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

Requirement of the Cytosolic Interaction between PATHOGENESIS-RELATED PROTEIN10 and LEUCINE-RICH REPEAT PROTEIN1 for Cell Death and Defense Signaling in Pepper[W

PubMed Central

Choi, Du Seok; Hwang, In Sun; Hwang, Byung Kook

2012-01-01

Plants recruit innate immune receptors such as leucine-rich repeat (LRR) proteins to recognize pathogen attack and activate defense genes. Here, we identified the pepper (Capsicum annuum) pathogenesis-related protein10 (PR10) as a leucine-rich repeat protein1 (LRR1)–interacting partner. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between LRR1 and PR10 in planta. Avirulent Xanthomonas campestris pv vesicatoria infection induces PR10 expression associated with the hypersensitive cell death response. Transient expression of PR10 triggers hypersensitive cell death in pepper and Nicotiana benthamiana leaves, which is amplified by LRR1 coexpression as a positive regulator. LRR1 promotes the ribonuclease activity and phosphorylation of PR10, leading to enhanced cell death signaling. The LRR1-PR10 complex is formed in the cytoplasm, resulting in its secretion into the apoplastic space. Engineered nuclear confinement of both proteins revealed that the cytoplasmic localization of the PR10-LRR1 complex is essential for cell death–mediated defense signaling. PR10/LRR1 silencing in pepper compromises resistance to avirulent X. campestris pv vesicatoria infection. By contrast, PR10/LRR1 overexpression in Arabidopsis thaliana confers enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that the cytosolic LRR-PR10 complex is responsible for cell death–mediated defense signaling. PMID:22492811

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

PubMed Central

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

2014-01-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

PubMed

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

2014-06-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

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.

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

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

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.

CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas

PubMed Central

Kinoshita, Ayumi; Niwa, Yoshimi; Onai, Kiyoshi; Fukuzawa, Hideya; Ishiura, Masahiro

2017-01-01

The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas. PMID:28333924

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.

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.

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

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

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.

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

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.

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

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

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

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.

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.

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 .

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.

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

[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.

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.

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.

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.

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.

CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

PubMed Central

King, Jeffrey B.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Galanko, Joseph A.

2012-01-01

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent

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.

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

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.

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.

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.

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.

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

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

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.

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.

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.

Cloning of Bordetella pertussis putative outer protein D (BopD) and Leucin/Isoleucine/Valin binding protein (LivJ)

NASA Astrophysics Data System (ADS)

Öztürk, Burcu Emine Tefon

2017-04-01

Whooping cough also known as pertussis is a contagious acute upper respiratory disease primarily caused by Bordetella pertussis. It is known that this disease may be fatal especially in infants and recently, the number of pertussis cases has been increased. Despite the fact that there are numbers of acellular vaccines on the market, the current acellular vaccine compositions are inadequate for providing sustainable immunity and avoiding subclinical disease cases. Hence, exploring novel proteins with high immune protective capacities is essential to enhance the clinical efficacy of current vaccines. In this study, genes of selected immunogenic proteins via -omics studies, namely Putative outer protein D (BopD) and Leucin/Isoleucine/Valin Binding Protein (LivJ) were first cloned into pGEM-T Easy vector and transformed to into E. coli DH5α cells and then cloned into the expression vector pET-28a(+) and transformed into E. coli BL21 (DE3) cells to express the proteins.

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.

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

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

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.

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.

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

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

PubMed

Kurucz, Eva; Márkus, Róbert; Zsámboki, János; Folkl-Medzihradszky, Katalin; Darula, Zsuzsanna; Vilmos, Péter; Udvardy, Andor; Krausz, Ildikó; Lukacsovich, Tamás; Gateff, Elisabeth; Zettervall, Carl-Johan; Hultmark, Dan; Andó, István

2007-04-03

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

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

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

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

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

PubMed

Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

2016-04-01

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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

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.

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

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

The putative leucine sensor Sestrin2 is hyperphosphorylated by acute resistance exercise but not protein ingestion in human skeletal muscle.

PubMed

Zeng, Nina; D'Souza, Randall F; Sorrenson, Brie; Merry, Troy L; Barnett, Matthew P G; Mitchell, Cameron J; Cameron-Smith, David

2018-06-01

Dietary protein and resistance exercise (RE) are both potent stimuli of the mammalian target of rapamycin complex 1 (mTORC1). Sestrins1, 2, 3 are multifunctional proteins that regulate mTORC1, stimulate autophagy and alleviate oxidative stress. Of this family, Sestrin2 is a putative leucine sensor implicated in mTORC1 and AMP-dependent protein kinase (AMPK) regulation. There is currently no data examining the responsiveness of Sestrin2 to dietary protein ingestion, with or without RE. In Study 1, 16 males ingested either 10 or 20 g of milk protein concentrate (MPC) with muscle biopsies collected pre, 90 and 210 min post-beverage consumption. In Study 2, 20 males performed a bout of RE immediately followed by the consumption of 9 g of MPC or carbohydrate placebo. Analysis of Sestrins, AMPK and antioxidant responses was examined. Dietary protein ingestion did not result in Sestrin2 mobility shift. After RE, Sestrin2 phosphorylation state was significantly altered and was not further modified by post-exercise protein or carbohydrate ingestion. With RE, AMPK phosphorylation remained stable, while the mRNA expressions of several antioxidants were upregulated. Dietary protein ingestion did not affect the signalling by the family of Sestrins. With RE, Sestrin2 was hyperphosphorylated, with no further evidence of a relationship to AMPK signalling.

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.

The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" encodes an afp-like prophage possibly used for protein secretion.

PubMed

Penz, Thomas; Horn, Matthias; Schmitz-Esser, Stephan

2010-01-01

The recently sequenced genome of the obligate intracellular amoeba symbiont 'Candidatus Amoebophilus asiaticus' is unique among prokaryotic genomes due to its extremely large fraction of genes encoding proteins harboring eukaryotic domains such as ankyrin-repeats, TPR/SEL1 repeats, leucine-rich repeats, as well as F- and U-box domains, most of which likely serve in the interaction with the amoeba host. Here we provide evidence for the presence of additional proteins which are presumably presented extracellularly and should thus also be important for host cell interaction. Surprisingly, we did not find homologues of any of the well-known protein secretion systems required to translocate effector proteins into the host cell in the A. asiaticus genome, and the type six secretion systems seems to be incomplete. Here we describe the presence of a putative prophage in the A. asiaticus genome, which shows similarity to the antifeeding prophage from the insect pathogen Serratia entomophila. In S. entomophila this system is used to deliver toxins into insect hosts. This putative antifeeding-like prophage might thus represent the missing protein secretion apparatus in A. asiaticus.

