysis of the RhVI1 promoter in a homologous system, we developed a direct method for stable transformation of rose cells. 5' deletions of the proximal promoter fused to the uidA reporter gene were inserted into the rose cell genome to study the cell's response to exogenous and endogenous stimuli. Deletion analysis revealed that the 468 bp promoter fragment is sufficient to trigger reporter gene activity in response to light, sugars and gibberellins. This region confers sucrose- and fructose-, but not glucose-, responsive activation in the dark. Inversely, the -595 to -468 bp region that carries the sugar-repressive element (SRE) is required to down-regulate the RhVI1 promoter in response to sucrose and fructose in the dark. We also demonstrate that sugar/light and gibberellin/light act synergistically to up-regulate β-glucuronidase (GUS) activity sharply under the control of the 595 bp pRhVI1 region. These results reveal that the 127 bp promoter fragment located between -595 and -468 bp is critical for light and sugar and light and gibberellins to act synergistically. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Enterobacter sp. I-3, a bio-herbicide inhibits gibberellins biosynthetic pathway and regulates abscisic acid and amino acids synthesis to control plant growth.

PubMed

Radhakrishnan, Ramalingam; Park, Jae-Man; Lee, In-Jung

2016-12-01

Very few bacterial species were identified as bio-herbicides for weed control. The present research was focused to elucidate the plant growth retardant properties of Enterobacter sp. I-3 during their interaction by determining the changes in endogenous photosynthetic pigments, plant hormones and amino acids. The two bacterial isolates I-4-5 and I-3 were used to select the superior bacterium for controlling weed seeds (Echinochloa crus-galli L. and Portulaca oleracea L.) germination. The post-inoculation of I-3 (Enterobacter sp. I-3) significantly inhibited the weeds seed germination than their controls. The mechanism of bacterium induced plant growth reduction was identified in lettuce treated with I-3 bacterium and compared their effects with known chemical herbicide, trinexapac-ethyl (TE). The treatment of I-3 and TE showed a significant inhibitory effect on shoot length, leaf number, leaf length, leaf width, shoot weight, root weight and chlorophyll content in lettuce seedlings. The endogenous gibberellins (GAs) and abscisic acid (ABA) analysis showed that Enterobacter sp. I-3 treated plants had lower levels of GAs (GA 12 , GA 19 , GA 20 and GA 8 ) and GAs/ABA ratio and then, the higher level of ABA when compared to their controls. Indeed, the individual amino acids ie., aspartic acid, glutamic acid, glycine, threonine, alanine, serine, leucine, isoleucine and tyrosine were declined in TE and I-3 exposed plants. Our results suggest that the utilization of Enterobacter sp. I-3 inhibits the GAs pathway and amino acids synthesis in weeds to control their growth can be an alternative to chemical herbicides. Copyright © 2016 Elsevier GmbH. All rights reserved.

Leucine increases mucin 2 and occludin production in LS174T cells partially via PI3K-Akt-mTOR pathway.

PubMed

Mao, Xiangbing; Hu, Haiyan; Tang, Jun; Chen, Daiwen; Yu, Bing

2016-09-01

Mucin 2 and occludin play a crucial role in preserving the intestinal mucosal integrity. However, the role for leucine mediating intestinal mucin 2 and occludin expression has little been investigated. The current study was conducted to test the hypothesis that leucine treatment could increase mucin 2 and occludin levels in LS174T cells. The LS174T cells were incubated in the Dulbecco's Modified Eagle Medium (DMEM) supplementing 0, 0.5 and 5 mmol/L L-leucine for the various durations. Two hours after the leucine treatment, the inhibitor of mammalian target of rapamycin (mTOR) and protein kinase B (Akt) phosphorylation in LS174T cells were significantly increased ( P  < 0.05), and the mucin 2 and occludin levels were also significantly enhanced ( P  < 0.05). However, the pretreatment of 10 nmol/L rapamycin, which was an mTOR inhibitor, or 1 μmol/L wortmanin, which was an inhibitor of phosphatidylinositol 3-kinase (PI3K), completely inhibited leucine-induced mTOR or Akt phosphorylation ( P  < 0.05), and significantly reduced leucine-stimulated mucin 2 and occludin levels ( P  < 0.05). These results suggest that leucine treatment promotes the mucin 2 and occludin levels in LS174T cells partially through the PI3K-Akt-mTOR signaling pathway.

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

PubMed

Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

2018-01-22

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

Prostate Cancer Cells in Different Androgen Receptor Status Employ Different Leucine Transporters.

PubMed

Otsuki, Hideo; Kimura, Toru; Yamaga, Takashi; Kosaka, Takeo; Suehiro, Jun-Ichi; Sakurai, Hiroyuki

2017-02-01

Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na + -independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear. LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new "LN-cr" cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of 14 C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na + . Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y + LAT2 on leucine uptake and cell viability in LN-cr. Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na + -independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC 50BCH :15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC 50BCH : ∼20 mM, IC 50JPH203 : ∼5 µM). In LN-cr cells, Na + -dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na + -independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (∼85%) Na + -dependent. y + LAT2 expression was confirmed in LN-cr. Knockdown of y + LAT2 lead to significant leucine uptake inhibition (40%) and cell

The Effect of Gibberellin on Plant Growth and Development

DTIC Science & Technology

1960-11-04

8217P? 1O cerh bi; ncv tnd the ntanber of berries In e-.c ch bunch. The &ver-r.j? c -vjeirht of-’a berry ::wes then derived for each bt/mchyaoD...gibberellin web strongly in evidence. In the " :* : control bunches/of the Mrran|P’variety the^er/age/weight : ,of .8. berry was 1.08 g,: fend

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.

Leucine Metabolism in T Cell Activation: mTOR Signaling and Beyond123

PubMed Central

Powell, Jonathan D; Hutson, Susan M

2016-01-01

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

Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

PubMed Central

Hong, S W; Jon, J H; Kwak, J M; Nam, H G

1997-01-01

A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

Functional Analysis of SPINDLY in Gibberellin Signaling in Arabidopsis1[C][W][OA

PubMed Central

Silverstone, Aron L.; Tseng, Tong-Seung; Swain, Stephen M.; Dill, Alyssa; Jeong, Sun Yong; Olszewski, Neil E.; Sun, Tai-ping

2007-01-01

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-Δ17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-Δ17 phenotype but does not reduce rga-Δ17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification. PMID:17142481

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.

Lifting DELLA repression of Arabidopsis seed germination by nonproteolytic gibberellin signaling

USDA-ARS?s Scientific Manuscript database

DELLA repression of Arabidopsis seed germination can be lifted through the ubiquitin-proteasome pathway and proteolysis-independent GA signaling. GA-binding to the GID1 (GIBBERELLIN-INSENSITIVE DWARF1) GA receptors stimulates GID1-GA-DELLA complex formation which in turn triggers DELLA protein ubiq...

Role of the inflammasome in acetaminophen-induced liver injury and acute liver failure.

PubMed

Woolbright, Benjamin L; Jaeschke, Hartmut

2017-04-01

Drug-induced acute liver failure carries a high morbidity and mortality rate. Acetaminophen overdose is the number one cause of acute liver failure and remains a major problem in Western medicine. Administration of N-acetyl cysteine is an effective antidote when given before the initial rise in toxicity; however, many patients present to the hospital after this stage occurs. As such, treatments which can alleviate late-stage acetaminophen-induced acute liver failure are imperative. While the initial mechanisms of toxicity are well described, a debate has recently occurred in the literature over whether there is a second phase of injury, mediated by inflammatory processes. Critical to this potential inflammatory process is the activation of caspase-1 and interleukin-1β by a molecular complex known as the inflammasome. Several different stimuli for the formation of multiple different inflammasome complexes have been identified. Formation of the NACHT, leucine-rich repeat (LRR) and pyrin (PYD) domains-containing protein 3 (Nalp3) inflammasome in particular, has directly been attributed to late-stage acetaminophen toxicity. In this review, we will discuss the mechanisms of acetaminophen-induced liver injury in mice and man with a particular focus on the role of inflammation and the inflammasome. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

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. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2013-01-01

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

Characterization of grape Gibberellin Insensitive 1 mutant alleles in transgenic Arabidopsis

USDA-ARS?s Scientific Manuscript database

We generated a dozen of different mutations in the grape Gibberellin Insensitive or GAI sequence, transformed them into Arabidopsis under the control of 35S, Arabidopsis or grape GAI promoter, and evaluated the impact of these mutant alleles on plant growth and development. These GAI sequence varian...

The Effect of the Oral Administration of Leucine on Endothelial Function, Glucose and Insulin Concentrations in Healthy Subjects.

PubMed

Argyrakopoulou, Georgia; Kontrafouri, Paraskevi; Eleftheriadou, Ioanna; Kokkinos, Alexander; Arapostathi, Christina; Kyriaki, Despoina; Perrea, Despoina; Revenas, Constantinos; Katsilambros, Nicholas; Tentolouris, Nicholas

2018-06-11

The aim of our study was to investigate the potential differential effect of hyperglycaemia and hyperinsulinaemia induced by glucose infusion alone and in combination with leucine consumption on endothelial function in healthy individuals. Ten male volunteers were examined in random order twice. In one visit, they consumed 250 ml water (baseline) and 30 min later glucose was infused iv. In the other visit, they consumed 250 ml water with 25 g of leucine and 30 min later the same amount of glucose was infused. Serum glucose and insulin were measured at baseline and every 10 min after glucose infusion for 1 h. Endothelial function was evaluated by measurement of flow mediated vasodilatation (FMD) at baseline, 10 and 60 min after glucose infusion. In both visits, glucose levels increased to the same degree, whereas insulin response was significantly higher after leucine administration. FMD values declined significantly compared to baseline 10 min after glucose infusion in the control visit (6.9±2.7 vs. 3.2±3.5%, respectively, p=0.006), while no significant change was observed when glucose infusion was followed by leucine consumption. Acute hyperglycaemia impairs endothelial function in healthy male individuals. Leucine administration prevents hyperglycaemia-mediated endothelial dysfunction probably due to enhanced insulin secretion. © Georg Thieme Verlag KG Stuttgart · New York.

Enhanced glucocorticoid-induced leucine zipper in dendritic cells induces allergen-specific regulatory CD4(+) T-cells in respiratory allergies.

PubMed

Karaki, S; Garcia, G; Tcherakian, C; Capel, F; Tran, T; Pallardy, M; Humbert, M; Emilie, D; Godot, V

2014-05-01

Respiratory allergies rely on a defect of IL-10-secreting regulatory CD4(+) T-cells (IL-10-Tregs ) leading to excessive Th2-biased immune responses to allergens. According to clinical data, the restoration of allergen-specific IL-10-Tregs is required to control respiratory allergies and cure patients. The discovery of mechanisms involved in the generation of IL-10-Tregs will thus help to provide effective treatments. We previously demonstrated that dendritic cells (DCs) expressing high levels of the glucocorticoid-induced leucine zipper protein (GILZ) generate antigen-specific IL-10-Tregs . We suspect a defective expression of GILZ in the DCs of respiratory allergic patients and speculate that increasing its expression might restore immune tolerance against allergens through the induction of IL-10-Tregs . We assessed GILZ expression in blood DCs of patients and healthy nonallergic donors by qPCR. We compared the ability of patients' DCs to induce allergen-specific IL-10-Tregs before and after an in vivo up-regulation of GILZ expression by steroid administration, steroids being inducers of GILZ. We report lower levels of GILZ in DCs of respiratory allergic patients that return to normal levels after steroid administration. We show that patients' DCs with increased levels of GILZ generate allergen-specific IL-10-Tregs again. We further confirm unequivocally that GILZ is required in patients' DCs to activate these IL-10-Tregs . This proof of concept study shows that the re-establishment of GILZ expression in patients' DCs to normal levels restores their capacity to activate allergen-specific IL-10-Tregs . We thus highlight the up-regulation of GILZ in DCs as a new interventional approach to restore the immune tolerance to allergens. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Medium-Chain Triglycerides in Combination with Leucine and Vitamin D Benefit Cognition in Frail Elderly Adults: A Randomized Controlled Trial.

PubMed

Abe, Sakiko; Ezaki, Osamu; Suzuki, Motohisa

2017-01-01

The combined supplementation of medium-chain triglycerides (MCTs), L-leucine-rich amino acids, and cholecalciferol (vitamin D 3 ) increase muscle strength and function in frail elderly individuals. However, their effects on cognition are unknown. We enrolled 38 elderly nursing home residents (mean age±SD, 86.6±4.8 y) in a 3-mo randomized, controlled, parallel group trial. The participants were randomly allocated to 3 groups: the first group received a L-leucine (1.2 g)- and cholecalciferol (20 μg)-enriched supplement with 6 g of MCT (LD+MCT); the second group received the same supplement with 6 g of long-chain triglycerides (LD+LCT); and the third group did not receive any supplements (control). Cognition was assessed at baseline and after the 3-mo intervention. The difference in changes among the groups was assessed with ANCOVA, adjusting for age and the baseline value as covariates. After 3 mo, the Mini-Mental State Examination (MMSE) score in the LD+MCT group increased by 10.6% (from 16.6 to 18.4 points, p<0.05). After 3 mo, the Nishimura geriatric rating scale for mental status (NM scale) score in the LD+MCT group increased by 30.6% (from 24.6 to 32.2 points, p<0.001), whereas that in the LD+LCT and control groups decreased by 11.2% (from 31.2 to 27.7 points, p<0.05) and 26.1% (from 27.2 to 20.1 points, p<0.001), respectively. The combined supplementation of MCTs (6 g), L-leucine-rich amino acids, and cholecalciferol may improve cognitive function in frail elderly individuals.

Leucine-rich alpha-2-glycoprotein-1 is up-regulated in colorectal cancer and is a tumor promoter

PubMed Central

Zhang, Qian; Huang, Rui; Tang, Qingchao; Yu, Yang; Huang, Quanlong; Chen, Yinggang; Wang, Guiyu; Wang, Xishan

2018-01-01

Background Leucine-rich α-2-glycoprotein-1 (LRG1) is differentially expressed in many kinds of diseases including cancer, however, it has not been thoroughly studied yet. Purpose The objective of this study was to detect the expression and potential mechanism of LRG1 in colorectal cancer (CRC). In our study, we examined LRG1 levels in CRC tissue and plasma with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The effect of LRG1 on cancer cells was detected with transwell and MTT assays. Results The average plasma LRG1 level in CRC was significantly higher than in polyp group (P=0.002) and healthy controls (P<0.001). Second, plasma LRG1 was positively associated with CA19-9 (r=0.133, P=0.039) and neutrophil ratio (r=0.403, P<0.001). Third, plasma LRG1 of stage IV patients was dramatically different from that of stage I, stage II or stage III patients (P<0.001). LRG1 mRNA expression levels were about 2-fold higher in CRCs compared to normal tissues (P<0.001). And levels of plasma LRG1 were found to be a risk factor in CRC in univariate survival analysis of colorectal prognosis (P=0.013, hazard ratio [HR]=1.803, 95% CI: 1.521–2.137), and multivariate analysis showed that LRG1 was an independent risk factor (P<0.001, HR=1.492, 95% CI: 1.223–1.820). The patients with higher plasma LRG1 value presented with poorer outcome (P=0.013). Functional experiments showed that LRG1 could promote the invasion and growth ability of cells. LRG1 was increased in plasma and tissue compared with that of controls and LRG1 may predict prognosis of CRC patients and LRG1 maybe a tumor promoter. Conclusion LRG1 is increased in CRC patients and might serve as a tumor promoter. PMID:29785123

Substituted Phthalimide AC94377 Is a Selective Agonist of the Gibberellin Receptor GID11[OPEN

PubMed Central

Otani, Masato; Shimotakahara, Hiroaki; Yoon, Jung-Min; Park, Seung-Hyun; Miyaji, Tomoko; Nakano, Takeshi; Nakamura, Hidemitsu; Nakajima, Masatoshi

2017-01-01

Gibberellin (GA) is a major plant hormone that regulates plant growth and development and is widely used as a plant growth regulator in agricultural production. There is an increasing demand for function-limited GA mimics due to the limitations on the agronomical application of GA to crops, including GA’s high cost of producing and its leading to the crops’ lodging. AC94377, a substituted phthalimide, is a chemical that mimics the growth-regulating activity of GAs in various plants, despite its structural difference. Although AC94377 is widely studied in many weeds and crops, its mode of action as a GA mimic is largely unknown. In this study, we confirmed that AC94377 displays GA-like activities in Arabidopsis (Arabidopsis thaliana) and demonstrated that AC94377 binds to the Arabidopsis GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor (AtGID1), forms the AtGID1-AC94377-DELLA complex, and induces the degradation of DELLA protein. Our results also indicated that AC94377 is selective for a specific subtype among three AtGID1s and that the selectivity of AC94377 is attributable to a single residue at the entrance to the hydrophobic pocket of GID1. We conclude that AC94377 is a GID1 agonist with selectivity for a specific subtype of GID1, which could be further developed and used as a function-limited regulator of plant growth in both basic study and agriculture. PMID:27899534

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

Reversal of haloperidol induced motor deficits in rats exposed to repeated immobilization stress.