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 ...

Unigenic Evolution: A Novel Genetic Method Localizes a Putative Leucine Zipper That Mediates Dimerization of the Saccharomyces Cerevisiae Regulator Gcr1p

PubMed Central

Deminoff, S. J.; Tornow, J.; Santangelo, G. M.

1995-01-01

The GCR1 gene of Saccharomyces cerevisiae encodes a transcriptional activator that complexes with Rap1p and, through UAS(RPG) elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes. To map the functionally important domains in Gcr1p, we combined multiple rounds of random mutagenesis in vitro with in vivo selection of functional genes to locate conserved, or hypomutable, regions. We name this method unigenic evolution, a statistical analysis of mutations in evolutionary variants of a single gene in an otherwise isogenic background. Examination of the distribution of 315 mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D). Dispensable N-terminal (intronic) and C-terminal portions of the evolved region of GCR1 were included in the analysis as controls and were, as expected, not hypomutable. The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains. In particular, we determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization. PMID:8601472

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

PubMed

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

2014-07-01

Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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.

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.

Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

PubMed

Rehm, Charlotte; Wurmthaler, Lena A; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S

2015-01-01

In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

PubMed Central

Rehm, Charlotte; Wurmthaler, Lena A.; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S.

2015-01-01

In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1–5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6–9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria. PMID:26695179

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 ...

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

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.

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.

Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe–host interactions

PubMed Central

Sequeira, Saannya; Kavanaugh, Devon; MacKenzie, Donald A.; Walpole, Samuel; Leclaire, Charlotte; Gunning, A. Patrick; Latousakis, Dimitrios; Willats, William G. T.; Dong, Changjiang; Juge, Nathalie

2018-01-01

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens. PMID:29507249

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

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

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

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

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.

Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense.

PubMed

Wiermer, Marcel; Cheng, Yu Ti; Imkampe, Julia; Li, Meilan; Wang, Dongmei; Lipka, Volker; Li, Xin

2012-06-01

In eukaryotic cells, transduction of external stimuli into the nucleus to induce transcription and export of mRNAs for translation in the cytoplasm is mediated by nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups). We previously reported that Arabidopsis MOS3, encoding the homolog of vertebrate Nup96, is required for plant immunity and constitutive resistance mediated by the de-regulated Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R gene snc1. In vertebrates, Nup96 is a component of the conserved Nup107-160 nuclear pore sub-complex, and implicated in immunity-related mRNA export. Here, we used a reverse genetics approach to examine the requirement for additional subunits of the predicted Arabidopsis Nup107-160 complex in plant immunity. We show that, among eight putative complex members, beside MOS3, only plants with defects in Nup160 or Seh1 are impaired in basal resistance. Constitutive resistance in the snc1 mutant and immunity mediated by TNL-type R genes also depend on functional Nup160 and have a partial requirement for Seh1. Conversely, resistance conferred by coiled coil-type immune receptors operates largely independently of both genes, demonstrating specific contributions to plant defense signaling. Our functional analysis further revealed that defects in nup160 and seh1 result in nuclear accumulation of poly(A) mRNA, and, in the case of nup160, considerable depletion of EDS1, a key positive regulator of basal and TNL-triggered resistance. These findings suggest that Nup160 is required for nuclear mRNA export and full expression of EDS1-conditioned resistance pathways in Arabidopsis. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

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.

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.

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.

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...

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.

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.

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.

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

Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

PubMed

Wagner, Ulrike; Hirzmann, Jörg; Hintz, Martin; Beck, Ewald; Geyer, Rudolf; Hobom, Gerd; Taubert, Anja; Zahner, Horst

2011-04-01

Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown. Copyright © 2011 Elsevier B.V. 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

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

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.

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

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

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.

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.

CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats.

PubMed

Grissa, Ibtissem; Vergnaud, Gilles; Pourcel, Christine

2007-07-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) constitute a particular family of tandem repeats found in a wide range of prokaryotic genomes (half of eubacteria and almost all archaea). They consist of a succession of highly conserved regions (DR) varying in size from 23 to 47 bp, separated by similarly sized unique sequences (spacer) of usually viral origin. A CRISPR cluster is flanked on one side by an AT-rich sequence called the leader and assumed to be a transcriptional promoter. Recent studies suggest that this structure represents a putative RNA-interference-based immune system. Here we describe CRISPRFinder, a web service offering tools to (i) detect CRISPRs including the shortest ones (one or two motifs); (ii) define DRs and extract spacers; (iii) get the flanking sequences to determine the leader; (iv) blast spacers against Genbank database and (v) check if the DR is found elsewhere in prokaryotic sequenced genomes. CRISPRFinder is freely accessible at http://crispr.u-psud.fr/Server/CRISPRfinder.php.

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

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

PubMed Central

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

2016-01-01

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

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.

A metal-linked gapped zipper model is proposed for the 90 kDa heat shock protein-estrogen receptor interface.

PubMed

Schwartz, J A; Mizukami, H

1991-06-01

A novel arrangement is proposed for the association of the 90 kDa heat shock protein (hsp 90) dimer and the human estrogen receptor (hER) monomer. Secondary structure analyses of the hsp 90 molecule reveal the presence of a cysteine-containing, leucine-rich, heptad repeat, which we refer to as region C. Similar analyses on the hER, at its hormone binding domain (HBD), have indicated the presence of a central subdomain bordered by 2 alpha-helical flanking segments which also display the heptad substructure. Due to its predicted potential for conformational change (1) we refer to this central subdomain as the Helix Conversion Unit or HCU. It contains an HX5C peptide and shares significant homology with the metal-binding domain of a gag-encoded HIV-LAV protein (2). We predict that, by virtue of its presence in duplicate, region C may be capable of simultaneous leucine zipper-like pairing with the hER at its flanking helices, as well as the formation of a shared CCHC-box-type metal binding link with the same hER at the putative HCU which lies in between.

Hal Is a Bacillus anthracis Heme Acquisition Protein

PubMed Central

Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

2012-01-01

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

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

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).

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 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

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).

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

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.

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

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.

Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis.

PubMed

Zhang, Shaohua; Cai, Xuepeng; Luo, Xuenong; Wang, Shuai; Guo, Aijiang; Hou, Junling; Wu, Run

2018-03-01

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn 2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention. Copyright © 2018 Elsevier Inc

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.

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

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Automated Neuropsychological Assessment Metrics: Repeated Assessment with Two Military Samples

DTIC Science & Technology

2011-01-01

to ra- diation, high altitude, undersea conditions, and toxins ( 10,14,20 ). The putative advantages of ANAM4 (com- pared to other...repeated measures ANOVA. Separate analyses were performed for each of the ANAM subtests. Session was treated as a repeated variable. Huynh-Feldt epsilon

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

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

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.

Neutral polymorphisms in putative housekeeping genes and tandem repeats unravels the population genetics and evolutionary history of Plasmodium vivax in India.

PubMed

Prajapati, Surendra K; Joshi, Hema; Carlton, Jane M; Rizvi, M Alam

2013-01-01

The evolutionary history and age of Plasmodium vivax has been inferred as both recent and ancient by several studies, mainly using mitochondrial genome diversity. Here we address the age of P. vivax on the Indian subcontinent using selectively neutral housekeeping genes and tandem repeat loci. Analysis of ten housekeeping genes revealed a substantial number of SNPs (n = 75) from 100 P. vivax isolates collected from five geographical regions of India. Neutrality tests showed a majority of the housekeeping genes were selectively neutral, confirming the suitability of housekeeping genes for inferring the evolutionary history of P. vivax. In addition, a genetic differentiation test using housekeeping gene polymorphism data showed a lack of geographical structuring between the five regions of India. The coalescence analysis of the time to the most recent common ancestor estimate yielded an ancient TMRCA (232,228 to 303,030 years) and long-term population history (79,235 to 104,008) of extant P. vivax on the Indian subcontinent. Analysis of 18 tandem repeat loci polymorphisms showed substantial allelic diversity and heterozygosity per locus, and analysis of potential bottlenecks revealed the signature of a stable P. vivax population, further corroborating our ancient age estimates. For the first time we report a comparable evolutionary history of P. vivax inferred by nuclear genetic markers (putative housekeeping genes) to that inferred from mitochondrial genome diversity.

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

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

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...

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.

The cysteine-rich domain regulates ADAM protease function in vivo.

PubMed

Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

2002-12-09

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

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.

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

Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogs cross-linked in vitro.

PubMed

Held, Michael A; Tan, Li; Kamyab, Abdolreza; Hare, Michael; Shpak, Elena; Kieliszewski, Marcia J

2004-12-31

Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further cross-linked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an inter-molecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.

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.

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…

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus ‘Robusta 5’ accessions

PubMed Central

2012-01-01

Background Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. Results When several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand ‘Malling 9’ X ‘Robusta 5’ population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein ( MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German ‘Idared’ X ‘Robusta 5’ population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene ( HSP90). In the US ‘Otawa3’ X ‘Robusta5’ population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor

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...

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

De Novo Assembly of the Japanese Flounder (Paralichthys olivaceus) Spleen Transcriptome to Identify Putative Genes Involved in Immunity

PubMed Central

Huang, Lin; Li, Guiyang; Mo, Zhaolan; Xiao, Peng; Li, Jie; Huang, Jie

2015-01-01

Background Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity. Methodology/Principal Findings A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14%) were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38%) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. Conclusions/Significance The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder. PMID:25723398

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.

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...

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

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

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

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.

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.

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...

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

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

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...

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.

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

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

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…

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.

Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode.

PubMed

van der Vossen, E A; van der Voort, J N; Kanyuka, K; Bendahmane, A; Sandbrink, H; Baulcombe, D C; Bakker, J; Stiekema, W J; Klein-Lankhorst, R M

2000-09-01

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.

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

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

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

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

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

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

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

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

Chen, J.; Varner, J.E.

1985-07-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 asmore » a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.« less

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

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.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

PubMed

Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

2013-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

Leucine Biosynthesis Is Involved in Regulating High Lipid Accumulation in Yarrowia lipolytica

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

Kerkhoven, Eduard J.; Kim, Young-Mo; Wei, Siwei

ABSTRACT The yeastYarrowia lipolyticais a potent accumulator of lipids, and lipogenesis in this organism can be influenced by a variety of factors, such as genetics and environmental conditions. Using a multifactorial study, we elucidated the effects of both genetic and environmental factors on regulation of lipogenesis inY. lipolyticaand identified how two opposite regulatory states both result in lipid accumulation. This study involved comparison of a strain overexpressing diacylglycerol acyltransferase (DGA1) with a control strain grown under either nitrogen or carbon limitation conditions. A strong correlation was observed between the responses on the transcript and protein levels. Combination ofDGA1overexpression with nitrogen limitationmore » resulted in a high level of lipid accumulation accompanied by downregulation of several amino acid biosynthetic pathways, including that of leucine in particular, and these changes were further correlated with a decrease in metabolic fluxes. This downregulation was supported by the measured decrease in the level of 2-isopropylmalate, an intermediate of leucine biosynthesis. Combining the multi-omics data with putative transcription factor binding motifs uncovered a contradictory role for TORC1 in controlling lipid accumulation, likely mediated through 2-isopropylmalate and a Leu3-like transcription factor. IMPORTANCEThe ubiquitous metabolism of lipids involves refined regulation, and an enriched understanding of this regulation would have wide implications. Various factors can influence lipid metabolism, including the environment and genetics. We demonstrated, using a multi-omics and multifactorial experimental setup, that multiple factors affect lipid accumulation in the yeastYarrowia lipolytica. Using integrative analysis, we identified novel interactions between nutrient restriction and genetic factors involving regulators that are highly conserved among eukaryotes. Given that lipid metabolism is involved in many

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

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.

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

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

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

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.

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

Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.