PubMed

Shireen, Erum; Pervez, Sidra; Masroor, Maria; Ali, Wafa Binte; Rais, Qudsia; Khalil, Samira; Tariq, Anum; Haleem, Darakshan Jabeen

2014-09-01

Stress is defined as a non specific response of body to any physiological and psychological demand. Preclinical studies have shown that an uncontrollable stress condition produces neurochemical and behavioral deficits. The present study was conducted to test the hypothesis that a decrease in the responsiveness of somatodendritic 5-hydroxytryptamine (5-HT)-1A receptors following adaptation to stress could attenuate haloperidol induced acute parkinsonian like effect. Results showed that single exposure (2h) to immobilization stress markedly decreased food intake, growth rate and locomotor activity but these stress-induced behavioral deficits were not observed following repeated (2h/day for 5 days) exposure of immobilization stress suggesting behavioral tolerance occurs to similar stress. An important finding of present study is a reversal of haloperidol-induced motor deficits in animals exposed to repeated immobilization stress than respective control animals. It is suggested that stress induced possible desensitization of somatodendritic 5-HT-1A as well as 5-HT-2C receptors could release dopamine system from the inhibitory influence of serotonin. On the other hand, an increase in the effectiveness of postsynaptic 5-HT-1A receptors elicits a direct stimulatory influence on the activity of dopaminergic neuron and is possibly involved in the reversal of haloperidol-induced parkinsonian like symptoms in repeatedly immobilized rats.

Neural basis of the potentiated inhibition of repeated haloperidol and clozapine treatment on the phencyclidine-induced hyperlocomotion

PubMed Central

Zhao, Changjiu; Sun, Tao; Li, Ming

2012-01-01

Clinical observations suggest that antipsychotic effect starts early and increases progressively over time. This time course of antipsychotic effect can be captured in a rat phencyclidine (PCP)-induced hyperlocomotion model, as repeated antipsychotic treatment progressively increases its inhibition of the repeated PCP-induced hyperlocomotion. Although the neural basis of acute antipsychotic action has been studied extensively, the system that mediates the potentiated effect of repeated antipsychotic treatment has not been elucidated. In the present study, we investigated the neuroanatomical basis of the potentiated action of haloperidol (HAL) and clozapine (CLZ) treatment in the repeated PCP-induced hyperlocomotion. Once daily for five consecutive days, adult Sprague-Dawley male rats were first injected with HAL (0.05 mg/kg, sc), CLZ (10.0 mg/kg, sc) or saline, followed by an injection of PCP (3.2 mg/kg, sc) or saline 30 min later, and motor activity was measured for 90 min after the PCP injection. C-Fos immunoreactivity was assessed either after the acute (day 1) or repeated (day 5) drug tests. Behaviorally, repeated HAL or CLZ treatment progressively increased the inhibition of PCP-induced hyperlocomotion throughout the five days of drug testing. Neuroanatomically, both acute and repeated treatment of HAL significantly increased PCP-induced c-Fos expression in the nucleus accumbens shell (NAs) and the ventral tegmental area (VTA), but reduced it in the central amygdaloid nucleus (CeA). Acute and repeated CLZ treatment significantly increased PCP-induced c-Fos expression in the ventral part of lateral septal nucleus (LSv) and VTA, but reduced it in the medial prefrontal cortex (mPFC). More importantly, the effects of HAL and CLZ in these brain areas underwent a time-dependent reduction from day 1 to day 5. These findings suggest that repeated HAL achieves its potentiated inhibition of the PCP-induced hyperlocomotion by acting on the NAs, CeA and VTA, while CLZ

Gibberellins and gravitropism in maize shoots: endogenous gibberellin-like substances and movement and metabolism of [3H]Gibberellin A20

NASA Technical Reports Server (NTRS)

Rood, S. B.; Kaufman, P. B.; Abe, H.; Pharis, R. P.

1987-01-01

[3H]Gibberellin A20 (GA20) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [3H]GA20 to [3H]GA1- and [3H]GA29-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [3H]GA1, [3H]GA29, and [3H]GA8. The tentative identification of these putative [3H]GA glucosyl conjugates was further supported by the release of the free [3H]GA moiety after cleavage with cellulase. Within 12 hours of the [3H]GA20 feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [3H]GA20, especially [3H]GA1, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the "Tanginbozu" dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA20-like substance and lower halves containing principally a GA20-like substance and lower halves containing mainly GA1-like and GA19-like substances. Gibberellins A1 (10 nanograms per gram) and A20 (5 nanograms per gram) were identified

Role of gibberellins in parthenocarpic fruit development induced by the genetic system pat-3/pat-4 in tomato.

PubMed

Fos, Mariano; Proaño, Karina; Nuez, Fernando; García-Martínez, José L.

2001-04-01

The role of gibberellins (GAs) in the induction of parthenocarpic fruit-set and growth by the pat-3/pat-4 genetic system in tomato (Lycopersicon esculentum Mill.) was investigated using wild type (WT; Cuarenteno) and a near-isogenic line derived from the German line RP75/59 (the source of pat-3/pat-4 parthenocarpy). Unpollinated WT ovaries degenerated but GA3 application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of pat-3/pat-4 fruits, which occurs in the absence of pollination and hormone treatment, was not affected by applied GA3. Unpollinated pat-3/pat-4 fruit growth was negated by paclobutrazol, an inhibitor of ent-kaurene oxidase, and this inhibitory effect was negated by GA3. The quantification of the main GAs of the early 13-hydroxylation pathway (GA1, GA8, GA19, GA20, GA29 and GA44) in unpollinated ovaries at 3 developmental stages (flower bud, FB; pre-anthesis, PR; and anthesis, AN), by gas chromatography-selected ion monitoring, showed that the concentration of most of them was higher in pat-3/pat-4 than in WT ovaries at PR and AN stages. The concentration of GA1, suggested previously to be the active GA in tomate, was 2-4 times higher. Unpollinated pat-3/pat-4 ovaries at FB, PR and AN stages also contained relatively high amounts (5-12 ng g-1) of GA3, a GA found at less than 0.5 ng g-1 in WT ovaries. It is concluded that the mutations pat-3/pat-4 may induce natural facultative parthenocarpy capacity in tomato by increasing the concentration of GA1 and GA3 in the ovaries before pollination.

Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice[C][W

PubMed Central

Hirano, Ko; Asano, Kenji; Tsuji, Hiroyuki; Kawamura, Mayuko; Mori, Hitoshi; Kitano, Hidemi; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

2010-01-01

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1G576V). The GA-dependent degradation of SLR1G576V was reduced in Slr1-d4, and compared with SLR1, SLR1G576V showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1G576V interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2. PMID:20716699

Cytoplasmic and nuclear localizations are important for the hypersensitive response conferred by maize autoactive Rp1-D21 protein

USDA-ARS?s Scientific Manuscript database

Disease resistance (R-) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich-repeat (NLR) proteins that trigger a rapid localized programmed cell death termed the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, d...

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

PubMed

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

2016-01-01

Eukaryotic cells coordinate growth with the availability of nutrients through the mechanistic target of rapamycin complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag guanosine triphosphatases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. Here we present the 2.7 angstrom 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. Copyright © 2016, American Association for the Advancement of Science.

Epidermal Cell Death in Rice Is Regulated by Ethylene, Gibberellin, and Abscisic Acid

PubMed Central

Steffens, Bianka; Sauter, Margret

2005-01-01

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth. PMID:16169967

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

Sestrin2 is a leucine sensor for the mTORC1 pathway.

PubMed

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

2016-01-01

Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, aGTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. Copyright © 2016, American Association for the Advancement of Science.

Acetone Formation in the Vibrio Family: a New Pathway for Bacterial Leucine Catabolism

PubMed Central

Nemecek-Marshall, Michele; Wojciechowski, Cheryl; Wagner, William P.; Fall, Ray

1999-01-01

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of l-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. l-Leucine, but not d-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of l-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only α-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d7)-l-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads. PMID:10601206

The effects of acute leucine or leucine-glutamine co-ingestion on recovery from eccentrically biased exercise.

PubMed

Waldron, Mark; Ralph, Cameron; Jeffries, Owen; Tallent, Jamie; Theis, Nicola; Patterson, Stephen David

2018-05-16

This study investigated the effects of leucine or leucine + glutamine supplementation on recovery from eccentric exercise. In a double-blind independent groups design, 23 men were randomly assigned to a leucine (0.087 g/kg; n = 8), leucine + glutamine (0.087 g/kg + glutamine 0.3 g/kg; n = 8) or placebo (0.3 g/kg maltodextrin; n = 7) group. Participants performed 5 sets of drop jumps, with each set comprising 20 repetitions. Isometric knee-extensor strength, counter-movement jump (CMJ) height, delayed-onset muscle soreness (DOMS) and creatine kinase (CK) were measured at baseline, 1, 24, 48 h and 72 h post-exercise. There was a time × group interaction for isometric strength, CMJ and CK (P < 0.05), with differences between the leucine + glutamine and placebo group at 48 h and 72 h for strength (P = 0.013; d = 1.43 and P < 0.001; d = 2.06), CMJ (P = 0.008; d = 0.87 and P = 0.019; d = 1.17) and CK at 24 h (P = 0.012; d = 0.54) and 48 h (P = 0.010; d = 1.37). The leucine group produced higher strength at 72 h compared to placebo (P = 0.007; d = 1.65) and lower CK at 24 h (P = 0.039; d = 0.63) and 48 h (P = 0.022; d = 1.03). Oral leucine or leucine + glutamine increased the rate of recovery compared to placebo after eccentric exercise. These findings highlight potential benefits of co-ingesting these amino acids to ameliorate recovery.

Acetohydroxy acid synthase is a target for leucine containing peptide toxicity in Escherichia coli.

PubMed Central

Gollop, N; Tavori, H; Barak, Z

1982-01-01

Acetohydroxy acid synthase from a mutant resistant to leucine-containing peptides was insensitive to leucine inhibition. It is concluded that acetohydroxy acid synthase is a target for the toxicity of the high concentrations of leucine brought into Escherichia coli K-12 by leucine-containing peptides. PMID:7033214

Attenuation of Cigarette Smoke-Induced Airway Mucus Production by Hydrogen-Rich Saline in Rats

PubMed Central

Zhang, Jingxi; Dong, Yuchao; Xu, Wujian; Li, Qiang

2013-01-01

Background Over-production of mucus is an important pathophysiological feature in chronic airway disease such as chronic obstructive pulmonary disease (COPD) and asthma. Cigarette smoking (CS) is the leading cause of COPD. Oxidative stress plays a key role in CS-induced airway abnormal mucus production. Hydrogen protected cells and tissues against oxidative damage by scavenging hydroxyl radicals. In the present study we investigated the effect of hydrogen on CS-induced mucus production in rats. Methods Male Sprague-Dawley rats were divided into four groups: sham control, CS group, hydrogen-rich saline pretreatment group and hydrogen-rich saline control group. Lung morphology and tissue biochemical changes were determined by immunohistochemistry, Alcian Blue/periodic acid-Schiff staining, TUNEL, western blot and realtime RT-PCR. Results Hydrogen-rich saline pretreatment attenuated CS-induced mucus accumulation in the bronchiolar lumen, goblet cell hyperplasia, muc5ac over-expression and abnormal cell apoptosis in the airway epithelium as well as malondialdehyde increase in the BALF. The phosphorylation of EGFR at Tyr1068 and Nrf2 up-regulation expression in the rat lungs challenged by CS exposure were also abrogated by hydrogen-rich saline. Conclusion Hydrogen-rich saline pretreatment ameliorated CS-induced airway mucus production and airway epithelium damage in rats. The protective role of hydrogen on CS-exposed rat lungs was achieved at least partly by its free radical scavenging ability. This is the first report to demonstrate that intraperitoneal administration of hydrogen-rich saline protected rat airways against CS damage and it could be promising in treating abnormal airway mucus production in COPD. PMID:24376700

Hydrogen-rich saline protects retina against glutamate-induced excitotoxic injury in guinea pig.

PubMed

Wei, Lihua; Ge, Li; Qin, Shucun; Shi, Yunzhi; Du, Changqing; Du, Hui; Liu, Liwei; Yu, Yang; Sun, Xuejun

2012-01-01

Molecular hydrogen (H(2)) is an efficient antioxidant that can selectively reduce hydroxyl radicals and inhibit oxidative stress-induced injuries. We investigated the protective effects and mechanism of hydrogen-rich saline in a glutamate-induced retinal injury model. Retinal excitotoxicity was induced in healthy guinea pigs by injecting glutamate into the vitreous cavity. After 30 min, hydrogen-rich saline was injected into the vitreous cavity, the peritoneal cavity or both. Seven days later, the retinal stress response was evaluated by examining the stress biomarkers, inducible nitric-oxide synthase (iNOS) and glucose-regulated protein 78 (GRP78). The impaired glutamate uptake was assessed by the expression of the excitatory amino acid transporter 1(EAAT-1). The retinal histopathological changes were investigated, focusing on the thicknesses of the entire retina and its inner layer, the number of cells in the retinal ganglion cell layer (GCL) and the ultrastructure of the retinal ganglion cells (RGCs) and glial cells. Compared with the glutamate-induced injury group, the hydrogen-rich saline treatment reduced the loss of cells in the GCL and thinning of the retina and attenuated cellular morphological damage. These improvements were greatest in animals that received H(2) injections into both the vitreous and the peritoneal cavities. The hydrogen-rich saline also inhibited the expression of glial fibrillary acidic protein (GFAP) in Müller cells, CD11b in microglia, and iNOS and GRP78 in glial cells. Moreover, the hydrogen-rich saline increased the expression of EAAT-1. In conclusion, the administration of hydrogen-rich saline through the intravitreal or/and intraperitoneal routes could reduce the retinal excitotoxic injury and promote retinal recovery. This result likely occurs by inhibiting the activation of glial cells, decreasing the production of the iNOS and GRP78 and promoting glutamate clearance. Copyright © 2011 Elsevier Ltd. All rights reserved.

Brassinosteroid and Gibberellin control of seedling traits in maize (Zea mays L.)

USDA-ARS?s Scientific Manuscript database

Brassinosteroids (BRs) and gibberellins (GAs) are two major plant hormones regulating various plant developmental processes. In maize, BRs and GAs have been shown to regulate field traits such as plant height and sex determination. This study used 207 doubled haploid maize lines and measured respons...

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Zhuang; Laboratory of System Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031; Zou, Xinle

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 (AAmore » 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.« less

Organization of a resistance gene cluster linked to rhizomania resistance in sugar beet

USDA-ARS?s Scientific Manuscript database

Genetic resistance to rhizomania has been in use for over 40 years. Characterization of the molecular basis for susceptibility and resistance has proved challenging. Nucleotide-binding leucine-rich-repeat-containing (NB-LRR) genes have been implicated in numerous gene-for-gene resistance interaction...

Rhizoctonia resistance conferred by a sugar beet polygalacturonase-inhibiting protein gene

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall leucine-rich repeat (LRR) proteins recognized as having a role in plant defense. PGIPs inhibit fungal polygalacturonase (PG) enzymes that break down the polygalacturonate chain in plant cell walls to initiate disease development. The inte...

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen

USDA-ARS?s Scientific Manuscript database

Disease resistance (R) genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLRs define the fastest evolving...

A Novel Class of Gibberellin 2-Oxidases Control Semidwarfism, Tillering, and Root Development in Rice[W

PubMed Central

Lo, Shuen-Fang; Yang, Show-Ya; Chen, Ku-Ting; Hsing, Yue-Ie; Zeevaart, Jan A.D.; Chen, Liang-Jwu; Yu, Su-May

2008-01-01

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2β-hydroxylation: a larger class of C19 GA2oxs and a smaller class of C20 GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C20 GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C20 GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C20 GA2oxs were found to cause less severe GA-defective phenotypes than C19 GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C20 GA2oxs. PMID:18952778

Determination of the tolerable upper intake level of leucine in acute dietary studies in young men.

PubMed

Elango, Rajavel; Chapman, Karen; Rafii, Mahroukh; Ball, Ronald O; Pencharz, Paul B

2012-10-01

Leucine has been suggested to improve athletic performance. Therefore, the branched-chain amino acids (BCAAs), especially leucine, are popular as dietary supplements in strength-training athletes; however, the intake of leucine in excess of requirements raises concerns regarding adverse effects. Currently, the tolerable upper intake level (UL) for leucine is unknown. The objective of the current study was to determine the UL for leucine in adult men under acute dietary conditions. Five healthy adults (20-35 y) each received graded stepwise increases in leucine intakes of 50, 150, 250, 500, 750, 1000, and 1250 mg · kg⁻¹ · d⁻¹, which corresponded to the Estimated Average Requirement (EAR) and the EAR ×3, ×5, ×10, ×15, ×20, and ×25 in a total of 29 studies. The UL of leucine was identified by the measurement of plasma and urinary biochemical variables and changes in leucine oxidation by using l-[1-¹³C]-leucine. A significant increase in blood ammonia concentrations above normal values, plasma leucine concentrations, and urinary leucine excretion were observed with leucine intakes >500 mg · kg⁻¹ · d⁻¹. The oxidation of l-[1-¹³C]-leucine expressed as label tracer oxidation in breath (F¹³CO₂), leucine oxidation, and α-ketoisocaproic acid (KIC) oxidation led to different results: a plateau in F¹³CO₂ observed after 500 mg · kg⁻¹ · d⁻¹, no clear plateau observed in leucine oxidation, and KIC oxidation appearing to plateau after 750 mg · kg⁻¹ · d⁻¹. On the basis of plasma and urinary variables, the UL for leucine in healthy adult men can be suggested at 500 mg · kg⁻¹ · d⁻¹ or ~35 g/d as a cautious estimate under acute dietary conditions.