PubMed

John, Ulrik P; Polotnianka, Renatam M; Sivakumaran, Kailayapillai A; Chew, Orinda; Mackin, Leanne; Kuiper, Micheal J; Talbot, Jonathan P; Nugent, Gregory D; Mautord, Julie; Schrauf, Gustavo E; Spangenberg, German C

2009-04-01

Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

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

PubMed

Prokop, Pavol

2015-08-01

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

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

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

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.

PubMed

Pittiruti, M; Siegel, J H; Sganga, G; Coleman, B; Wiles, C E; Belzberg, H; Wedel, S; Placko, R

1985-09-01

The body clearance of 10 plasma amino acids (AA) was determined from the rate of compared muscle-released AA and AA administered by infusion of total parenteral nutrition (TPN) compared to their estimated extracellular (ECW) pool in patients with multiple trauma with (n = 10) or without (n = 16) sepsis at 8-hour intervals. In both nonseptic and septic trauma, increasing TPN increased the mean clearance rate of all infused AA. When the individual AA clearance rates were normalized by the total AA infusion rate, regression-covariance analysis revealed that patients with sepsis had relatively impaired clearances of alanine (p less than 0.01) and methionine, proline, phenylalanine, and tyrosine p less than 0.05 for all). In contrast, the clearances of branched-chain AA (BCAA) valine and isoleucine were maintained, and the clearance of leucine was higher (p less than 0.05) in trauma patients with sepsis than in those without. At any AA infusion rate, compared with surviving patients with sepsis (p less than 0.05), patients who developed fatal multiple organ failure syndrome (MOFS) showed increased clearances of all BCAA with further impaired clearance of tyrosine. The clearance ratio of leucine/tyrosine was increased in MOFS at any AA infusion rate (p less than 0.0001), was an indicator of severity, and, if persistent, was a manifestation of a fatal outcome. Because tyrosine metabolism occurs almost entirely in the liver while leucine can be utilized by viscera and muscle, these data suggest early and progressive septic impairment of the pattern of hepatic uptake and oxidation of AA with a greater body dependence on BCAA, especially leucine, as septic MOFS develops.

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...

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

PubMed

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

2013-11-01

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

Urinary tract effects of HPSE2 mutations.

PubMed

Stuart, Helen M; Roberts, Neil A; Hilton, Emma N; McKenzie, Edward A; Daly, Sarah B; Hadfield, Kristen D; Rahal, Jeffery S; Gardiner, Natalie J; Tanley, Simon W; Lewis, Malcolm A; Sites, Emily; Angle, Brad; Alves, Cláudia; Lourenço, Teresa; Rodrigues, Márcia; Calado, Angelina; Amado, Marta; Guerreiro, Nancy; Serras, Inês; Beetz, Christian; Varga, Rita-Eva; Silay, Mesrur Selcuk; Darlow, John M; Dobson, Mark G; Barton, David E; Hunziker, Manuela; Puri, Prem; Feather, Sally A; Goodship, Judith A; Goodship, Timothy H J; Lambert, Heather J; Cordell, Heather J; Saggar, Anand; Kinali, Maria; Lorenz, Christian; Moeller, Kristina; Schaefer, Franz; Bayazit, Aysun K; Weber, Stefanie; Newman, William G; Woolf, Adrian S

2015-04-01

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS. Copyright © 2015 by the American Society of Nephrology.

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.

Leucine Deprivation Stimulates Fat Loss via Increasing CRH Expression in the Hypothalamus and Activating The Sympathetic Nervous System

PubMed Central

Cheng, Ying; Zhang, Qian; Meng, Qingshu; Xia, Tingting; Huang, Zhiying; Wang, Chunxia; Liu, Bin; Chen, Shanghai; Xiao, Fei; Du, Ying

2011-01-01

We previously showed that leucine deprivation decreases abdominal fat mass largely by increasing energy expenditure, as demonstrated by increased lipolysis in white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). The goal of the present study was to investigate the possible involvement of central nervous system (CNS) in this regulation and elucidate underlying molecular mechanisms. For this purpose, levels of genes and proteins related to lipolysis in WAT and UCP1 expression in BAT were analyzed in wild-type mice after intracerebroventricular administration of leucine or corticotrophin-releasing hormone antibodies, or in mice deleted for three β-adrenergic receptors, after being maintained on a leucine-deficient diet for 7 d. Here, we show that intracerebroventricular administration of leucine significantly attenuates abdominal fat loss and blocks activation of hormone sensitive lipase in WAT and induction of UCP1 in BAT in leucine-deprived mice. Furthermore, we provide evidence that leucine deprivation stimulates fat loss by increasing expression of corticotrophin-releasing hormone in the hypothalamus via activation of stimulatory G protein/cAMP/protein kinase A/cAMP response element-binding protein pathway. Finally, we show that the effect of leucine deprivation on fat loss is mediated by activation of the sympathetic nervous system. These results suggest that CNS plays an important role in regulating fat loss under leucine deprivation and thereby provide novel and important insights concerning the importance of CNS leucine in the regulation of energy homeostasis. PMID:21719534

Leucine deprivation stimulates fat loss via increasing CRH expression in the hypothalamus and activating the sympathetic nervous system.

PubMed

Cheng, Ying; Zhang, Qian; Meng, Qingshu; Xia, Tingting; Huang, Zhiying; Wang, Chunxia; Liu, Bin; Chen, Shanghai; Xiao, Fei; Du, Ying; Guo, Feifan

2011-09-01

We previously showed that leucine deprivation decreases abdominal fat mass largely by increasing energy expenditure, as demonstrated by increased lipolysis in white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). The goal of the present study was to investigate the possible involvement of central nervous system (CNS) in this regulation and elucidate underlying molecular mechanisms. For this purpose, levels of genes and proteins related to lipolysis in WAT and UCP1 expression in BAT were analyzed in wild-type mice after intracerebroventricular administration of leucine or corticotrophin-releasing hormone antibodies, or in mice deleted for three β-adrenergic receptors, after being maintained on a leucine-deficient diet for 7 d. Here, we show that intracerebroventricular administration of leucine significantly attenuates abdominal fat loss and blocks activation of hormone sensitive lipase in WAT and induction of UCP1 in BAT in leucine-deprived mice. Furthermore, we provide evidence that leucine deprivation stimulates fat loss by increasing expression of corticotrophin-releasing hormone in the hypothalamus via activation of stimulatory G protein/cAMP/protein kinase A/cAMP response element-binding protein pathway. Finally, we show that the effect of leucine deprivation on fat loss is mediated by activation of the sympathetic nervous system. These results suggest that CNS plays an important role in regulating fat loss under leucine deprivation and thereby provide novel and important insights concerning the importance of CNS leucine in the regulation of energy homeostasis.