Spatio-temporal expression of miRNA159 family members and their GAMYB target gene during the modulation of gibberellin-induced grapevine parthenocarpy.

PubMed

Wang, Chen; Jogaiah, Sudisha; Zhang, WenYing; Abdelrahman, Mostafa; Fang, Jing Gui

2018-06-27

Grapevine, Vitis vinifera, is an important economic fruit crop that is highly sensitive to gibberellin (GA), and the exogenous application of GA can efficiently induce grapevine parthenocarpy. However, the molecular mechanisms underlying this process remain elusive. In this study, morphological changes during flower development in response to GA treatments were examined in the 'Zuijinxiang' cultivar. To obtain insights into the roles of miRNA159s in GA-induced grapevine parthenocarpy, VvmiR159a, VvmiR159b, VvmiR159c, and their target gene VvGAMYB were isolated, sequenced and characterized. Spatial-temporal expression analyses showed that VvmiR159c exhibited the highest expression levels at 4 d before flowering, followed by a gradual decrease, while VvGAMYB displayed an opposite pattern of expression with the lowest expression at the corresponding stage in response to GA treatment. A cleavage interaction between VvmiR159s and VvGAMYB and variations of their cleavage roles were confirmed in grapevine floral development. In addition, the potential roles of VvmiR159s in GA signaling were investigated through DELLA-protein repressors, indicating that GA-DELLA (SLR1)-VvmiR159c-VvGAMYB is the key signaling regulatory module in grapevine. Our findings provide novel insights into the GA-responsive roles of VvmiR159s in modulating grapevine floral development, which have important implications for the molecular breeding of high-quality seedless grapevine berry.

Genetic overexpression of glutathione peroxidase-1 attenuates microcystin-leucine-arginine-induced memory impairment in mice.

PubMed

Shin, Eun-Joo; Hwang, Yeong Gwang; Pham, Duc Toan; Lee, Ji Won; Lee, Yu Jeung; Pyo, Dongjin; Lei, Xin Gen; Jeong, Ji Hoon; Kim, Hyoung-Chun

2018-06-13

Microcystin-leucine-arginine (MCLR) is the most common form of microcystins, which are environmental toxins produced by cyanobacteria, and its hepatotoxicity has been well-documented. However, the neurotoxic potential of MCLR remains to be further elucidated. In the present study, we investigated whether intracerebroventricular (i.c.v.) infusion of MCLR induces mortality and neuronal loss in the hippocampus of mice. Because we found that MCLR impairs memory function in the hippocampus at a low dose (4 ng/μl/mouse, i.c.v.) without a significant neuronal loss, we focused on this dose for further analyses. Results showed that MCLR (4 ng/μl/mouse, i.c.v.) significantly increased oxidative stress (i.e., malondialdehyde, protein carbonyl, and synaptosomal ROS) in the hippocampus. In addition, MCLR significantly increased superoxide dismutase (SOD) activity without corresponding induction of glutathione peroxidase (GPx) activity, and thus led to significant decrease in the ratio of GPx/SODs activity. The GSH/GSSG ratio was also significantly reduced after MCLR treatment. GPx-1 overexpressing transgenic mice (GPx-1 Tg) were significantly protected from MCLR-induced memory impairment and oxidative stress. The DNA binding activity of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in these mice was significantly enhanced, and the ratios of GPx/SODs activity and GSH/GSSG returned to near control levels in the hippocampus. Importantly, memory function exhibited a significant positive correlation with the ratios of GPx/SODs activity and GSH/GSSG in the hippocampus of MCLR-treated non-transgenic (non-Tg)- and GPx-1 Tg-mice. Combined, our results suggest that MCLR induces oxidative stress and memory impairment without significant neuronal loss, and that GPx-1 gene constitutes an important protectant against MCLR-induced memory impairment and oxidative stress via maintaining antioxidant defense system homeostasis, possibly through the induction of Nrf2

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

Brassinosteroid regulates cell elongation by modulating gibberellin metabolism in rice.

PubMed

Tong, Hongning; Xiao, Yunhua; Liu, Dapu; Gao, Shaopei; Liu, Linchuan; Yin, Yanhai; Jin, Yun; Qian, Qian; Chu, Chengcai

2014-11-01

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA1 levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana. © 2014 American Society of Plant Biologists. All rights reserved.

Gene expression and metabolite profiling of gibberellin biosynthesis during induction of somatic embryogenesis in Medicago truncatula Gaertn

PubMed Central

Igielski, Rafał

2017-01-01

Gibberellins (GAs) are involved in the regulation of numerous developmental processes in plants including zygotic embryogenesis, but their biosynthesis and role during somatic embryogenesis (SE) is mostly unknown. In this study we show that during three week- long induction phase, when cells of leaf explants from non-embryogenic genotype (M9) and embryogenic variant (M9-10a) were forming the callus, all the bioactive gibberellins from non-13-hydroxylation (GA4, GA7) and 13-hydroxylation (GA1, GA5, GA3, GA6) pathways were present, but the contents of only a few of them differed between the tested lines. The GA53 and GA19 substrates synthesized by the 13-hydroxylation pathway accumulated specifically in the M9-10a line after the first week of induction; subsequently, among the bioactive gibberellins detected, only the content of GA3 increased and appeared to be connected with acquisition of embryogenic competence. We fully annotated 20 Medicago truncatula orthologous genes coding the enzymes which catalyze all the known reactions of gibberellin biosynthesis. Our results indicate that, within all the genes tested, expression of only three: MtCPS, MtGA3ox1 and MtGA3ox2, was specific to embryogenic explants and reflected the changes observed in GA53, GA19 and GA3 contents. Moreover, by analyzing expression of MtBBM, SE marker gene, we confirmed the inhibitory effect of manipulation in GAs metabolism, applying exogenous GA3, which not only impaired the production of somatic embryos, but also significantly decreased expression of this gene. PMID:28750086

Incorporation of C-Kaurene into the Gibberellin of a Higher Plant (Pharbitis nil Chois).

PubMed

Barendse, G W; Kok, N J

1971-10-01

Enzymic formation of (14)C-kaurene from 2-(14)C-mevalonate was carried out with a cell-free system of Cucurbita pepo L. It was shown that either heating of the enzyme system or the addition of the growth retardants (2-chloroethyl)-trimethylammonium chloride and 2'-isopropyl-4' (trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate prevented the synthesis of (14)C-kaurene. Experiments in which (14)C-kaurene was applied to seedlings of Pharbitis nil revealed that the kaurene is converted to at least two compounds present in the acidic ethyl acetate fraction, containing free gibberellins, as well as in the second acidic ethyl acetate fraction, containing the released bound gibberellins. One of the compounds cochromatographed with gibberellic acid; the other compound is possibly a break-down product of gibberellic acid with no biological activity.

Natural Variation in Small Molecule–Induced TIR-NB-LRR Signaling Induces Root Growth Arrest via EDS1- and PAD4-Complexed R Protein VICTR in Arabidopsis[C][W

PubMed Central

Kim, Tae-Houn; Kunz, Hans-Henning; Bhattacharjee, Saikat; Hauser, Felix; Park, Jiyoung; Engineer, Cawas; Liu, Amy; Ha, Tracy; Parker, Jane E.; Gassmann, Walter; Schroeder, Julian I.

2012-01-01

In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- and ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstream of EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thaliana accession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR (VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary for DFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0 VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 and PAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and their cochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acid signaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 in a nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4 and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest. PMID:23275581

A dual role for glucocorticoid-induced leucine zipper in glucocorticoid function: tumor growth promotion or suppression?

PubMed

Ayroldi, Emira; Cannarile, Lorenza; Delfino, Domenico V; Riccardi, Carlo

2018-04-26

Glucocorticoids (GCs), important therapeutic tools to treat inflammatory and immunosuppressive diseases, can also be used as part of cancer therapy. In oncology, GCs are used as anticancer drugs for lymphohematopoietic malignancies, while in solid neoplasms primarily to control the side effects of chemo/radiotherapy treatments. The molecular mechanisms underlying the effects of GCs are numerous and often overlapping, but not all have been elucidated. In normal, cancerous, and inflammatory tissues, the response to GCs differs based on the tissue type. The effects of GCs are dependent on several factors: the tumor type, the GC therapy being used, the expression level of the glucocorticoid receptor (GR), and the presence of any other stimuli such as signals from immune cells and the tumor microenvironment. Therefore, GCs may either promote or suppress tumor growth via different molecular mechanisms. Stress exposure results in dysregulation of the hypothalamic-pituitary-adrenal axis with increased levels of endogenous GCs that promote tumorigenesis, confirming the importance of GCs in tumor growth. Most of the effects of GCs are genomic and mediated by the modulation of GR gene transcription. Moreover, among the GR-induced genes, glucocorticoid-induced leucine zipper (GILZ), which was cloned and characterized primarily in our laboratory, mediates many GC anti-inflammatory effects. In this review, we analyzed the possible role for GILZ in the effects GCs have on tumors cells. We also suggest that GILZ, by affecting the immune system, tumor microenvironment, and directly cancer cell biology, has a tumor-promoting function. However, it may also induce apoptosis or decrease the proliferation of cancer cells, thus inhibiting tumor growth. The potential therapeutic implications of GILZ activity on tumor cells are discussed here.

Prevalence of Abortion and Contraceptive Practice among Women Seeking Repeat Induced Abortion in Western Nigeria.

PubMed

Lamina, Mustafa Adelaja

2015-01-01

Induced abortion contributes significantly to maternal mortality in developing countries yet women still seek repeat induced abortion in spite of availability of contraceptive services. The aim of this study is to determine the rate of abortion and contraceptive use among women seeking repeat induced abortion in Western Nigeria. A prospective cross-sectional study utilizing self-administered questionnaires was administered to women seeking abortion in private hospitals/clinics in four geopolitical areas of Ogun State, Western Nigeria, from January 1 to December 31 2012. Data were analyzed using SPSS 17.0. The age range for those seeking repeat induced abortion was 15 to 51 years while the median age was 25 years. Of 2934 women seeking an abortion, 23% reported having had one or more previous abortions. Of those who had had more than one abortion, the level of awareness of contraceptives was 91.7% while only 21.5% used a contraceptive at their first intercourse after the procedure; 78.5% of the pregnancies were associated with non-contraceptive use while 17.5% were associated with contraceptive failure. The major reason for non-contraceptive use was fear of side effects. The rate of women seeking repeat abortions is high in Nigeria. The rate of contraceptive use is low while contraceptive failure rate is high.

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway

DOE PAGES

Saxton, Robert A.; Knockenhauer, Kevin E.; Wolfson, Rachel L.; ...

2015-11-19

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 leucinemore » leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. Lastly, these results provide a structural mechanism of amino acid sensing by the mTORC1 pathway.« less

Characterization of a Beta vulgaris PGIP defense gene promoter in transgenic plants

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting protein (BvPGIP) genes were cloned from a sugar beet breeding line F1016 with increased tolerance to the sugar beet root maggot. Polygalacturonase-inhibiting proteins are cell wall leucine-rich repeat (LRR) proteins with crucial roles in development, pathogen defense an...

Sugar beet polygalacturonase-inhibiting proteins with 11 LRRs confer Rhizoctonia, Fusarium and Botrytis resistance in Nicotiana plants

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit polygalacturonase (PG) enzymes secreted by pathogens to break down plant cell walls during early stage of disease development. Sugar beet (Beta vulgaris L.) PGIP genes (BvPGIPs) have 11 LRR domains as ...

Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

ERIC Educational Resources Information Center

Zhang, Xiaorong

2009-01-01

This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR…

Leucine acts as a nutrient signal to stimulate protein synthesis

USDA-ARS?s Scientific Manuscript database

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...

Repeated restraint stress exposure during early withdrawal accelerates incubation of cue-induced cocaine craving.

PubMed

Glynn, Ryan M; Rosenkranz, J Amiel; Wolf, Marina E; Caccamise, Aaron; Shroff, Freya; Smith, Alyssa B; Loweth, Jessica A

2018-01-01

A major challenge for treating cocaine addiction is the propensity for abstinent users to relapse. Two important triggers for relapse are cues associated with prior drug use and stressful life events. To study their interaction in promoting relapse during abstinence, we used the incubation model of craving and relapse in which cue-induced drug seeking progressively intensifies ('incubates') during withdrawal from extended-access cocaine self-administration. We tested rats for cue-induced cocaine seeking on withdrawal day (WD) 1. Rats were then subjected to repeated restraint stress or control conditions (seven sessions held between WD6 and WD14). All rats were tested again for cue-induced cocaine seeking on WD15, 1 day after the last stress or control session. Although controls showed a time-dependent increase in cue-induced cocaine seeking (incubation), rats exposed to repeated stress in early withdrawal exhibited a more robust increase in seeking behavior between WD1 and WD15. In separate stressed and control rats, equivalent cocaine seeking was observed on WD48. These results indicate that repeated stress in early withdrawal accelerates incubation of cocaine craving, although craving plateaus at the same level were observed in controls. However, 1 month after the WD48 test, rats subjected to repeated stress in early withdrawal showed enhanced cue-induced cocaine seeking following acute (24 hours) food deprivation stress. Together, these data indicate that chronic stress exposure enhances the initial rate of incubation of craving during early withdrawal, resulting in increased vulnerability to cue-induced relapse during this period, and may lead to a persistent increase in vulnerability to the relapse-promoting effects of stress. © 2016 Society for the Study of Addiction.

Repeated blood flow restriction induces muscle fiber hypertrophy.

PubMed

Sudo, Mizuki; Ando, Soichi; Kano, Yutaka

2017-02-01

We recently developed an animal model to investigate the effects of eccentric contraction (ECC) and blood flow restriction (BFR) on muscle tissue at the cellular level. This study clarified the effects of repeated BFR, ECC, and BFR combined with ECC (BFR+ECC) on muscle fiber hypertrophy. Male Wistar rats were assigned to 3 groups: BFR, ECC, and BFR+ECC. The contralateral leg in the BFR group served as a control (CONT). Muscle fiber cross-sectional area (CSA) of the tibialis anterior was determined after the respective treatments for 6 weeks. CSA was greater in the BFR+ECC group than in the CONT (P < 0.01) and ECC (P < 0.05) groups. CSA was greater in the BFR group than that in the CONT group (P < 0.05). These results suggest that repeated BFR alone as well as BFR+ECC induces muscle fiber hypertrophy at the cellular level. Muscle Nerve 55: 274-276, 2017. © 2016 Wiley Periodicals, Inc.

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. 2011 Elsevier Ltd. All rights reserved.

Long Glucocorticoid-induced Leucine Zipper (L-GILZ) Protein Interacts with Ras Protein Pathway and Contributes to Spermatogenesis Control*

PubMed Central

Bruscoli, Stefano; Velardi, Enrico; Di Sante, Moises; Bereshchenko, Oxana; Venanzi, Alessandra; Coppo, Maddalena; Berno, Valeria; Mameli, Maria Grazia; Colella, Renato; Cavaliere, Antonio; Riccardi, Carlo

2012-01-01

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis. PMID:22110132

Repeated administration of amitriptyline reduces oxaliplatin-induced mechanical allodynia in rats.

PubMed

Sada, Hikaru; Egashira, Nobuaki; Ushio, Soichiro; Kawashiri, Takehiro; Shirahama, Masafumi; Oishi, Ryozo

2012-01-01

Oxaliplatin is a key drug in the treatment of colorectal cancer, but it causes acute and chronic neuropathies in patients. Amitriptyline has widely been used in patients with painful neuropathy. In this study, we investigated the effect of amitriptyline on the oxaliplatin-induced neuropathy in rats. Repeated administration of amitriptyline (5 and 10 mg/kg, p.o., once a day) reduced the oxaliplatin-induced mechanical allodynia but not cold hyperalgesia and reversed the oxaliplatin-induced increase in the expression of NR2B protein and mRNA in rat spinal cord. These results suggest that amitriptyline is useful for the treatment of oxaliplatin-induced neuropathy clinically.

Polymerization- and Solvent-Induced Phase Separation in Hydrophilic-rich Dentin Adhesive Mimic

PubMed Central

Abedin, Farhana; Ye, Qiang; Good, Holly J; Parthasarathy, Ranganathan; Spencer, Paulette

2014-01-01

Current dental resin undergoes phase separation into hydrophobic-rich and hydrophilic-rich phases during infiltration of the over-wet demineralized collagen matrix. Such phase separation undermines the integrity and durability of the bond at the composite/tooth interface. This study marks the first time that the polymerization kinetics of model hydrophilic-rich phase of dental adhesive has been determined. Samples were prepared by adding varying water content to neat resins made from 95 and 99wt% hydroxyethylmethacrylate (HEMA) and 5 and 1wt% (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl1]-propane (BisGMA) prior to light curing. Viscosity of the formulations decreased with increased water content. The photo-polymerization kinetics study was carried out by time-resolved FTIR spectrum collector. All of the samples exhibited two-stage polymerization behavior which has not been reported previously for dental resin formulation. The lowest secondary rate maxima were observed for water content of 10-30%wt. Differential scanning calorimetry (DSC) showed two glass transition temperatures for the hydrophilic-rich phase of dental adhesive. The DSC results indicate that the heterogeneity within the final polymer structure decreased with increased water content. The results suggest a reaction mechanism involving both polymerization-induced phase separation (PIPs) and solvent-induced phase separation (SIPs) for the model hydrophilic-rich phase of dental resin. PMID:24631658

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

PubMed Central

Kirchman, D; K'nees, E; Hodson, R

1985-01-01

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems. PMID:3994368

Leucine supplementation attenuates macrophage foam-cell formation: Studies in humans, mice, and cultured macrophages.

PubMed

Grajeda-Iglesias, Claudia; Rom, Oren; Hamoud, Shadi; Volkova, Nina; Hayek, Tony; Abu-Saleh, Niroz; Aviram, Michael

2018-02-05

Whereas atherogenicity of dietary lipids has been largely studied, relatively little is known about the possible contribution of dietary amino acids to macrophage foam-cell formation, a hallmark of early atherogenesis. Recently, we showed that leucine has antiatherogenic properties in the macrophage model system. In this study, an in-depth investigation of the role of leucine in macrophage lipid metabolism was conducted by supplementing humans, mice, or cultured macrophages with leucine. Macrophage incubation with serum obtained from healthy adults supplemented with leucine (5 g/d, 3 weeks) significantly decreased cellular cholesterol mass by inhibiting the rate of cholesterol biosynthesis and increasing cholesterol efflux from macrophages. Similarly, leucine supplementation to C57BL/6 mice (8 weeks) resulted in decreased cholesterol content in their harvested peritoneal macrophages (MPM) in relation with reduced cholesterol biosynthesis rate. Studies in J774A.1 murine macrophages revealed that leucine dose-dependently decreased cellular cholesterol and triglyceride mass. Macrophages treated with leucine (0.2 mM) showed attenuated uptake of very low-density lipoproteins and triglyceride biosynthesis rate, with a concurrent down-regulation of diacylglycerol acyltransferase-1, a key enzyme catalyzing triglyceride biosynthesis in macrophages. Similar effects were observed when macrophages were treated with α-ketoisocaproate, a key leucine metabolite. Finally, both in vivo and in vitro leucine supplementation significantly improved macrophage mitochondrial respiration and ATP production. The above studies, conducted in human, mice, and cultured macrophages, highlight a protective role for leucine attenuating macrophage foam-cell formation by mechanisms related to the metabolism of cholesterol, triglycerides, and energy production. © 2018 BioFactors, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Endogenous Gibberellins of Pine Pollen

PubMed Central

Kamienska, Aniela; Pharis, Richard P.

1975-01-01

The endogenous gibberellins (GAs) of pollen of Pinus attenuata, P. coulteri, and P. ponderosa were bioassayed at hour 0, 3, 15, 24, 48 and 72 of germination. Dormant pollen showed relatively high GA activity throughout the elution spectrum (i.e. ranging from relatively nonpolar to highly polar). The maximum GA activity was obtained at hour 15 in more polar regions and especially in the zone corresponding to GA3 (for P. attenuata estimated as 250 micrograms of GA3/kilogram pollen). It is probable that the “nonpolar” GAs present in high quantities in dormant pollen and in early stages of germination were converted to “more polar” GAs as germination progressed. The amount of all GAs decreased after hour 15 of germination and by hour 72 no GAs could be detected. Among the species tested P. attenuata showed the highest over-all GA activity. PMID:16659365

Characteristics of a leucine aminoacyl transfer RNA synthetase from Tritrichomonas augusta.

PubMed

Horner, J; Champney, W S; Samuels, R

1991-04-01

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37 degrees C for 20 min, with protein at 180 micrograms ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

Leucine-rich amelogenin peptide (LRAP) as a surface primer for biomimetic remineralization of superficial enamel defects: An in vitro study.

PubMed

Shafiei, Farhad; Hossein, Bagheri G; Farajollahi, Mohammad M; Fathollah, Moztarzadeh; Marjan, Behroozibakhsh; Tahereh, Jafarzadeh Kashi

2015-01-01

This study was carried out to obtain more information about the assembly of hydroxyapatite bundles formed in the presence of Leucine-Rich Amelogenin Peptide (LRAP) and to evaluate its effect on the remineralization of enamel defects through a biomimetic approach. One or 2 mg/mL LRAP solutions containing 2.5 mM of Ca(+2) and 1.5 mM phosphate were prepared (pH = 7.2) and stored at 37 °C for 24 h. The products of the reaction were studied using atomic force microscopy (AFM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). Vickers surface microhardness recovery (SMR%) of acid-etched bovine enamel, with or without LRAP surface treatment, were calculated to evaluate the influence of peptide on the lesion remineralization. Distilled water and 1 or 2 mg/mL LRAP solution (pH = 7.2) were applied on the lesions and the specimens were incubated in mineralization solution (2.5mM Ca(+2) , 1.5mM PO4 (-3) , pH = 7.2) for 24 h. One-way ANOVA and Tukey's multi-comparison tests were used for statistical analysis. The pattern of enamel surface repair was studied using FE-SEM. AFM showed the formation of highly organized hierarchical structures, composed of hydroxyapatite (HA) crystals, similar to the dental enamel microstructure. ANOVA procedure showed significant effect of peptide treatment on the calculated SMR% (p < 0.001). Tukey's test revealed that peptide treated groups had significantly higher values of SMR%. In conclusion, LRAP is able to regulate the formation of HA and enhances the remineralization of acid-etched enamel as a surface treatment agent. © Wiley Periodicals, Inc.

Functional importance of EAK1 tyrosine phosphorylation in vivo

USDA-ARS?s Scientific Manuscript database

The plant receptor kinase BRASSINOSTEROID ASSOCIATED KINASE 1 (BAK1) is known as a partner of several ligand-binding leucine-rich repeat receptor kinases, including BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLS2. Autophosphorylation of receptor kinases is recognized to be an i...

Maize homologs of HCT, a key enzyme in lignin biosynthesis, bind the NLR Rp1 proteins to modulate the defense response

USDA-ARS?s Scientific Manuscript database

In plants, most disease resistance (R) genes encode nucleotide binding leucine-rich-repeat 42 (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) 43 upon pathogen recognition. The maize NLR protein Rp1-D21 derives from an intragenic 44 recombination between...

Identification and distribution of the NBS-LRR gene family in the cassava genome

USDA-ARS?s Scientific Manuscript database

Plant resistance genes (R genes) exist in large families and usually contain both a nucleotide-binding site domain and a leucine-rich repeat domain, denoted NBS-LRR. The genome sequence of cassava (Manihot esculenta) is a valuable resource for analyzing the genomic organization of resistance genes i...

Gibberellin inhibitors improve embryogenic tissue initiation in conifers.

PubMed

Pullman, Gerald S; Mein, J; Johnson, S; Zhang, Y

2005-02-01

Somatic embryogenesis (SE), the most promising technology to multiply high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and uniformity of future forests in the United States. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (LP; Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation of LP, slash pine (SP; Pinus elliottii), Douglas-fir (DF; Pseudotsuga menziesii), and Norway spruce (NS; Picea abies) were improved through the use of paclobutrazol, a gibberellin synthesis inhibitor. Paclobutrazol was effective at concentrations ranging from 0.25 mg/l to 3.0 mg/l (0.85-10.2 microM) and optimal in LP at 1.0 mg/l. Using control media (no paclobutrazol) and 0.33-1.0 mg/l paclobutrazol, initiation percentages in LP, SP, DF, and NS were improved from 37.7% to 44.2% (across experiments), 19.3% to 28.5%, 16.9% to 23.7%, and 38.8% to 48.5%, respectively. Other gibberellin inhibitors such as flurprimidol, chlormequat-Cl, and daminozide also caused statistically significant increases in LP initiation when added to the medium at concentrations of 0.34, 10.0, and 1.0 microM, respectively. No detrimental effects on subsequent embryo development were observed when 29 new initiations from medium without GA inhibitor and 28 new initiations from medium containing paclobutrazol were tracked through culture capture, liquid culture establishment, cotyledonary embryo development, and germination.

Overexpression of the gibberellin 2-oxidase gene from Torenia fournieri induces dwarf phenotypes in the liliaceous monocotyledon Tricyrtis sp.

PubMed

Otani, Masahiro; Meguro, Shuhei; Gondaira, Haruka; Hayashi, Megumi; Saito, Misaki; Han, Dong-Sheng; Inthima, Phithak; Supaibulwatana, Kanyaratt; Mori, Shiro; Jikumaru, Yusuke; Kamiya, Yuji; Li, Tuoping; Niki, Tomoya; Nishijima, Takaaki; Koshioka, Masaji; Nakano, Masaru

2013-11-01

Gibberellins (GAs) are the plant hormones that control many aspects of plant growth and development, including stem elongation. Genes encoding enzymes related to the GA biosynthetic and metabolic pathway have been isolated and characterized in many plant species. Gibberellin 2-oxidase (GA2ox) catalyzes bioactive GAs or their immediate precursors to inactive forms; therefore, playing a direct role in determining the levels of bioactive GAs. In the present study, we produced transgenic plants of the liliaceous monocotyledon Tricyrtis sp. overexpressing the GA2ox gene from the linderniaceous dicotyledon Torenia fournieri (TfGA2ox2). All six transgenic plants exhibited dwarf phenotypes, and they could be classified into two classes according to the degree of dwarfism: three plants were moderately dwarf and three were severely dwarf. All of the transgenic plants had small or no flowers, and smaller, rounder and darker green leaves. Quantitative real-time reverse transcription-polymerase chain reaction (PCR) analysis showed that the TfGA2ox2 expression level generally correlated with the degree of dwarfism. The endogenous levels of bioactive GAs, GA1 and GA4, largely decreased in transgenic plants as shown by liquid chromatography-mass spectrometry (LC-MS) analysis, and the level also correlated with the degree of dwarfism. Exogenous treatment of transgenic plants with gibberellic acid (GA3) resulted in an increased shoot length, indicating that the GA signaling pathway might normally function in transgenic plants. Thus, morphological changes in transgenic plants may result from a decrease in the endogenous levels of bioactive GAs. Finally, a possibility of molecular breeding for plant form alteration in liliaceous ornamental plants by genetically engineering the GA metabolic pathway is discussed. Copyright © 2013 Elsevier GmbH. All rights reserved.

Prevalence of Abortion and Contraceptive Practice among Women Seeking Repeat Induced Abortion in Western Nigeria

PubMed Central

Lamina, Mustafa Adelaja

2015-01-01

Background. Induced abortion contributes significantly to maternal mortality in developing countries yet women still seek repeat induced abortion in spite of availability of contraceptive services. The aim of this study is to determine the rate of abortion and contraceptive use among women seeking repeat induced abortion in Western Nigeria. Method. A prospective cross-sectional study utilizing self-administered questionnaires was administered to women seeking abortion in private hospitals/clinics in four geopolitical areas of Ogun State, Western Nigeria, from January 1 to December 31 2012. Data were analyzed using SPSS 17.0. Results. The age range for those seeking repeat induced abortion was 15 to 51 years while the median age was 25 years. Of 2934 women seeking an abortion, 23% reported having had one or more previous abortions. Of those who had had more than one abortion, the level of awareness of contraceptives was 91.7% while only 21.5% used a contraceptive at their first intercourse after the procedure; 78.5% of the pregnancies were associated with non-contraceptive use while 17.5% were associated with contraceptive failure. The major reason for non-contraceptive use was fear of side effects. Conclusion. The rate of women seeking repeat abortions is high in Nigeria. The rate of contraceptive use is low while contraceptive failure rate is high. PMID:26078881

Bioinformatics Analysis of NBS-LRR Encoding Resistance Genes in Setaria italica.

PubMed

Zhao, Yan; Weng, Qiaoyun; Song, Jinhui; Ma, Hailian; Yuan, Jincheng; Dong, Zhiping; Liu, Yinghui

2016-06-01

In plants, resistance (R) genes are involved in pathogen recognition and subsequent activation of innate immune responses. The nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes family forms the largest R-gene family among plant genomes and play an important role in plant disease resistance. In this paper, comprehensive analysis of NBS-encoding genes is performed in the whole Setaria italica genome. A total of 96 NBS-LRR genes are identified, and comprehensive overview of the NBS-LRR genes is undertaken, including phylogenetic analysis, chromosome locations, conserved motifs of proteins, and gene expression. Based on the domain, these genes are divided into two groups and distributed in all Setaria italica chromosomes. Most NBS-LRR genes are located at the distal tip of the long arms of the chromosomes. Setaria italica NBS-LRR proteins share at least one nucleotide-biding domain and one leucine-rich repeat domain. Our results also show the duplication of NBS-LRR genes in Setaria italica is related to their gene structure.

Role of chirality in peptide-induced formation of cholesterol-rich domains

PubMed Central

2005-01-01

The chiral specificity of the interactions of peptides that induce the formation of cholesterol-rich domains has not been extensively investigated. Both the peptide and most lipids are chiral, so there is a possibility that interactions between peptide and lipid could require chiral recognition. On the other hand, in our models with small peptides, the extent of folding of the peptide to form a specific binding pocket is limited. We have determined that replacing cholesterol with its enantiomer, ent-cholesterol, alters the modulation of lipid organization by peptides. The phase-transition properties of SOPC (1-stearoyl-2-oleoylphosphatidylcholine):cholesterol [in a 6:4 ratio with 0.2 mol% PtdIns(4,5)P2] are not significantly altered when ent-cholesterol replaces cholesterol. However, in the presence of 10 mol% of a 19-amino-acid, N-terminally myristoylated fragment (myristoyl-GGKLSKKKKGYNVNDEKAK-amide) of the protein NAP-22 (neuronal axonal membrane protein), the lipid mixture containing cholesterol undergoes separation into cholesterol-rich and cholesterol-depleted domains. This does not occur when ent-cholesterol replaces cholesterol. In another example, when N-acetyl-Leu-Trp-Tyr-Ile-Lys-amide (N-acetyl-LWYIK-amide) is added to SOPC:cholesterol (7:3 ratio), there is a marked increase in the transition enthalpy of the phospholipid, indicating separation of a cholesterol-depleted domain of SOPC. This phenomenon completely disappears when ent-cholesterol replaces cholesterol. The all-D-isomer of N-acetyl-LWYIK-amide also induces the formation of cholesterol-rich domains with natural cholesterol, but does so to a lesser extent with ent-cholesterol. Thus specific peptide chirality is not required for interaction with cholesterol-containing membranes. However, a specific chirality of membrane lipids is required for peptide-induced formation of cholesterol-rich domains. PMID:15929726

Methods for sequencing GC-rich and CCT repeat DNA templates

DOEpatents

Robinson, Donna L.

2007-02-20

The present invention is directed to a PCR-based method of cycle sequencing DNA and other polynucleotide sequences having high CG content and regions of high GC content, and includes for example DNA strands with a high Cytosine and/or Guanosine content and repeated motifs such as CCT repeats.

Leucine and HMB differentially modulate proteasome system in skeletal muscle under different sarcopenic conditions.

PubMed

Baptista, Igor L; Silva, Willian J; Artioli, Guilherme G; Guilherme, Joao Paulo L F; Leal, Marcelo L; Aoki, Marcelo S; Miyabara, Elen H; Moriscot, Anselmo S

2013-01-01

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.

Leucine and HMB Differentially Modulate Proteasome System in Skeletal Muscle under Different Sarcopenic Conditions

PubMed Central

Baptista, Igor L.; Silva, Willian J.; Artioli, Guilherme G.; Guilherme, Joao Paulo L. F.; Leal, Marcelo L.; Aoki, Marcelo S.; Miyabara, Elen H.; Moriscot, Anselmo S.

2013-01-01

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB. PMID:24124592

Incorporation of [h]leucine and [h]valine into protein of freshwater bacteria: field applications.

PubMed

Jørgensen, N O

1992-11-01

Incorporation of leucine and valine into proteins of freshwater bacteria as a measure of bacterial production was tested in two eutrophic Danish lakes and was related to bacterial production measured by thymidine incorporation. In a depth profile (0 to 8 m) in Frederiksborg Castle Lake, incorporation of 100 nM leucine and valine gave similar rates of protein production. In terms of carbon, this production was about 50% lower than incorporation of 10 nM thymidine. In another depth profile in the same lake, incorporations of 10 nM valine and 100 nM leucine were identical, but differed from incorporations of 10 nM leucine and 100 nM valine. Bacterial carbon production calculated from incorporations of 10 nM thymidine and 10 nM leucine was similar, whereas 10 nM valine and 100 nM leucine and valine indicated an up to 2.4-fold-higher rate of carbon production. In a diel study in Lake Bagsvaerd, incorporation of 100 nM leucine and valine indicated a similar protein production, but the calculated carbon production was about 1.9-fold higher than the production based on uptake of 10 nM thymidine. Different diel changes in incorporation of the two amino acids and in incorporation of thymidine were observed. In both lakes, concentrations of naturally occurring leucine and valine were <5 nM in most samples. This means that the specific activity of a H isotope added at a concentration of 100 nM usually was diluted a maximum of 5%. Net assimilation of natural free amino acids in the lakes sustained 8 to 69% of the net bacterial carbon requirement, estimated from incorporation of leucine, valine, or thymidine. The present results indicate that incorporation of leucine and valine permits realistic measurements of bacterial production in freshwater environments.

Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity

PubMed Central

Su, Jianbin; Yang, Liuyi; Zhu, Qiankun; Wu, Hongjiao; He, Yi; Liu, Yidong; Xu, Juan; Jiang, Dean

2018-01-01

Extensive research revealed tremendous details about how plants sense pathogen effectors during effector-triggered immunity (ETI). However, less is known about downstream signaling events. In this report, we demonstrate that prolonged activation of MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MPKs), is essential to ETI mediated by both coiled coil-nucleotide binding site-leucine rich repeats (CNLs) and toll/interleukin-1 receptor nucleotide binding site-leucine rich repeats (TNLs) types of R proteins. MPK3/MPK6 activation rapidly alters the expression of photosynthesis-related genes and inhibits photosynthesis, which promotes the accumulation of superoxide (O2•−) and hydrogen peroxide (H2O2), two major reactive oxygen species (ROS), in chloroplasts under light. In the chemical-genetically rescued mpk3 mpk6 double mutants, ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation are compromised, which correlates with delayed hypersensitive response (HR) cell death and compromised resistance. Furthermore, protection of chloroplasts by expressing a plastid-targeted cyanobacterial flavodoxin (pFLD) delays photosynthetic inhibition and compromises ETI. Collectively, this study highlights a critical role of MPK3/MPK6 in manipulating plant photosynthetic activities to promote ROS accumulation in chloroplasts and HR cell death, which contributes to the robustness of ETI. Furthermore, the dual functionality of MPK3/MPK6 cascade in promoting defense and inhibiting photosynthesis potentially allow it to orchestrate the trade-off between plant growth and defense in plant immunity. PMID:29723186

Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase, GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins*

PubMed Central

Shi, Wei-Wei; Jiang, Yong-Liang; Zhu, Fan; Yang, Yi-Hu; Shao, Qiu-Yan; Yang, Hong-Bo; Ren, Yan-Min; Wu, Hui; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides. PMID:24936067

Group B Streptococcal Serine-Rich Repeat Proteins Promote Interaction With Fibrinogen and Vaginal Colonization

PubMed Central

Wang, Nai-Yu; Patras, Kathryn A.; Seo, Ho Seong; Cavaco, Courtney K.; Rösler, Berenice; Neely, Melody N.; Sullam, Paul M.; Doran, Kelly S.

2014-01-01

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 “latching” domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr–fibrinogen interactions in the female reproductive tract. PMID:24620021

Comparison of simple sequence repeats in 19 Archaea.

PubMed

Trivedi, S

2006-12-05

All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome.

Concentration-Dependent Patterns of Leucine Incorporation by Coastal Picoplankton

PubMed Central

Alonso, Cecilia; Pernthaler, Jakob

2006-01-01

Coastal pelagic environments are believed to feature concentration gradients of dissolved organic carbon at a microscale, and they are characterized by pronounced seasonal differences in substrate availability for the heterotrophic picoplankton. Microbial taxa that coexist in such habitats might thus differ in their ability to incorporate substrates at various concentrations. We investigated the incorporation patterns of leucine in four microbial lineages from the coastal North Sea at concentrations between 0.1 and 100 nM before and during a spring phytoplankton bloom. Community bulk incorporation rates and the fraction of leucine-incorporating cells in the different populations were analyzed. Significantly fewer bacterial cells incorporated the amino acid before (13 to 35%) than during (23 to 47%) the bloom at all but the highest concentration. The incorporation rate per active cell in the prebloom situation was constant above 0.1 nM added leucine, whereas it increased steeply with substrate concentration during the bloom. At both time points, a high proportion of members of the Roseobacter clade incorporated leucine at all concentrations (55 to 80% and 86 to 94%, respectively). In contrast, the fractions of leucine-incorporating cells increased substantially with substrate availability in bacteria from the SAR86 clade (8 to 31%) and from DE cluster 2 of the Flavobacteria-Sphingobacteria (14 to 33%). The incorporation patterns of marine Euryarchaeota were between these extremes (30 to 56% and 48 to 70%, respectively). Our results suggest that the contribution of microbial taxa to the turnover of particular substrates may be concentration dependent. This may help us to understand the specific niches of coexisting populations that appear to compete for the same resources. PMID:16517664

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 1...

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 1...

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 1...

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 1...

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

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

PubMed

Dong, Wen-Wen; Zhang, Yun-Qian; Zhu, Xiao-Yan; Mao, Yan-Fei; Sun, Xue-Jun; Liu, Yu-Jian; Jiang, Lai

2017-05-19

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Light Dependence of [3H]Leucine Incorporation in the Oligotrophic North Pacific Ocean†

PubMed Central

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

2004-01-01

The influence of irradiance on bacterial incorporation of [3H]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 [3H]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 [14C]bicarbonate) in both the upper and lower photic zones. Rates of [3H]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. [3H]leucine incorporation rates were analyzed with a photosynthesis-irradiance model for a quantitative description of the relationships between [3H]leucine incorporation and irradiance. Maximum rates of [3H]leucine incorporation in the upper photic zone increased 48 to 92% relative to those of dark-incubated samples, with [3H]leucine incorporation saturating at light intensities between 58 and 363 μmol of quanta m−2 s−1. Rates of [3H]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 μmol of quanta m−2 s−1. The results of these experiments revealed that sunlight directly influences bacterial growth in this open-ocean ecosystem. PMID:15240286

Brassinosteroids Are Master Regulators of Gibberellin Biosynthesis in Arabidopsis

PubMed Central

Unterholzner, Simon J.; Rozhon, Wilfried; Papacek, Michael; Ciomas, Jennifer; Lange, Theo; Kugler, Karl G.; Mayer, Klaus F.; Sieberer, Tobias; Poppenberger, Brigitte

2015-01-01

Plant growth and development are highly regulated processes that are coordinated by hormones including the brassinosteroids (BRs), a group of steroids with structural similarity to steroid hormones of mammals. Although it is well understood how BRs are produced and how their signals are transduced, BR targets, which directly confer the hormone’s growth-promoting effects, have remained largely elusive. Here, we show that BRs regulate the biosynthesis of gibberellins (GAs), another class of growth-promoting hormones, in Arabidopsis thaliana. We reveal that Arabidopsis mutants deficient in BR signaling are severely impaired in the production of bioactive GA, which is correlated with defective GA biosynthetic gene expression. Expression of the key GA biosynthesis gene GA20ox1 in the BR signaling mutant bri1-301 rescues many of its developmental defects. We provide evidence that supports a model in which the BR-regulated transcription factor BES1 binds to a regulatory element in promoters of GA biosynthesis genes in a BR-induced manner to control their expression. In summary, our study underscores a role of BRs as master regulators of GA biosynthesis and shows that this function is of major relevance for the growth and development of vascular plants. PMID:26243314

Antineoplastic activity of linear leucine homodipeptides and their potential mechanisms of action.

PubMed

Lei, Yun; Yang, Xiao-Xia; Guo, Wei; Zhang, Fu-Yong; Liao, Xiao-Jian; Yang, Hui-Fu; Xu, Shi-Hai; Xiong, Sheng

2018-07-01

Galaxamide is a rare cyclic homopentapeptide composed of three leucines and two N-methyl leucines isolated from marine algae Galaxaura filamentosa. The strong antitumor activity of this compound makes it a promising candidate for tumor therapy. The synthesis of galaxamide, however, is a complex process, and it has poor water solubility. On the basis of its special chemical composition, we designed a series of linear leucine homopeptides. Among seven dipeptide derivatives, five compounds with terminal protection groups and methyl substitution of the hydrogen in the amido group showed remarkable inhibitory effects against various cancer cells. N-tertbutyl-D-leucine-N-methyl-D-leucinebenzyl (A7), the only stereomer condensed by two D-leucines, showed the highest antineoplastic activity. A7-treated cells showed cell cycle arrest and morphological changes typical of cells undergoing apoptosis. The population of Annexin-V positive/propidium iodide-negative cells also increased, indicating the induction of early apoptosis. A7 promoted the cleavage of caspase-9 and caspase-3, as well as increased intracellular Ca levels and decreased the mitochondrial membrane potential. Collectively, certain linear leucine dipeptides derived from cyclic pentapeptide are able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis induction. The N-methyl group in the side chain and the D/L conformation of the amino-acid residue are critical for their activity.

Are we there yet for rice disease control

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes play an important role in fighting against plant pathogens. For the past two decades, significant efforts have been directed to map and clone R genes. Most of the cloned plant R genes encode proteins with leucine rich repeat and nucleotide binding sites (NLR), their cellul...

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity

USDA-ARS?s Scientific Manuscript database

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NB-LRR or NLR) proteins, which trigger a hypersensitive response (HR), a rapid, localized cell death upon recognition of specific pathogens. The maize NLR-encoding Rp1-D21 gene is the result of an intergenic recomb...

Long-term treatment of hydrogen-rich saline abates testicular oxidative stress induced by nicotine in mice.

PubMed

Li, Shu; Lu, DanDan; Zhang, Yaling; Zhang, Yi

2014-01-01

The present study was designed to test the hypothesis that long-term treatment with hydrogen-rich saline abated testicular oxidative stress induced by nicotine in mice. The effects of hydrogen-rich saline (6 ml/kg, i.p.), vitamin C (60 mg/kg, i.p.) and vitamin E (100 mg/kg, i.p.) on reproductive system and testicular oxidative levels in nicotine-treated (4.5 mg/kg, s.b.) mice were investigated. It was found that vitamin C and vitamin E attenuated serum oxidative level, but did not lower testicular oxidative levels in mice subjected to chronic nicotine treatment, and did not improve the male reproductive damage and apoptosis induced by nicotine. Different from normal antioxidants, vitamin C and vitamin E, hydrogen-rich saline abated oxidative stress in testis, and protected against nicotine-induced male reproductive damages. Our results first demonstrated that long-term treatment with hydrogen-rich saline attenuated testicular oxidative level and improved male reproductive function in nicotine-treated mice.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

PubMed

Zhang, Lu; Mo, Jinyao; Swanson, Karen V; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom; Shu, Hong-Bing; Duncan, Joseph A; Ting, Jenny P-Y

2014-03-20

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP. Copyright © 2014 Elsevier Inc. All rights reserved.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING

PubMed Central

Zhang, Lu; Mo, Jinyao; Swanson, Karen V.; Wen, Haitao; Petrucelli, Alex; Gregory, Sean M.; Zhang, Zhigang; Schneider, Monika; Jiang, Yan; Fitzgerald, Katherine A.; Ouyang, Songying; Liu, Zhi-Jie; Damania, Blossom A; Shu, Hong-Bing; Duncan, Joseph A.; Ting, Jenny P-Y.

2014-01-01

SUMMARY Stimulator of interferon genes (STING, also named MITA, MYPS or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich repeat containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses. NLRC3 associated with both STING and TBK1, and impeded STING-TBK1 interaction and downstream type I interferon production. Using purified recombinant proteins NLRC3 was found to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3−/− mice exhibited enhanced innate immunity, reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus and c-di-GMP PMID:24560620

Muscle damage and repeated bout effect induced by enhanced eccentric squats.

PubMed

Coratella, Giuseppe; Chemello, Alessandro; Schena, Federico

2016-12-01

Muscle damage and repeated bout effect have been studied after pure eccentric-only exercise. The aim of this study was to evaluate muscle damage and repeated bout effect induced by enhanced eccentric squat exercise using flywheel device. Thirteen healthy males volunteered for this study. Creatine kinase blood activity (CK), quadriceps isometric peak torque and muscle soreness were used as markers of muscle damage. The dependent parameters were measured at baseline, immediately after and each day up to 96 hours after the exercise session. The intervention consisted of 100 repetitions of enhanced eccentric squat exercise using flywheel device. The same protocol was repeated after 4 weeks. After the first bout, CK and muscle soreness were significantly greater (P<0.05) than baseline respectively up to 72 and 96 hours. Isometric peak torque was significantly lower (P<0.05) up to 72 hours. After the second bout, CK showed no significant increase (P>0.05), while isometric peak torque and muscle soreness returned to values similar to baseline after respectively 48 and 72 hours. All muscle damage markers were significantly lower after second compared to first bout. The enhanced eccentric exercise induced symptoms of muscle damage up to 96 hours. However, it provided muscle protection after the second bout, performed four weeks later. Although it was not eccentric-only exercise, the enhancement of eccentric phase provided muscle protection.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

PubMed Central

Toyomasu, Tomonobu; Kawaide, Hiroshi; Mitsuhashi, Wataru; Inoue, Yasunori; Kamiya, Yuji

1998-01-01

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action. PMID:9847128

Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area.

PubMed

Sotomayor-Zárate, Ramón; Abarca, Jorge; Araya, Katherine A; Renard, Georgina M; Andrés, María E; Gysling, Katia

2015-11-01

A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20μM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10μM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100μM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diet-induced thermogenesis and substrate oxidation are not different between lean and obese women after two different isocaloric meals, one rich in protein and one rich in fat.

PubMed

Tentolouris, Nicholas; Pavlatos, Spyridon; Kokkinos, Alexander; Perrea, Despoina; Pagoni, Stamata; Katsilambros, Nicholas

2008-03-01

Reduction in diet-induced thermogenesis (DIT) may promote weight gain and maintenance. Data on differences in DIT and macronutrient oxidation between lean and obese subjects are conflicting. In this study, we sought for differences in DIT and macronutrient oxidation between lean and obese women after consumption of 2 different isocaloric meals, one rich in protein and one rich in fat. Fifteen lean and 15 obese women were studied on 2 occasions, 1 week apart. In one visit, they consumed a protein-rich meal; in the other visit, a fat-rich meal. The 2 meals were isocaloric ( approximately 2026 kJ each), of equal volume, and given in random order. Resting energy expenditure and macronutrient oxidation rates were measured and calculated in the fasting state and every 1 hour for 3 hours after meal consumption. Diet-induced thermogenesis was not significantly different between lean and obese subjects after consumption of either the protein-rich (P = .59) or the fat-rich meal (P = .68). Diet-induced thermogenesis was significantly higher (by almost 3-fold) after consumption of the protein-rich meal in comparison with the fat-rich meal in both study groups. In addition, no significant differences in macronutrient oxidation rates were found between lean and obese women after the test meals. The results indicate that DIT is higher after protein intake than after fat intake in both lean and obese participants; however, DIT and macronutrient oxidation rate are not different between lean and obese subjects after consumption of either a protein-rich or a fat-rich meal. Over the long term, a low DIT after regular or frequent fat intake may contribute to the development and maintenance of obesity.

Ursolic Acid Inhibits Leucine-Stimulated mTORC1 Signaling by Suppressing mTOR Localization to Lysosome

PubMed Central

Ou, Xiang; Liu, Meilian; Luo, Hairong; Dong, Lily Q.; Liu, Feng

2014-01-01

Ursolic acid (UA), a pentacyclic triterpenoid widely found in medicinal herbs and fruits, has been reported to possess a wide range of beneficial properties including anti-hyperglycemia, anti-obesity, and anti-cancer. However, the molecular mechanisms underlying the action of UA remain largely unknown. Here we show that UA inhibits leucine-induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in C2C12 myotubes. The UA-mediated inhibition of mTORC1 is independent of Akt, tuberous sclerosis complex 1/2 (TSC1/2), and Ras homolog enriched in brain (Rheb), suggesting that UA negatively regulates mTORC1 signaling by targeting at a site downstream of these mTOR regulators. UA treatment had no effect on the interaction between mTOR and its activator Raptor or inhibitor Deptor, but suppressed the binding of RagB to Raptor and inhibited leucine-induced mTOR lysosomal localization. Taken together, our study identifies UA as a direct negative regulator of the mTORC1 signaling pathway and suggests a novel mechanism by which UA exerts its beneficial function. PMID:24740400

Expression and regulation of glucocorticoid-induced leucine zipper in the developing anterior pituitary gland.

PubMed

Ellestad, Laura E; Malkiewicz, Stefanie A; Guthrie, H David; Welch, Glenn R; Porter, Tom E

2009-02-01

The expression profile of glucocorticoid-induced leucine zipper (GILZ) in the anterior pituitary during the second half of embryonic development in the chick is consistent with in vivo regulation by circulating corticosteroids. However, nothing else has been reported about the presence of GILZ in the neuroendocrine system. We sought to characterize expression and regulation of GILZ in the chicken embryonic pituitary gland and determine the effect of GILZ overexpression on anterior pituitary hormone levels. Pituitary GILZ mRNA levels increased during embryogenesis to a maximum on the day of hatch, and decreased through the first week after hatch. GILZ expression was rapidly upregulated by corticosterone in embryonic pituitary cells. To determine whether GILZ regulates hormone gene expression in the developing anterior pituitary, we overexpressed GILZ in embryonic pituitary cells and measured mRNA for the major pituitary hormones. Exogenous GILZ increased prolactin mRNA above basal levels, but not as high as that in corticosterone-treated cells, indicating that GILZ may play a small role in lactotroph differentiation. The largest effect we observed was a twofold increase in FSH beta subunit in cells transfected with GILZ but not treated with corticosterone, suggesting that GILZ may positively regulate gonadotroph development in a manner not involving glucocorticoids. In conclusion, this is the first report to characterize avian GILZ and examine its regulation in the developing neuroendocrine system. We have shown that GILZ is upregulated by glucocorticoids in the embryonic pituitary gland and may regulate expression of several pituitary hormones.