Genome Wide Characterization of Short Tandem Repeat Markers in Sweet Orange (Citrus sinensis)

PubMed Central

Biswas, Manosh Kumar; Xu, Qiang; Mayer, Christoph; Deng, Xiuxin

2014-01-01

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community. PMID:25148383

Genome wide characterization of short tandem repeat markers in sweet orange (Citrus sinensis).

PubMed

Biswas, Manosh Kumar; Xu, Qiang; Mayer, Christoph; Deng, Xiuxin

2014-01-01

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii

PubMed Central

Krishnan, Neeraja M.

2017-01-01

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers. PMID:29230357

Leptin and leucine synergistically regulate protein metabolism in C2C12 myotubes and mouse skeletal muscles.

PubMed

Mao, Xiangbing; Zeng, Xiangfang; Huang, Zhimin; Wang, Junjun; Qiao, Shiyan

2013-07-28

Leucine and leptin play important roles in regulating protein synthesis and degradation in skeletal muscles in vitro and in vivo. However, the objective of the present study was to determine whether leptin and leucine function synergistically in regulating protein metabolism of skeletal muscles. In the in vitro experiment, C2C12 myotubes were cultured for 2 h in the presence of 5 mm-leucine and/or 50 ng/ml of leptin. In the in vivo experiment, C57BL/6 and ob/ob mice were randomly assigned to be fed a non-purified diet supplemented with 3 % L-leucine or 2·04 % L-alanine (isonitrogenous control) for 14 d. Ob/ob mice were injected intraperitoneally with sterile PBS or recombinant mouse leptin (0·1 μg/g body weight) for 14 d. In C57BL/6 mice, dietary leucine supplementation increased (P< 0·05) plasma leptin, leptin receptor expression and protein synthesis in skeletal muscles, but reduced (P< 0·05) plasma urea and protein degradation in skeletal muscles. Dietary leucine supplementation and leptin injection increased the relative weight of the gastrocnemius and soleus muscles in ob/ob mice. Moreover, leucine and leptin treatments stimulated (P< 0·05) protein synthesis and inhibited (P< 0·05) protein degradation in C2C12 myotubes and skeletal muscles of ob/ob mice. There were interactions (P< 0·05) between the leucine and leptin treatments with regard to protein metabolism in C2C12 myotubes and soleus muscles of ob/ob mice but not in the gastrocnemius muscles of ob/ob mice. Collectively, these results suggest that leptin and leucine synergistically regulate protein metabolism in skeletal muscles both in vitro and in vivo.

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

PubMed

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

2015-02-01

Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

Controlling Mechanical Properties of Bis-leucine Oxalyl Amide Gels

NASA Astrophysics Data System (ADS)

Chang, William; Carvajal, Daniel; Shull, Kenneth

2011-03-01

is-leucine oxalyl amide is a low molecular weight gelator capable of gelling polar and organic solvents. A fundamental understanding of self-assembled systems can lead to new methods in drug delivery and the design of new soft material systems. An important feature of self-assembled systems are the intermolecular forces between solvent and gelator molecule; by changing the environment the gel is in, the mechanical properties also change. In this project two variables were considered: the degree of neutralization present for the gelator molecule from neutral to completely ionized, and the concentration of the gelator molecule, from 1 weight percent to 8 weight percent in 1-butanol. Mechanical properties were studied using displacement controlled indentation techniques and temperature sweep rheometry. It has been found that properties such as the storage modulus, gelation temperature and maximum stress allowed increase with bis-leucine oxalyl amide concentration. The results from this study establish a 3-d contour map between the gelator concentration, the gelator degree of ionization and mechanical properties such as storage modulus and maximum stress allowed. The intermolecular forces between the bis-leucine low molecular weight gelator and 1-butanol govern the mechanical properties of the gel system, and understanding these interactions will be key to rationally designed self-assembled systems.

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

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

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.

Amyloid β (1-40) Toxicity Depends on the Molecular Contact between Phenylalanine 19 and Leucine 34.

PubMed

Korn, Alexander; McLennan, Steffane; Adler, Juliane; Krueger, Martin; Surendran, Dayana; Maiti, Sudipta; Huster, Daniel

2018-04-18

The formation of the hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) of amyloid β (1-40) (Aβ(1-40)) is known to be an important step in the fibrillation of Aβ(1-40) peptides. Mutations of this putatively early molecular contact were shown to strongly influence the toxicity of Aβ(1-40) ( Das et al. ( 2015 ) ACS Chem. Neurosci. 6 , 1290 - 1295 ). Any mutation of residue F19 completely abolished the toxicity of Aβ(1-40), suggesting that a proper F19-L34 contact is crucial also for the formation of transient oligomers. In this work, we investigate a series of isomeric substitutions of L34, namely, d-leucine, isoleucine, and valine, to study further details of this molecular contact. These replacements represent very minor alterations in the Aβ(1-40) structure posing the question how these alterations challenge the fibrillation kinetics, structure, dynamics, and toxicity of the Aβ(1-40) aggregates. Our work involves kinetic studies using thioflavin T, transmission electron microscopy, X-ray diffraction for the analysis of the fibril morphology, and nuclear magnetic resonance experiments for local structure and molecular dynamics investigations. Combined with cell toxicity assays of the mutated Aβ(1-40) peptides, the physicochemical and biological importance of the early folding contact between F19 and L34 in Aβ(1-40) is underlined. This implies that the F19-L34 contact influences a broad range of different processes including the initiation of fibrillation, oligomer stability, fibril elongation, local fibril structure, and dynamics and cellular toxicity. These processes do not only cover a broad range of diverse mechanisms, but also proved to be highly sensitive to minor modulations of this crucial contact. Furthermore, our work shows that the contact is not simply mediated by general hydrophobic interactions, but also depends on stereospecific mechanisms.