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

PubMed Central

Wendt, H.; Dürr, E.; Thomas, R. M.; Przybylski, M.; Bosshard, H. R.

1995-01-01

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains. PMID:8520482

Gene Isolation Using Degenerate Primers Targeting Protein Motif: A Laboratory Exercise

ERIC Educational Resources Information Center

Yeo, Brandon Pei Hui; Foong, Lian Chee; Tam, Sheh May; Lee, Vivian; Hwang, Siaw San

2018-01-01

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the…

Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice.

PubMed

Suzuki, Yohei; Sato, Tadashi; Sugimoto, Masataka; Baskoro, Hario; Karasutani, Keiko; Mitsui, Aki; Nurwidya, Fariz; Arano, Naoko; Kodama, Yuzo; Hirano, Shin-Ichi; Ishigami, Akihito; Seyama, Kuniaki; Takahashi, Kazuhisa

2017-10-07

Chronic obstructive pulmonary disease (COPD) is predominantly a cigarette smoke (CS)-triggered disease with features of chronic systemic inflammation. Oxidants derived from CS can induce DNA damage and stress-induced premature cellular senescence in the respiratory system, which play significant roles in COPD. Therefore, antioxidants should provide benefits for the treatment of COPD; however, their therapeutic potential remains limited owing to the complexity of this disease. Recently, molecular hydrogen (H 2 ) has been reported as a preventive and therapeutic antioxidant. Molecular H 2 can selectively reduce hydroxyl radical accumulation with no known side effects, showing potential applications in managing oxidative stress, inflammation, apoptosis, and lipid metabolism. However, there have been no reports on the efficacy of molecular H 2 in COPD patients. In the present study, we used a mouse model of COPD to investigate whether CS-induced histological damage in the lungs could be attenuated by administration of molecular H 2 . We administered H 2 -rich pure water to senescence marker protein 30 knockout (SMP30-KO) mice exposed to CS for 8 weeks. Administration of H 2 -rich water attenuated the CS-induced lung damage in the SMP30-KO mice and reduced the mean linear intercept and destructive index of the lungs. Moreover, H 2 -rich water significantly restored the static lung compliance in the CS-exposed mice compared with that in the CS-exposed H 2 -untreated mice. Moreover, treatment with H 2 -rich water decreased the levels of oxidative DNA damage markers such as phosphorylated histone H2AX and 8-hydroxy-2'-deoxyguanosine, and senescence markers such as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1, and β-galactosidase in the CS-exposed mice. These results demonstrated that H 2 -rich pure water attenuated CS-induced emphysema in SMP30-KO mice by reducing CS-induced oxidative DNA damage and premature cell senescence in the lungs. Our

Investigation of L-leucine in reducing the moisture-induced deterioration of spray-dried salbutamol sulfate power for inhalation.

PubMed

Li, Liang; Leung, Sharon Shui Yee; Gengenbach, Thomas; Yu, Jiaqi; Gao, Ge Fiona; Tang, Patricia; Zhou, Qi Tony; Chan, Hak-Kim

2017-09-15

The aim of this study was to investigate the ability of L-leucine (LL) in preventing moisture-induced deterioration in the in vitro aerosolization performance of spray-dried (SD) salbutamol sulfate (SS). Increasing mass fraction of LL (5-80%) were co-spray dried with SS, and the physicochemical properties of the powders were characterized by laser diffraction, X-ray powder diffraction (XRD) and dynamic vapour sorption (DVS). Furthermore, the surface morphology and chemistry of fine particles was analyzed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The in vitro aerosolization performance of powders stored at different relative humidity (RH) was evaluated by a next generation impactor (NGI). The SD SS powders were moderately hygroscopic and amorphous, of which the uptake of moisture upon storage caused a drop in the aerosolization performance. The results showed that 40% (w/w) LL was sufficient to eliminate the effect of moisture on the aerosolization performance at 60% RH. The formulation containing 40% (w/w) LL also maximized the aerosolization performance of SD SS powders (stored in desiccator) with the emitted fraction being 90.0±1.8%, and the fine particle fraction based on the recovered dose (FPF recovered ) and emitted dose (FPF emitted ) being 78.0±3.7% and 86.6±2.9%, respectively. The underlying mechanisms were that the crystalline LL increased the degree of particle surface corrugation, and reduced particle fusion and cohesiveness to facilitate dispersion. However, there is still a great challenge to prevent the moisture-induced deterioration in the aerosolization performance at 75% RH due to the recrystallization of SD SS. In conclusion, LL is a potential excipient for reducing moisture-induced deterioration in the aerosolization performance of SD amorphous powders, but still has drawbacks in preventing the recrystallization-induced deterioration. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana).

PubMed

Baurens, Franc-Christophe; Bocs, Stéphanie; Rouard, Mathieu; Matsumoto, Takashi; Miller, Robert N G; Rodier-Goud, Marguerite; MBéguié-A-MBéguié, Didier; Yahiaoui, Nabila

2010-07-16

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. A

Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana)

PubMed Central

2010-01-01

Background Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Results Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M

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.

Effects of free leucine supplementation and resistance training on muscle strength and functional status in older adults: a randomized controlled trial

PubMed Central

Trabal, Joan; Forga, Maria; Leyes, Pere; Torres, Ferran; Rubio, Jordi; Prieto, Esther; Farran-Codina, Andreu

2015-01-01

Objective To assess the effect of free leucine supplementation combined with resistance training versus resistance training only on muscle strength and functional status in older adults. Methods This was a randomized, double-blind, placebo-controlled, parallel study with two intervention groups. Thirty older adults were randomly assigned to receive either 10 g leucine/day (leucine group [LG], n=15) or a placebo (control group [CG], n=15), plus resistance training over a 12-week period. Maximal overcoming isometric leg strength, functional status, nutritional status, body composition, health-related quality of life, depression, and dietary intake were assessed at 4 and 12 weeks. Missing data at 12 weeks were handled using mixed models for repeated measurements for data imputation. Results Twenty-four subjects completed the 4-week assessment and eleven completed the 12-week intervention. Clinically significant gains were found in isometric leg strength at both assessment time points. Analysis of the effect size also showed how participants in LG outperformed those in CG for chair stands and the timed up and go test. No significant changes were observed for the rest of the outcomes. Conclusion Our combined analysis showed moderate changes in isometric leg muscle strength and certain components of functional status. The magnitude of changes found on these outcomes should be qualified as a positive effect of the concomitant intervention. PMID:25926725

Effects of free leucine supplementation and resistance training on muscle strength and functional status in older adults: a randomized controlled trial.

PubMed

Trabal, Joan; Forga, Maria; Leyes, Pere; Torres, Ferran; Rubio, Jordi; Prieto, Esther; Farran-Codina, Andreu

2015-01-01

To assess the effect of free leucine supplementation combined with resistance training versus resistance training only on muscle strength and functional status in older adults. This was a randomized, double-blind, placebo-controlled, parallel study with two intervention groups. Thirty older adults were randomly assigned to receive either 10 g leucine/day (leucine group [LG], n=15) or a placebo (control group [CG], n=15), plus resistance training over a 12-week period. Maximal overcoming isometric leg strength, functional status, nutritional status, body composition, health-related quality of life, depression, and dietary intake were assessed at 4 and 12 weeks. Missing data at 12 weeks were handled using mixed models for repeated measurements for data imputation. Twenty-four subjects completed the 4-week assessment and eleven completed the 12-week intervention. Clinically significant gains were found in isometric leg strength at both assessment time points. Analysis of the effect size also showed how participants in LG outperformed those in CG for chair stands and the timed up and go test. No significant changes were observed for the rest of the outcomes. Our combined analysis showed moderate changes in isometric leg muscle strength and certain components of functional status. The magnitude of changes found on these outcomes should be qualified as a positive effect of the concomitant intervention.

Rapamycin blocks leucine-induced protein synthesis by suppressing mTORC1 activation in skeletal muscle of neonatal pigs

USDA-ARS?s Scientific Manuscript database

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...

Light Regulation of Gibberellin Biosynthesis and Mode of Action.

PubMed

García-Martinez, José Luis; Gil, Joan

2001-12-01

Some phenotypic effects produced in plants by light are very similar to those induced by hormones. In this review, the light-gibberellin (GA) interaction in germination, de-etiolation, stem growth, and tuber formation (process regulated by GAs) are discussed. Germination of lettuce and Arabidopsis seeds depends on red irradiation (R), which enhances the expression of GA 3-oxidase genes (GA3ox) and leads to an increase in active GA content. De-etiolation of pea seedling alters the expression of GA20ox and GA3ox genes and induces a rapid decrease of GA1 content. Stem growth of green plants is also affected by diverse light irradiation characteristics. Low light intensity increases stem elongation and active GA content in pea and Brassica. Photoperiod controls active GA levels in long-day rosette (spinach and Silene) and in woody plants (Salix and hybrid aspen) by regulating different steps of GA biosynthesis, mainly through transcript levels of GA20ox and GA3ox genes. Light modulation of stem elongation in light-grown plants is controlled by phytochrome, which modifies GA biosynthesis and catabolism (tobacco, potato, cowpea, Arabidopsis) and GA-response (pea, cucumber, Arabidopsis). In Arabidopsis and tobacco, ATH1 (a gene encoding an homeotic transcription factor) is a positive mediator of a phyB-specific signal transduction cascade controlling GA levels by regulating the expression of GA20ox and GA3ox. Tuber formation in potato is controlled by photoperiod (through phyB) and GAs. Inductive short-day conditions alter the diurnal rhythm of GA20ox transcript abundance, and increases the expression of a new protein (PHOR1) that plays a role in the photoperiod-GA interaction.

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.

PCR Cloning of Partial "nbs" Sequences from Grape ("Vitis aestivalis" Michx)

ERIC Educational Resources Information Center

Chang, Ming-Mei; DiGennaro, Peter; Macula, Anthony

2009-01-01

Plants defend themselves against pathogens via the expressions of disease resistance (R) genes. Many plant R gene products contain the characteristic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. There are highly conserved motifs within the NBS domain which could be targeted for polymerase chain reaction (PCR) cloning of R…

The Leucine Incorporation Method Estimates Bacterial Growth Equally Well in Both Oxic and Anoxic Lake Waters

PubMed Central

Bastviken, David; Tranvik, Lars

2001-01-01

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O2 contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O2 contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O2 contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O2-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace. PMID:11425702

R-spondin3-LGR4 signaling protects hepatocytes against DMOG-induced hypoxia/reoxygenation injury through activating β-catenin.

PubMed

Liu, Shiying; Yin, Yue; Yu, Ruili; Li, Yin; Zhang, Weizhen

2018-04-30

Leucine-rich repeat G-protein-coupled receptor 4 (LGR4) and its ligands R-spondin1-4 (Rspos) have been vastly investigated in embryonic development. The biological functions of Rspos-LGR4 system in liver remains largely unknown. Here, we explored whether it protects hepatocytes against hypoxia/reoxygenation (H/R) induced damage. H/R injury was induced by dimethyloxalylglycine (DMOG) in AML12 cells and the effects of Rspo3 on cell proliferation and apoptosis were assessed. Specific shRNAs were used to interfere LGR4 or β-catenin. DMOG caused hepatocytes damage evidenced by increase in HIF-1α, cell death and apoptosis genes p27 and Bax, with concurrent decrease of cell proliferation genes PCNA and CyclinD1. Of all the Rspos, Rspo3 is predominantly expressed in AML12 hepatocytes. Importantly, Rspo3 demonstrated an alteration in a manner similar to proliferation-related genes during H/R injury. Rspo3 pretreatment rendered hepatocytes less vulnerable to DMOG induced H/R injury. Ablation of LGR4 using shRNA attenuated the protective effects of Rspo3. Wnt3a also protected AML12 cells from damages caused by H/R, showing enhanced proliferation activity. Notably, knockdown of β-catenin in hepatocytes completely abolished the effect of Rspo3 pretreatment on the expression levels of PCNA and CyclinD1. Rspo3-LGR4 axis protects hepatocytes from H/R injury via activating β-catenin. Copyright © 2018. Published by Elsevier Inc.

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

Repeat induced abortions in Georgia, characteristics of women with multiple pregnancy terminations: secondary analysis of the Reproductive Health Survey 2010.

PubMed

Pestvenidze, Ekaterine; Berdzuli, Nino; Lomia, Nino; Gagua, Tinatin; Umikashvili, Lia; Stray-Pedersen, Babill

2016-10-01

To examine the multi-faceted characteristics of women with repeat induced abortions and assess post-abortion family planning service provision in Georgia. We performed secondary analysis of the data from the Georgian Reproductive Health Survey 2010. A logistic regression model was used to assess the socio-demographic and behavioral factors, contraceptive practices in relation to repeat induced abortions for 2203 women of reproductive age with at least one induced abortion. The Chi-Square test was used to evaluate provision of post-abortion family planning services. Among the targeted women, 70% (n=1539) had repeat induced abortions. The odds of terminating pregnancy raised exponentially with age (OR 3.12, 95% CI: 2.11-4.61), number of complete pregnancies (3 vs. 0-1 complete pregnancies: OR 3.25, 95% CI: 2.36-4.48) and lower education (OR 1.38, 95% CI: 1.10-1.73). The current use of contraception had a protective effect on the occurrence of repeat induced abortions (OR 0.69, 95% CI: 0.53-0.89 for modern and OR 0.68, 95% CI: 0.50-0.92 for traditional methods). The contraceptive counseling and family planning method was provided only to 32% and 6% of post-abortion women, respectively before discharge from the clinic. Repeat induced abortions were found to be significantly more common (P<0.05) among women who did not receive any post-abortion contraceptive at the site of care (n=1627/1929) compared to those who left the abortion facility with family planning method (n=94/125). Low education, higher age, high parity and non-usage of contraceptives carry an increased risk of repeat induced abortions. Post-abortion family planning service delivery is limited in Georgia. Mandating provision of universal post-abortion contraception at the sites of care has a potential to reduce repeat induced abortions and should become a standard of practice for all clinics providing abortion services in Georgia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A gibberellin-stimulated transcript, OsGASR1, controls seedling growth and α-amylase expression in rice.

PubMed

Lee, Sang-Choon; Kim, Soo-Jin; Han, Soon-Ki; An, Gynheung; Kim, Seong-Ryong

2017-07-01

From a T-DNA-tagging population in rice, we identified OsGASR1 (LOC_Os03g55290), a member of the GAST (gibberellin (GA)-Stimulated Transcript) family that is induced by salt stress and ABA treatment. This gene was highly expressed in the regions of cell proliferation and panicle development, as revealed by a GUS assay of the mutant line. In the osgasr1 mutants, the second leaf blades were much longer than those of the segregating wild type due to an increase in cell length. In addition, five α-amylase genes were up-regulated in the mutants, implying that OsGASR1 is a negative regulator of those genes. These results suggest that OsGASR1 plays important roles in seedling growth and α-amylase gene expression. Copyright © 2017 Elsevier GmbH. All rights reserved.

Molecular cloning, characterization, and functional analysis of pigeon (Columba livia) Toll-like receptor 5.

PubMed

Xiong, Dan; Song, Li; Pan, Zhiming; Jiao, Xinan

2018-06-26

Toll-like receptors (TLRs) are pattern recognition receptors that are vital for the recognition of pathogen-associated molecular patterns. TLR5 is responsible for the recognition of bacterial flagellin to induce the NF-κB activation and innate immune responses. In this study, we cloned and identified the TLR5 gene from the King pigeon (Columba livia) designated as PiTLR5. Full-length PiTLR5 cDNA (2583 bp) encoded an 860-amino acid protein containing a signal peptide sequence, 10 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. Pigeon TLR5 mRNA expression was quantified by performing quantitative real-time PCR (qRT-PCR), which showed that PiTLR5 was broadly expressed in all examined tissues, with the highest expression in the liver, peripheral blood mononuclear cells, and spleen. PiTLR5-mediated innate immune responses were measured by determining its effects on NF-κB activation and cytokine expression. The results showed that HEK293T cells transfected with PiTLR5 robustly activated the NF-κB response to flagellin, but not other TLR stimuli, and induced significant upregulation of IL-1β, IL-8, TNF-α, and IFN-γ, indicating that PiTLR5 is a functional TLR5 homolog. Additionally, following flagellin stimulation of pigeon splenic lymphocytes, the levels of TLR5, NF-κB, IL-6, IL-8, CCL5, and IFN-γ mRNA, assessed using qRT-PCR, were significantly upregulated. Besides, TLR5 knockdown resulted in the significantly downregulated expression of NF-κB and related cytokines/chemokines. Triggering pigeon TLR5 contributes to significant upregulation of inflammatory cytokines and chemokines, suggesting that pigeon TLR5 plays an important role in the innate immune responses.

Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea.