Putative fossil life in a hydrothermal system of the Dellen impact structure, Sweden

NASA Astrophysics Data System (ADS)

Lindgren, Paula; Ivarsson, Magnus; Neubeck, Anna; Broman, Curt; Henkel, Herbert; Holm, Nils G.

2010-07-01

Impact-generated hydrothermal systems are commonly proposed as good candidates for hosting primitive life on early Earth and Mars. However, evidence of fossil microbial colonization in impact-generated hydrothermal systems is rarely reported in the literature. Here we present the occurrence of putative fossil microorganisms in a hydrothermal system of the 89 Ma Dellen impact structure, Sweden. We found the putative fossilized microorganisms hosted in a fine-grained matrix of hydrothermal alteration minerals set in interlinked fractures of an impact breccia. The putative fossils appear as semi-straight to twirled filaments, with a thickness of 1-2 μm, and a length between 10 and 100 μm. They have an internal structure with segmentation, and branching of filaments occurs frequently. Their composition varies between an outer and an inner layer of a filament, where the inner layer is more iron rich. Our results indicate that hydrothermal systems in impact craters could potentially be capable of supporting microbial life. This could have played an important role for the evolution of life on early Earth and Mars.

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

PubMed Central

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

2015-01-01

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

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.

Potential of mean force analysis of the self-association of leucine-rich transmembrane α-helices: difference between atomistic and coarse-grained simulations.

PubMed

Nishizawa, Manami; Nishizawa, Kazuhisa

2014-08-21

Interaction of transmembrane (TM) proteins is important in many biological processes. Large-scale computational studies using coarse-grained (CG) simulations are becoming popular. However, most CG model parameters have not fully been calibrated with respect to lateral interactions of TM peptide segments. Here, we compare the potential of mean forces (PMFs) of dimerization of TM helices obtained using a MARTINI CG model and an atomistic (AT) Berger lipids-OPLS/AA model (AT(OPLS)). For helical, tryptophan-flanked, leucine-rich peptides (WL15 and WALP15) embedded in a parallel configuration in an octane slab, the AT(OPLS) PMF profiles showed a shallow minimum (with a depth of approximately 3 kJ/mol; i.e., a weak tendency to dimerize). A similar analysis using the CHARMM36 all-atom model (AT(CHARMM)) showed comparable results. In contrast, the CG analysis generally showed steep PMF curves with depths of approximately 16-22 kJ/mol, suggesting a stronger tendency to dimerize compared to the AT model. This CG > AT discrepancy in the propensity for dimerization was also seen for dilauroylphosphatidylcholine (DLPC)-embedded peptides. For a WL15 (and WALP15)/DLPC bilayer system, AT(OPLS) PMF showed a repulsive mean force for a wide range of interhelical distances, in contrast to the attractive forces observed in the octane system. The change from the octane slab to the DLPC bilayer also mitigated the dimerization propensity in the CG system. The dimerization energies of CG (AALALAA)3 peptides in DLPC and dioleoylphosphatidylcholine bilayers were in good agreement with previous experimental data. The lipid headgroup, but not the length of the lipid tails, was a key causative factor contributing to the differences between octane and DLPC. Furthermore, the CG model, but not the AT model, showed high sensitivity to changes in amino acid residues located near the lipid-water interface and hydrophobic mismatch between the peptides and membrane. These findings may help interpret CG

Rat leucine-rich protein binds and activates the promoter of the beta isoform of Ca2+/calmodulin-dependent protein kinase II gene.

PubMed

Ochiai, Nagahiro; Masumoto, Shuji; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Yamauchi, Takashi

2007-05-01

We previously found the neuronal cell-type specific promoter and binding partner of the beta isoform of Ca(2+)/calmodulin-dependent protein kinase II (beta CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of beta isoform of Ca(2+)/calmodulin-dependent protein kinase II of rat brain. Mol. Brain Res. 94, 35-47]. In the present study, we purified a protein that binds specifically a promoter region of beta CaM kinase II gene from a nuclear extract of the rat cerebellum using DEAE-cellulose column chromatography, ammonium sulfate fractionation, gel filtration and polyacrylamide gel electrophoresis. The purified protein was identified as rat leucine-rich protein 157 (rLRP157) using tandem mass spectrometry. Then, we prepared its cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly(A)(+)RNA of rat cerebellum. The rLRP157 cDNA was introduced into mouse neuroblastomaxrat glioma hybrid NG108-15 cells, and cells stably expressing rLRP157 (NG/LRP cells) were isolated. Binding of rLRP157 with the promoter sequence was confirmed by electrophoretic mobility shift assay using nuclear extract of NG/LRP cells. A luciferase reporter gene containing a promoter of beta CaM kinase II was transiently expressed in NG/LRP cells. Under the conditions, the promoter activity was enhanced about 2.6-fold in NG/LRP cells as compared with wild-type cells. The expression of rLRP157 mRNA was paralleled with that of beta CaM kinase II in the adult and embryo rat brain detected by in situ hybridization. Nuclear localization of rLRP157 was confirmed using GFP-rLRP157 fusion protein investigated under a confocal microscope. These results indicate that rLRP157 is one of the proteins binding to, and regulating the activity of, the promoter of beta CaM kinase II.

The Influence of 8 Weeks of Whey-Protein and Leucine Supplementation on Physical and Cognitive Performance

DTIC Science & Technology

2010-01-01

Influence of 8 Weeks of Whey Protein and Leucine Supplementation on Physical and Cognitive Performance 5a. GONTRAGT NUMBER FA8650-04-D-6472 5b. GRANT NUMBER...investigate the ability of whey -protein and leucine supplementation to enhance physical and cognitive performance and body composition. Thirty moderately fit...composition before and after supplementing their daily diet for 8 wk with either 19.7 g of whey protein and 6.2 g leucine (WPL) or a calorie-equivalent placebo

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses

[Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

PubMed

Petrova, L P; Prilipov, A G; Katsy, E I

2017-01-01

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

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

PubMed

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

2016-02-20

Here, we report the complete genome sequence of Corynebacterium glutamicum CP, an industrial l-leucine producing strain in China. The whole genome consists of a circular chromosome and a plasmid. The comparative genomics analysis shows that there are many mutations in the key enzyme coding genes relevant to l-leucine biosynthesis compared to C. glutamicum ATCC 13032. Copyright © 2016 Elsevier B.V. All rights reserved.

Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

PubMed Central

Sun, Meihong; Liu, Shiyang; Qi, Baoxiu; Li, Xinzheng

2013-01-01

Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family. PMID:24155879

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

PubMed

Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

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

Isolation of a full-length CC-NBS-LRR resistance gene analog candidate from sugar pine showing low nucleotide diversity.

Treesearch

K.D. Jermstad; L.A. Sheppard; B.B. Kinloch; A. Delfino-Mix; E.S. Ersoz; K.V. Krutovsky; D.B Neale

2006-01-01

The nucleotide-binding-site and leucine-rich-repeat (NBS–LRR) class of R proteins is abundant and widely distributed in plants. By using degenerate primers designed on the NBS domain in lettuce, we amplified sequences in sugar pine that shared sequence identity with many of the NBS–LRR class resistance genes catalogued in GenBank. The polymerase chain reaction products...

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...

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

Association of circulating pro-inflammatory marker, Leucine-Rich-α2-Glycoprotein (LRG1), with obesity and changes following metabolic/bariatric surgery.

PubMed

Pek, Sharon Li Ting; Cheng, Anton Kui Sing; Lin, Michelle Xueqin; Wong, Moh Sim; Liang, Eric Chan Zit; Moh, Angela Mei Chung; Sum, Chee Fang; Lim, Su Chi; Tavintharan, Subramaniam

2018-06-08

Obesity confers substantial excess risk for morbidity and mortality, especially for Type 2 diabetes (T2D). Leucine-Rich-α2-Glycoprotein 1 (LRG1), a novel pro-inflammatory factor, was recently reported to be higher in patients with T2D with complications of peripheral arterial disease. Association of LRG1, obesity and weight loss is unknown. We examined whether plasma LRG1 is associated with obesity in health screening participants and if it predicts future weight loss in morbidly obese patients after metabolic/bariatric surgery. Cohort 1 was a cross-sectional study from a Health Screening program (n=616) in a tertiary hospital. Cohort 2 was a prospective study of morbidly obese patients (n=231) who underwent metabolic/bariatric surgery with follow-up weight measurements. Anthropometric data, baseline fasting glucose, plasma adiponectin, high sensitivity C-reactive protein (HsCRP) and LRG1 were measured. Post-surgery blood, after metabolic/bariatric surgery, were available for LRG1and HsCRP measurements in 57 patients. In the group with highest tertile of LRG1, body mass index (BMI), waist circumference and HsCRP were significantly higher, while total cholesterol, HDL, LDL and adiponectin were lower than tertile 2 and tertile 1. Generalized linear model analysis showed that female gender (p<0.0001), non-Chinese ethnicity (p<0.019) and higher HsCRP (p<0.0001) levels were independent and significant determinants of higher plasma LRG1 levels. After adjustment for age, gender, ethnicity and baseline BMI, female gender (p=0.020), higher pre-surgery BMI (p=0.001) and lower pre-surgery LRG1 (p=0.002) remained statistically significant predictors for greater weight loss. Plasma LRG1 increased significantly [from 28.2 (21.9 - 36.8) to 34.9 (22.6 - 49.5)]μg/ml (p=0.003) within 1.5 months, after metabolic/bariatric surgery. Our study demonstrates that LRG1 level is positively associated with obesity and a lower level of plasma LRG1 predicts weight loss in metabolic

21 CFR 862.1460 - Leucine aminopeptidase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

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

Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis.

PubMed

Roodbarkelari, Farshad; Groot, Edwin P

2017-01-01

Homeodomain-leucine zipper proteins (HD-ZIPs) form a plant-specific family of transcription factors functioning as homo- or heterodimers. Certain members of all four classes of this family are involved in embryogenesis, the focus of this review. They support auxin biosynthesis, transport and response, which are in turn essential for the apical-basal patterning of the embryo, radicle formation and outgrowth of the cotyledons. They transcriptionally regulate meristem regulators to maintain the shoot apical meristem once it is initiated. Some members are specific to the protoderm, the outermost layer of the embryo, and play a role in shoot apical meristem function. Within classes, homeodomain-leucine zippers tend to act redundantly during embryo development, and there are many examples of regulation within and between classes of homeodomain-leucine zippers. This indicates a complex network of regulation that awaits future experiments to uncover. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Supplementation of Diabetic Rats with Leucine, Zinc, and Chromium: Effects on Function and Histological Structure of Testes.

PubMed

Kolahian, Saeed; Sadri, Hassan; Larijani, Amir; Hamidian, Gholamreza; Davasaz, Afshin

2015-12-01

The objective was to study whether leucine, zinc, and chromium supplementations influence function and histological structure of testes in a rat model of type 2 diabetes. Seventy seven adult male rats were categorized into 11 groups of 7 animals each: (1) nondiabetic (negative control); (2) non-treated (positive control); (3) treated with insulin; (4) treated with glibenclamide; (5) treated with leucine; (6) treated with zinc; (7) treated with chromium; (8) treated with leucine + zinc; (9) treated with leucine + chromium; (10) treated with zinc + chromium; (11) treated with leucine + zinc + chromium. In the non-treated group, hyperglycemia severely damaged testes morphology as well as the spermatogenic process. Diabetes induction decreased testicular length, height, width, volume, total number of epididymal sperm, and number of live sperm. Seminiferous tubules of diabetic rats showed a decrease in diameter of tubules and height of epithelium. Diabetes induction decreased the number of cells (spermatogonia, spermatocyte, spermatid, and Sertoli) in cross sections of seminiferous tubules. Administration of nutritional supplements to the diabetic rats improved testes morphology and reversed, although not completely, impairment of spermatogenesis. Treatment with nutritional supplements increased testicular length, height, width, and volume. All treatments increased the number of live sperm and the total number of epididymal sperm. Furthermore, nutritional supplements increased diameter of tubules, height of epithelium, and the number of cells in seminiferous tubules. These alleviating effects were more pronounced in animals treated with the leucine-zinc-chromium combination. The present results demonstrate beneficial effects of zinc, leucine, and chromium supplements to improve testes morphology and to restore spermatogenesis in type 2 diabetic rats.

Physico-Chemical Properties, Aerosolization and Dissolution of Co-Spray Dried Azithromycin Particles with L-Leucine for Inhalation.