PubMed

Foo, Eloise; McAdam, Erin L; Weller, James L; Reid, James B

2016-04-01

The regulation of arbuscular mycorrhizal development and nodulation involves complex interactions between the plant and its microbial symbionts. In this study, we use the recently identified ethylene-insensitive ein2 mutant in pea (Pisum sativum L.) to explore the role of ethylene in the development of these symbioses. We show that ethylene acts as a strong negative regulator of nodulation, confirming reports in other legumes. Minor changes in gibberellin1 and indole-3-acetic acid levels in ein2 roots appear insufficient to explain the differences in nodulation. Double mutants produced by crosses between ein2 and the severely gibberellin-deficient na and brassinosteroid-deficient lk mutants showed increased nodule numbers and reduced nodule spacing compared with the na and lk single mutants, but nodule numbers and spacing were typical of ein2 plants, suggesting that the reduced number of nodules innaandlkplants is largely due to the elevated ethylene levels previously reported in these mutants. We show that ethylene can also negatively regulate mycorrhizae development when ethylene levels are elevated above basal levels, consistent with a role for ethylene in reducing symbiotic development under stressful conditions. In contrast to the hormone interactions in nodulation, ein2 does not override the effect of lk or na on the development of arbuscular mycorrhizae, suggesting that brassinosteroids and gibberellins influence this process largely independently of ethylene. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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

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. 

Thermodynamic analysis of the heterodimerization of leucine zippers of Jun and Fos transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seldeen, Kenneth L.; McDonald, Caleb B.; Deegan, Brian J.

2008-10-31

Jun and Fos are components of the AP1 family of transcription factors and bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. Here, using the powerful technique of isothermal titration calorimetry, we characterize the thermodynamics of heterodimerization of leucine zippers of Jun and Fos. Our data suggest that the heterodimerization of leucine zippers is driven by enthalpic forces with unfavorable entropy change at physiological temperatures. Furthermore, the basic regions appear to modulate the heterodimerization of leucine zippers and may undergo atmore » least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain {alpha}-helical conformations in isolation and that the formation of the native coiled-coil {alpha}-helical dimer is attained through a coupled folding-dimerization mechanism.« less

A leu-rich repeat receptor-like protein kinase, FaRIPK1, interacts with the ABA receptor, FaABAR, to regulate fruit ripening in strawberry.

PubMed

Hou, Bing-Zhu; Xu, Cheng; Shen, Yuan-Yue

2018-03-24

Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA); however, its exact molecular mechanisms are yet not fully understood. In this study, a predicted leu-rich repeat (LRR) receptor-like kinase in strawberry, red-initial protein kinase 1 (FaRIPK1), was screened and, using a yeast two-hybrid assay, was shown to interact with a putative ABA receptor, FaABAR. This association was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation assays, and shown to occur in the nucleus. Expression analysis by real-time PCR showed that FaRIPK1 is expressed in roots, stems, leaves, flowers, and fruit, with a particularly high expression in white fruit at the onset of coloration. Down-regulation of FaRIPK1 expression in strawberry fruit, using Tobacco rattle virus-induced gene silencing, inhibited ripening, as evidenced by suppression of ripening-related physiological changes and reduced expression of several genes involved in softening, sugar content, pigmentation, and ABA biosynthesis and signaling. The yeast-expressed LRR and STK (serine/threonine protein kinase) domains of FaRIPK1 bound ABA and showed kinase activity, respectively. A fruit disc-incubation test revealed that FaRIPK1 expression was induced by ABA and ethylene. The synergistic action of FaRIPK1 with FaABAR in regulation of strawberry fruit ripening is discussed.

Autoimmune encephalitis associated with voltage-gated potassium channels-complex and leucine-rich glioma-inactivated 1 antibodies - a national cohort study.

PubMed

Celicanin, M; Blaabjerg, M; Maersk-Moller, C; Beniczky, S; Marner, L; Thomsen, C; Bach, F W; Kondziella, D; Andersen, H; Somnier, F; Illes, Z; Pinborg, L H

2017-08-01

The aim of this study was to describe clinical and paraclinical characteristics of all Danish patients who tested positive for anti-voltage-gated potassium channels (VGKC)-complex, anti-leucine-rich glioma-inactivated 1 (LGI1) and anti-contactin-associated protein-2 antibodies in the serum/cerebrospinal fluid between 2009 and 2013 with follow-up interviews in 2015 and 2016. We evaluated antibody status, symptoms leading to testing, course of disease, suspected diagnosis and time of admission as well as diagnosis and treatment. All magnetic resonance imaging, electroencephalography and 18 F-fluorodeoxyglucose positron emission tomography scans were re-evaluated by experts in the field. A total of 28/192 patients tested positive for VGKC-complex antibodies by radioimmunoassay and indirect immunofluorescence; 17 had antibodies to LGI1 and 6/7 of the available cerebrospinal fluids from these patients were seropositive. These 17 patients all had a clinical phenotype appropriate to LGI1 antibodies. The remaining 11 were LGI1 negative (n = 4) or not tested (n = 7). Of these, two had a phenotype consistent with limbic encephalitis. The remaining phenotypes were Guillain-Barré syndrome, Creutzfeldt-Jakob disease, neuromyotonia and anti-N-methyl-D-aspartate receptor encephalitis. Magnetic resonance imaging abnormalities were demonstrated in 69% of the LGI1-positive patients. Two patients with normal magnetic resonance imaging demonstrated temporal lobe hypermetabolism using 18 F-fluorodeoxyglucose positron emission tomography. Abnormal electroencephalography recordings were found in 86% of the patients. Upon follow-up (median 3.2 years), the median modified Rankin Scale score of anti-LGI1-positive patients was 2 and only two patients reported seizures in the past year. Patients diagnosed with anti-LGI1 autoimmune encephalitis increased significantly from 2009 to 2014, probably due to increased awareness. In contrast to seropositive anti-VGKC-complex patients, all anti-LGI1

Intrathecal Infusion of Hydrogen-Rich Normal Saline Attenuates Neuropathic Pain via Inhibition of Activation of Spinal Astrocytes and Microglia in Rats

PubMed Central

Sun, Xuejun; Xiang, Zhenghua; Yang, Liqun; Huang, Shengdong; Lu, Zhijie; Sun, Yuming; Yu, Wei-Feng

2014-01-01

Background Reactive oxygen and nitrogen species are key molecules that mediate neuropathic pain. Although hydrogen is an established antioxidant, its effect on chronic pain has not been characterized. This study was to investigate the efficacy and mechanisms of hydrogen-rich normal saline induced analgesia. Methodology/Principal findings In a rat model of neuropathic pain induced by L5 spinal nerve ligation (L5 SNL), intrathecal injection of hydrogen-rich normal saline relieved L5 SNL-induced mechanical allodynia and thermal hyperalgesia. Importantly, repeated administration of hydrogen-rich normal saline did not lead to tolerance. Preemptive treatment with hydrogen-rich normal saline prevented development of neuropathic pain behavior. Immunofluorochrome analysis revealed that hydrogen-rich normal saline treatment significantly attenuated L5 SNL-induced increase of 8-hydroxyguanosine immunoreactive cells in the ipsilateral spinal dorsal horn. Western blot analysis of SDS/PAGE-fractionated tyrosine-nitrated proteins showed that L5 SNL led to increased expression of tyrosine-nitrated Mn-containing superoxide dismutase (MnSOD) in the spinal cord, and hydrogen-rich normal saline administration reversed the tyrosine-nitrated MnSOD overexpression. We also showed that the analgesic effect of hydrogen-rich normal saline was associated with decreased activation of astrocytes and microglia, attenuated expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord. Conclusion/Significance Intrathecal injection of hydrogen-rich normal saline produced analgesic effect in neuropathic rat. Hydrogen-rich normal saline-induced analgesia in neuropathic rats is mediated by reducing the activation of spinal astrocytes and microglia, which is induced by overproduction of hydroxyl and peroxynitrite. PMID:24857932

Mechanism of internal browning of pineapple: The role of gibberellins catabolism gene (AcGA2ox) and GAs

PubMed Central

Zhang, Qin; Rao, Xiuwen; Zhang, Lubin; He, Congcong; Yang, Fang; Zhu, Shijiang

2016-01-01

Internal browning (IB), a physiological disorder (PD) that causes severe losses in harvested pineapple, can be induced by exogenous gibberellins (GAs). Over the years, studies have focused on roles of Gibberellin 2-oxidase (GA2oxs), the major GAs catabolic enzyme in plants, in the regulation of changes in morphology or biomass. However, whether GA2oxs could regulate PD has not been reported. Here, a full-length AcGA2ox cDNA was isolated from pineapple, with the putative protein sharing 23.59% to 72.92% identity with GA2oxs from five other plants. Pineapples stored at 5 °C stayed intact, while those stored at 20 °C showed severe IB. Storage at 5 °C enhanced AcGA2ox expression and decreased levels of a GAs (GA4) ‘compared with storage at 20 °C. However, at 20 °C, exogenous application of abscisic acid (ABA) significantly suppressed IB. ABA simultaneously upregulated AcGA2ox and reduced GA4. Ectopic expression of AcGA2ox in Arabidopsis resulted in reduced GA4, lower seed germination, and shorter hypocotyls and roots, all of which were restored by exogenous GA4/7. Moreover, in pineapple, GA4/7 upregulated polyphenol oxidase, while storage at 5 °C and ABA downregulated it. These results strongly suggest the involvement of AcGA2ox in regulation of GAs levels and a role of AcGA2ox in regulating IB. PMID:27982026

Hydrogen-rich saline ameliorates the severity of L-arginine-induced acute pancreatitis in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Han; Sun, Yan Ping; Li, Yang

2010-03-05

Molecular hydrogen, which reacts with the hydroxyl radical, has been considered as a novel antioxidant. Here, we evaluated the protective effects of hydrogen-rich saline on the L-arginine (L-Arg)-induced acute pancreatitis (AP). AP was induced in Sprague-Dawley rats by giving two intraperitoneal injections of L-Arg, each at concentrations of 250 mg/100 g body weight, with an interval of 1 h. Hydrogen-rich saline (>0.6 mM, 6 ml/kg) or saline (6 ml/kg) was administered, respectively, via tail vein 15 min after each L-Arg administration. Severity of AP was assessed by analysis of serum amylase activity, pancreatic water content and histology. Samples of pancreasmore » were taken for measuring malondialdehyde and myeloperoxidase. Apoptosis in pancreatic acinar cell was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL). Expression of proliferating cell nuclear antigen (PCNA) and nuclear factor kappa B (NF-{kappa}B) were detected with immunohistochemistry. Hydrogen-rich saline treatment significantly attenuated the severity of L-Arg-induced AP by ameliorating the increased serum amylase activity, inhibiting neutrophil infiltration, lipid oxidation and pancreatic tissue edema. Moreover, hydrogen-rich saline treatment could promote acinar cell proliferation, inhibit apoptosis and NF-{kappa}B activation. These results indicate that hydrogen treatment has a protective effect against AP, and the effect is possibly due to its ability to inhibit oxidative stress, apoptosis, NF-{kappa}B activation and to promote acinar cell proliferation.« less

Autoimmune encephalitis with anti-leucine-rich glioma-inactivated 1 or anti-contactin-associated protein-like 2 antibodies (formerly called voltage-gated potassium channel-complex antibodies).

PubMed

Bastiaansen, Anna E M; van Sonderen, Agnes; Titulaer, Maarten J

2017-06-01

Twenty years since the discovery of voltage-gated potassium channel (VGKC)-related autoimmunity; it is currently known that the antibodies are not directed at the VGKC itself but to two closely associated proteins, anti-leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-like 2 (Caspr2). Antibodies to LGI1 and Caspr2 give well-described clinical phenotypes. Anti-LGI1 encephalitis patients mostly have limbic symptoms, and anti-Caspr2 patients have variable syndromes with both central and peripheral symptoms. A large group of patients with heterogeneous symptoms are VGKC positive but do not have antibodies against LGI1 or Caspr2. The clinical relevance of VGKC positivity in these 'double-negative' patients is questionable. This review focusses on these three essentially different subgroups. The clinical phenotypes of anti-LGI1 encephalitis and anti-Caspr2 encephalitis have been described in more detail including data on treatment and long-term follow-up. A specific human leukocyte antigen (HLA) association was found in nontumor anti-LGI1 encephalitis, but not clearly in those with tumors. There has been increasing interest in the VGKC patients without LGI1/Caspr2 antibodies questioning its relevance in clinical practice. Anti-LGI1 encephalitis and anti-Caspr2 encephalitis are separate clinical entities. Early recognition and treatment is necessary and rewarding. The term VGKC-complex antibodies, lumping patients with anti-LGI1, anti-Caspr2 antibodies or lacking both, should be considered obsolete.

Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor

PubMed Central

2011-01-01

Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps) synthesizes PHA copolymer containing 3-hydroxybutyrate (3HB) and a small amount (0.5 mol%) of 3-hydroxy-4-methylvalerate (3H4MV) from fructose as a carbon source. In this study, enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural and self-producible precursor. We found that the incorporation of 3H4MV was enhanced by the supplementation of excess amount (10 g/L) of leucine in the culture medium. This finding indicates that 3H4MV can be derived from leucine. To increase metabolic flux to leucine biosynthesis in the host strain by eliminating the feedback inhibition, the cells were subjected to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and leucine analog resistant mutants were generated. The mutants showed statistically higher 3H4MV fraction than the parent strain without supplementing leucine. Additionally, by supplying excess amount of leucine, the mutants synthesized 3HB-based PHA copolymer containing 3.1 mol% 3H4MV and 1.2 mol% 3-hydroxyvalerate (3HV) as minor constituents, which significantly affected the thermal properties of the copolymer. This study demonstrates that it is possible to enhance the monomer supply of 3H4MV into PHA by manipulating leucine metabolism. PMID:21906338

Examination of two lowland rice cultivars reveals that gibberellin-dependent early response to submergence is not necessarily mediated by ethylene.

PubMed

Dubois, Vincent; Moritz, Thomas; García-Martínez, José L

2011-01-01

Using two lowland rice (Oryza sativa L.) cultivars we found that in both cases submerged-induced elongation early after germination depends on gibberellins (GAs). Submergence increases the content of the active GA 1 by enhancing the expression of GA biosynthesis genes, thus facilitating the seedlings to escape from the water and preventing asphyxiation. However, the two cultivars differ in their response to ethylene. The cultivar Senia (short), by contrast to cultivar Bomba (tall), does not elongate after ethylene application, and submerged-induced elongation is not negated by an inhibitor of ethylene perception. Also, while ethylene emanation in Senia is not altered by submergence, Bomba seedlings emanate more ethylene upon de-submergence, associated with enhanced expression of the ethylene biosynthesis gene OsACS5. The cultivar Senia thus allows the possibility of clarifying the role of ethylene and other factors as triggers of GA biosynthesis enhancement in rice seedlings under submergence.

Examination of two lowland rice cultivars reveals that gibberellin-dependent early response to submergence is not necessarily mediated by ethylene

PubMed Central

Dubois, Vincent; Moritz, Thomas

2011-01-01

Using two lowland rice (Oryza sativa L.) cultivars we found that in both cases submerged-induced elongation early after germination depends on gibberellins (GAs). Submergence increases the content of the active GA1 by enhancing the expression of GA biosynthesis genes, thus facilitating the seedlings to escape from the water and preventing asphyxiation. However, the two cultivars differ in their response to ethylene. The cultivar Senia (short), by contrast to cultivar Bomba (tall), does not elongate after ethylene application, and submerged-induced elongation is not negated by an inhibitor of ethylene perception. Also, while ethylene emanation in Senia is not altered by submergence, Bomba seedlings emanate more ethylene upon desubmergence, associated with enhanced expression of the ethylene biosynthesis gene OsACS5. The cultivar Senia thus allows the possibility of clarifying the role of ethylene and other factors as triggers of GA biosynthesis enhancement in rice seedlings under submergence. PMID:21224726

Sensitivity of Female Inbreds of Cucumis sativus to Sex Reversion by Gibberellin.

PubMed

Shifriss, O; George, W L

1964-03-27

Two female inbred cucumbers were developed by substituting gene Acr for acr in the genetic backgrounds of the monoecious races Marketer and Tokyo, which exhibit weak and strong male tendency respectively. Marketer females are resistant and Tokyo females are sensitive to sex reversion in response to treatments with gibberellin A(3). Resistance and sensitivity of this type appear to depend upon the genetic system which controls sex tendency.

The conformational and subcellular compartmental dance of plant NLRs during viral recognition and defense signaling

PubMed Central

Padmanabhan, Meenu S; Dinesh-Kumar, Savithramma P

2014-01-01

Plant innate immune response against viruses utilizes intracellular Nucleotide Binding domain Leucine Rich Repeat (NLR) class of receptors. NLRs recognize different viral proteins termed elicitors and initiate diverse signaling processes that induce programmed cell death (PCD) in infected cells and restrict virus spread. In this review we describe the recent advances made in the study of plant NLRs that detect viruses. We describe some of the physical and functional interactions these NLRs undertake. We elaborate on the intra-molecular and homotypic association of NLRs that function in self-regulation and activation. Nuclear role for some viral NLRs is discussed as well as the emerging importance of the RNAi pathway in regulating the NLR family. PMID:24906192

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

PubMed

Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

2015-02-01

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

PubMed

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana

PubMed Central

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts. PMID:26451798

Evolution of Alternative Adaptive Immune Systems in Vertebrates.

PubMed

Boehm, Thomas; Hirano, Masayuki; Holland, Stephen J; Das, Sabyasachi; Schorpp, Michael; Cooper, Max D

2018-04-26

Adaptive immunity in jawless fishes is based on antigen recognition by three types of variable lymphocyte receptors (VLRs) composed of variable leucine-rich repeats, which are differentially expressed by two T-like lymphocyte lineages and one B-like lymphocyte lineage. The T-like cells express either VLRAs or VLRCs of yet undefined antigen specificity, whereas the VLRB antibodies secreted by B-like cells bind proteinaceous and carbohydrate antigens. The incomplete VLR germline genes are assembled into functional units by a gene conversion-like mechanism that employs flanking variable leucine-rich repeat sequences as templates in association with lineage-specific expression of cytidine deaminases. B-like cells develop in the hematopoietic typhlosole and kidneys, whereas T-like cells develop in the thymoid, a thymus-equivalent region at the gill fold tips. Thus, the dichotomy between T-like and B-like cells and the presence of dedicated lymphopoietic tissues emerge as ancestral vertebrate features, whereas the somatic diversification of structurally distinct antigen receptor genes evolved independently in jawless and jawed vertebrates.

Variation and Evolution in the Glutamine-Rich Repeat Region of Drosophila Argonaute-2

PubMed Central

Palmer, William H.; Obbard, Darren J.

2016-01-01

RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel. PMID:27317784

Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis).

PubMed

Schmiderer, Corinna; Grausgruber-Gröger, Sabine; Grassi, Paolo; Steinborn, Ralf; Novak, Johannes

2010-07-01

Common sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, composed mainly of the monoterpenes 1,8-cineole, alpha-thujone, beta-thujone and camphor, is responsible for some of these effects. Gibberellins regulate diverse physiological processes in plants, such as seed germination, shoot elongation and cell division. In this study, we analyzed the effect of exogenously applied plant growth regulators, namely gibberellic acid (GA(3)) and daminozide, on leaf morphology and essential oil formation of two leaf stages during the period of leaf expansion. Essential oil content increased with increasing levels of gibberellins and decreased when gibberellin biosynthesis was blocked with daminozide. With increasing levels of gibberellins, 1,8-cineole and camphor contents increased. Daminozide blocked the accumulation of alpha- and beta-thujone. GA(3) at the highest level applied also led to a significant decrease of alpha- and beta-thujone. Monoterpene synthases are a class of enzymes responsible for the first step in monoterpene biosynthesis, competing for the same substrate geranylpyrophosphate. The levels of gene expression of the three most important monoterpene synthases in sage were investigated, 1,8-cineole synthase leading directly to 1,8-cineole, (+)-sabinene synthase responsible for the first step in the formation of alpha- and beta-thujone, and (+)-bornyl diphosphate synthase, the first step in camphor biosynthesis. The foliar application of GA(3) increased, while daminozide significantly decreased gene expression of the monoterpene synthases. The amounts of two of the end products, 1,8-cineole and camphor, were directly correlated with the levels of gene expression of the respective monoterpene synthases, indicating transcriptional control, while the formation of alpha- and beta

Overexpression of Jatropha Gibberellin 2-oxidase 6 (JcGA2ox6) Induces Dwarfism and Smaller Leaves, Flowers and Fruits in Arabidopsis and Jatropha

PubMed Central

Hu, Ying-Xiong; Tao, Yan-Bin; Xu, Zeng-Fu

2017-01-01

Gibberellins (GAs) are plant hormones that play fundamental roles in plant growth and development. Gibberellin 2-oxidase (GA2ox) plays a direct role in determining the levels of bioactive GAs by catalyzing bioactive GAs or their immediate precursors to inactive forms. In this study, a GA2ox gene, designated JcGA2ox6, was isolated from Jatropha curcas. JcGA2ox6 is expressed in all tissues of adult Jatropha, with the highest expression level in male flowers and the lowest expression level in young leaves. Overexpression of JcGA2ox6 in Arabidopsis resulted in a typical dwarf phenotype, along with late flowering, smaller leaves and flowers, shorter siliques and smaller seeds. Similarly, when JcGA2ox6 was overexpressed in Jatropha, the transgenic plants exhibited a dwarf phenotype with dark-green leaves and smaller inflorescences, flowers, fruits and seeds. However, the flowering time of Jatropha was not affected by overexpression of JcGA2ox6, unlike that in the transgenic Arabidopsis. Moreover, the number of flowers per inflorescence, the weight of 10 seeds and the seed oil content were significantly decreased in transgenic Jatropha. The results indicated that overexpression of JcGA2ox6 had a great impact on the vegetative and reproductive growth of transgenic Jatropha. Furthermore, we found that the dwarf phenotype of transgenic Jatropha was caused by a decrease in endogenous bioactive GA4, which was correlated with the degree of dwarfism. PMID:29312375

[Primary investigation of the relationship between glucocorticoid induced leucine zipper and inflammatory reaction].

PubMed

Bai, Xiang-jun; Li, Bo; Wang, Hai-ping; Yang, Zhao-hui; Li, Si-qi

2007-01-01

To investigate the mechanism of the action of glucocorticoid induced leucine zipper (GILZ) in inflammatory reaction. Human monocyte cell line THP-1 cells were divided into two groups and cultured in non-serum RPMI1640 medium.In one group the cells were treated with dexamethasone (DEX). Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by reserve transcriptase-polymerase chain reaction (RT-PCR). Protein expression of nuclear factor-KappaB (NF-KappaB) p65 and activator protein-1 (AP-1) were assessed by Western blotting. Peripheral blood of 10 trauma patients [injury severity score (ISS) >or=16 scores] were collected and the leukocytes were isolated within 24 hours after trauma. The leukocytes were divided into two groups and cultured in non-serum medium. In one group the cells were treated with DEX. Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by RT-PCR. Protein expression of NF-KappaB p65 and AP-1 were assessed by Western blotting. Stimulated by DEX, the expression of GILZ mRNA was increased both in THP-1 cells and the leukocytes of trauma patients compared with those of control groups (both P<0.01). Whereas, protein expressions of NF-KappaB p65 and AP-1 of THP-1 cells and leukocytes in peripheral blood of trauma patients were decreased in the stimulation groups compared with those of control groups (all P<0.01). The expression of GILZ gene is up-regulated by glucocorticoid. Overexpression of GILZ inhibits NF-KappaB and AP-1 activities, suggesting that GILZ possesses anti-inflammatory function.

Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

PubMed

Yano, Kenji; Aya, Koichiro; Hirano, Ko; Ordonio, Reynante Lacsamana; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

2015-02-01

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells. © 2015 American Society of Plant Biologists. All Rights Reserved.

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

Effects of abscisic acid, gibberellin, ethylene and their interactions on production of phenolic acids in salvia miltiorrhiza bunge hairy roots.

PubMed

Liang, Zongsuo; Ma, Yini; Xu, Tao; Cui, Beimi; Liu, Yan; Guo, Zhixin; Yang, Dongfeng

2013-01-01

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Phenolic acids mainly including caffeic acid, rosmarinic acid and salvianolic acid B are a group of active ingredients in S. miltiorrhiza. Abscisic acid (ABA), gibberellin (GA) and ethylene are three important phytohormones. In this study, effects of the three phytohormones and their interactions on phenolic production in S. miltiorrhiza hairy roots were investigated. The results showed that ABA, GA and ethylene were all effective to induce production of phenolic acids and increase activities of PAL and TAT in S. miltiorrhiza hairy roots. Effects of phytohormones were reversed by their biosynthetic inhibitors. Antagonistic actions between the three phytohormones played important roles in the biosynthesis of phenolic acids. GA signaling is necessary for ABA and ethylene-induced phenolic production. Yet, ABA and ethylene signaling is probably not necessary for GA3-induced phenolic production. The complex interactions of phytohormones help us reveal regulation mechanism of secondary metabolism and scale-up production of active ingredients in plants.

Effects of Abscisic Acid, Gibberellin, Ethylene and Their Interactions on Production of Phenolic Acids in Salvia miltiorrhiza Bunge Hairy Roots

PubMed Central

Xu, Tao; Cui, Beimi; Liu, Yan; Guo, Zhixin; Yang, Dongfeng

2013-01-01

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Phenolic acids mainly including caffeic acid, rosmarinic acid and salvianolic acid B are a group of active ingredients in S. miltiorrhiza. Abscisic acid (ABA), gibberellin (GA) and ethylene are three important phytohormones. In this study, effects of the three phytohormones and their interactions on phenolic production in S. miltiorrhiza hairy roots were investigated. The results showed that ABA, GA and ethylene were all effective to induce production of phenolic acids and increase activities of PAL and TAT in S. miltiorrhiza hairy roots. Effects of phytohormones were reversed by their biosynthetic inhibitors. Antagonistic actions between the three phytohormones played important roles in the biosynthesis of phenolic acids. GA signaling is necessary for ABA and ethylene-induced phenolic production. Yet, ABA and ethylene signaling is probably not necessary for GA3-induced phenolic production. The complex interactions of phytohormones help us reveal regulation mechanism of secondary metabolism and scale-up production of active ingredients in plants. PMID:24023778

Use of the [(14)C]leucine incorporation technique to measure bacterial production in river sediments and the epiphyton.

PubMed

Fischer, H; Pusch, M

1999-10-01

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [(14)C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 microM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 microM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.

Use of the [14C]Leucine Incorporation Technique To Measure Bacterial Production in River Sediments and the Epiphyton

PubMed Central

Fischer, Helmut; Pusch, Martin

1999-01-01

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [14C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 μM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 μM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined. PMID:10508068

Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway.

PubMed

Lee, Minji; Kim, Jong Hyun; Yoon, Ina; Lee, Chulho; Fallahi Sichani, Mohammad; Kang, Jong Soon; Kang, Jeonghyun; Guo, Min; Lee, Kang Young; Han, Gyoonhee; Kim, Sunghoon; Han, Jung Min

2018-06-05

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating "ON" switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an "OFF" switch by controlling GTP hydrolysis of RagB in the Rag GTPase-mTORC1 axis. The LRS-RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP-GDP cycle of the RagD-RagB pair, rather than the RagC-RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD-RagB pair can overcome the absence of the RagC-RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD-RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

PubMed

Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

2014-09-15

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-01

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

An engineered allosteric switch in leucine-zipper oligomerization.

PubMed

Gonzalez, L; Plecs, J J; Alber, T

1996-06-01

Controversy remains about the role of core side-chain packing in specifying protein structure. To investigate the influence of core packing on the oligomeric structure of a coiled coil, we engineered a GCN4 leucine zipper mutant that switches from two to three strands upon binding the hydrophobic ligands cyclohexane and benzene. In solution these ligands increased the apparent thermal stability and the oligomerization order of the mutant leucine zipper. The crystal structure of the peptide-benzene complex shows a single benzene molecule bound at the engineered site in the core of the trimer. These results indicate that coiled coils are well-suited to function as molecular switches and emphasize that core packing is an important determinant of oligomerization specificity.

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling.

PubMed

Konishi, Hirosato; Yamane, Hisakazu; Maeshima, Masayoshi; Komatsu, Setsuko

2004-12-01

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.

Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice[C][W][OPEN

PubMed Central

Tong, Hongning; Xiao, Yunhua; Liu, Dapu; Gao, Shaopei; Liu, Linchuan; Yin, Yanhai; Jin, Yun; Qian, Qian; Chu, Chengcai

2014-01-01

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA1 levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana. PMID:25371548

MADS-Box Genes and Gibberellins Regulate Bolting in Lettuce (Lactuca sativa L.)

PubMed Central

Han, Yingyan; Chen, Zijing; Lv, Shanshan; Ning, Kang; Ji, Xueliang; Liu, Xueying; Wang, Qian; Liu, Renyi; Fan, Shuangxi; Zhang, Xiaolan

2016-01-01

Bolting in lettuce is promoted by high temperature and bolting resistance is of great economic importance for lettuce production. But how bolting is regulated at the molecular level remains elusive. Here, a bolting resistant line S24 and a bolting sensitive line S39 were selected for morphological, physiological, transcriptomic and proteomic comparisons. A total of 12204 genes were differentially expressed in S39 vs. S24. Line S39 was featured with larger leaves, higher levels of chlorophyll, soluble sugar, anthocyanin and auxin, consistent with its up-regulation of genes implicated in photosynthesis, oxidation-reduction and auxin actions. Proteomic analysis identified 30 differentially accumulated proteins in lines S39 and S24 upon heat treatment, and 19 out of the 30 genes showed differential expression in the RNA-Seq data. Exogenous gibberellins (GA) treatment promoted bolting in both S39 and S24, while 12 flowering promoting MADS-box genes were specifically induced in line S39, suggesting that although GA regulates bolting in lettuce, it may be the MADS-box genes, not GA, that plays a major role in differing the bolting resistance between these two lettuce lines. PMID:28018414

Arabidopsis scaffold protein RACK1A modulates rare sugar D-allose regulated gibberellin signaling.

PubMed

Fennell, Herman; Olawin, Abdulquadri; Mizanur, Rahman M; Izumori, Ken; Chen, Jin-Gui; Ullah, Hemayet

2012-11-01

As energy sources and structural components, sugars are the central regulators of plant growth and development. In addition to the abundant natural sugars in plants, more than 50 different kinds of rare sugars exist in nature, several of which show distinct roles in plant growth and development. Recently, one of the rare sugars, D-allose, an epimer of D-glucose at C3, is found to suppress plant hormone gibberellin (GA) signaling in rice. Scaffold protein RACK1A in the model plant Arabidopsis is implicated in the GA pathway as rack1a knockout mutants show insensitivity to GA in GA-induced seed germination. Using genetic knockout lines and a reporter gene, the functional role of RACK1A in the D-allose pathway was investigated. It was found that the rack1a knockout seeds showed hypersensitivity to D-allose-induced inhibition of seed germination, implicating a role for RACK1A in the D-allose mediated suppression of seed germination. On the other hand, a functional RACK1A in the background of the double knockout mutations in the other two RACK1 isoforms, rack1b/rack1c, showed significant resistance to the D-allose induced inhibition of seed germination. The collective results implicate the RACK1A in the D-allose mediated seed germination inhibition pathway. Elucidation of the rare sugar signaling mechanism will help to advance understanding of this less studied but important cellular signaling pathway.

Arabidopsis scaffold protein RACK1A modulates rare sugar D-allose regulated gibberellin signaling

PubMed Central

Fennell, Herman; Olawin, Abdulquadri; Mizanur, Rahman M.; Izumori, Ken; Chen, Jin-Gui; Ullah, Hemayet

2012-01-01

As energy sources and structural components, sugars are the central regulators of plant growth and development. In addition to the abundant natural sugars in plants, more than 50 different kinds of rare sugars exist in nature, several of which show distinct roles in plant growth and development. Recently, one of the rare sugars, D-allose, an epimer of D-glucose at C3, is found to suppress plant hormone gibberellin (GA) signaling in rice. Scaffold protein RACK1A in the model plant Arabidopsis is implicated in the GA pathway as rack1a knockout mutants show insensitivity to GA in GA-induced seed germination. Using genetic knockout lines and a reporter gene, the functional role of RACK1A in the D-allose pathway was investigated. It was found that the rack1a knockout seeds showed hypersensitivity to D-allose-induced inhibition of seed germination, implicating a role for RACK1A in the D-allose mediated suppression of seed germination. On the other hand, a functional RACK1A in the background of the double knockout mutations in the other two RACK1 isoforms, rack1b/rack1c, showed significant resistance to the D-allose induced inhibition of seed germination. The collective results implicate the RACK1A in the D-allose mediated seed germination inhibition pathway. Elucidation of the rare sugar signaling mechanism will help to advance understanding of this less studied but important cellular signaling pathway. PMID:22951405

False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene.

PubMed

Koita, Ousmane A; Doumbo, Ogobara K; Ouattara, Amed; Tall, Lalla K; Konaré, Aoua; Diakité, Mahamadou; Diallo, Mouctar; Sagara, Issaka; Masinde, Godfred L; Doumbo, Safiatou N; Dolo, Amagana; Tounkara, Anatole; Traoré, Issa; Krogstad, Donald J

2012-02-01

We identified 480 persons with positive thick smears for asexual Plasmodium falciparum parasites, of whom 454 had positive rapid diagnostic tests (RDTs) for the histidine-rich protein 2 (HRP2) product of the hrp2 gene and 26 had negative tests. Polymerase chain reaction (PCR) amplification for the histidine-rich repeat region of that gene was negative in one-half (10/22) of false-negative specimens available, consistent with spontaneous deletion. False-negative RDTs were found only in persons with asymptomatic infections, and multiplicities of infection (MOIs) were lower in persons with false-negative RDTs (both P < 0.001). These results show that parasites that fail to produce HRP2 can cause patent bloodstream infections and false-negative RDT results. The importance of these observations is likely to increase as malaria control improves, because lower MOIs are associated with false-negative RDTs and false-negative RDTs are more frequent in persons with asymptomatic infections. These findings suggest that the use of HRP2-based RDTs should be reconsidered.

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

Increased dependence of leucine in posttraumatic sepsis: leucine/tyrosine clearance ratio as an indicator of hepatic impairment in septic multiple organ failure syndrome.