PubMed

Mangal, Sharad; Nie, Haichen; Xu, Rongkun; Guo, Rui; Cavallaro, Alex; Zemlyanov, Dmitry; Zhou, Qi Tony

2018-01-08

Inhalation therapy is popular to treat lower respiratory tract infections. Azithromycin is effective against some bacteria that cause respiratory tract infections; but it has poor water solubility that may limit its efficacy when administrated as inhalation therapy. In this study, dry powder inhaler formulations were developed by co-spray drying azithromycin with L-leucine with a purpose to improve dissolution. The produced powder formulations were characterized regarding particle size, morphology, surface composition and in-vitro aerosolization performance. Effects of L-leucine on the solubility and in-vitro dissolution of azithromycin were also evaluated. The spray dried azithromycin alone formulation exhibited a satisfactory aerosol performance with a fine particle fraction (FPF) of 62.5 ± 4.1%. Addition of L-leucine in the formulation resulted in no significant change in particle morphology and FPF, which can be attributed to enrichment of azithromycin on the surfaces of composite particles. Importantly, compared with the spray-dried amorphous azithromycin alone powder, the co-spray dried powder formulations of azithromycin and L-leucine demonstrated a substantially enhanced in-vitro dissolution rate. Such enhanced dissolution of azithromycin could be attributed to the formation of composite system and the acidic microenvironment around azithromycin molecules created by the dissolution of acidic L-leucine in the co-spray dried powder. Fourier transform infrared spectroscopic data showed intermolecular interactions between azithromycin and L-leucine in the co-spray dried formulations. We developed the dry powder formulations with satisfactory aerosol performance and enhanced dissolution for a poorly water soluble weak base, azithromycin, by co-spray drying with an amino acid, L-leucine.

Comparative Physiological Studies of the Yeast and Mycelial Forms of Histoplasma capsulatum: Uptake and Incorporation of l-Leucine

PubMed Central

Gupta, Rishab K.; Howard, Dexter H.

1971-01-01

l-Leucine entered the cells of both morphological forms of Histoplasma capsulatum by a permease-like system at low external concentrations of substrate. However, at levels greater than 5 × 10−5m l-leucine, the amino acid entered the cells both through a simple diffusion-like process and the permease-like system. The rate of the amino acid diffusion into yeast and mycelial forms appeared to be the same, whereas the initial rate of accumulation through the permease-like system was 5 to 10 times faster in the mycelial phase than it was in the yeast phase. The Michaelis constants were 2.2 × 10−5m in yeast phase and 2 × 10−5m in mycelial phase cells. Transport of l-leucine at an external concentration of 10−5m showed all of the characteristics of a system of active transport, which was dependent on temperature and pH. Displacement or removal of the α-amino group, or modification of the α-carboxyl group abolished amino acid uptake. The process was competitively inhibited by neutral aliphatic side-chain amino acids (inhibition constants ranged from 1.5 × 10−5 to 6.2 × 10−5m). Neutral aromatic side-chain amino acids and the d-isomers of leucine and valine did not inhibit l-leucine uptake. These data were interpreted to mean that the l-leucine transport system is stereospecific and is highly specific for neutral aliphatic side-chain amino acids. Incorporation of l-leucine into macromolecules occurred at almost the same rate in both morphological forms of the fungus. The mycelial phase but not the yeast phase showed a slight initial lag in incorporation. In both morphological forms the intracellular pool of l-leucine was of limited capacity, and the total uptake of the amino acid was a function of intracellular pool size. The initial rate of l-leucine uptake was independent of the level of intracellular pool. Both morphological forms deaminated and degraded only a minor fraction of the accumulated leucine. PMID:4323295

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

PubMed

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

2016-05-01

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

Framework for measuring adaptive knowledge-rich systems performance.

PubMed

Bushko, Renata G

2005-01-01

The universe is non repeatable in nature--most of events cannot be prestated and do not repeat themselves. The only way to create systems that are truly useful is to make them adaptive (able to reason by analogy and learn) and rich in knowledge (including common sense knowledge). Adaptive and knowledge-rich health management could get us closer to errorless health care where small incremental adjustments happen all the time preventing occurrence of an error. In the era of adaptive systems we need to have a way to evaluate their performance. Are they truly adaptive? How adaptive are they? Are they accurate enough? Are they fast enough? Are they cost effective? This chapter presents general framework for measuring adaptive knowledge-rich systems' performance and includes among others definitions of adaptiveness factor, britt (a unit of brittleness) and uso-quant (unit of usefulness of a piece of knowledge). Measuring adaptive knowledge-rich systems performance is one of the most important research areas that can have a big pay-off in healthcare now and in the future.

[3H]Leucine incorporation method as a tool to measure secondary production by periphytic bacteria associated to the roots of floating aquatic macrophyte.

PubMed

Miranda, M R; Guimarães, J R D; Coelho-Souza, A S

2007-10-01

The present study assessed the application of [(3)H]Leucine incorporation into protein by periphytic bacteria associated with the roots of the floating aquatic macrophyte Eichornia crassipes. Basic assumptions underlying the method, such as linearity of leucine incorporation, saturation level of incorporation rates, incorporation into other macromolecules, specificity of incorporation for bacterial assemblages and [(3)H]Leucine degradation during samples storage were tested, and two procedures for extracting the incorporated leucine were compared. Both methods gave the same results, however, the hot TCA extraction method was less time consuming than the alkaline extraction method. Incorporation of [(3)H]Leucine was linear for up to 40 min. Saturation concentration of [(3)H]Leucine incorporation into protein was 1500 nM. An experiment with prokaryotic and eukaryotic inhibitors showed no significant [(3)H]Leucine incorporation into eukaryotes even in high leucine concentrations. No significant amounts of radiolabel were incorporated into other macromolecules. The maximum time of sample storage after the incubation is 15 days. The leucine incorporation method can be a reliable tool to measure bacterial production in the periphyton root-associated bacteria.

Acetyl-DL-leucine improves gait variability in patients with cerebellar ataxia-a case series.

PubMed

Schniepp, Roman; Strupp, Michael; Wuehr, Max; Jahn, Klaus; Dieterich, Marianne; Brandt, Thomas; Feil, Katharina

2016-01-01

Acetyl-DL-leucine is a modified amino acid that was observed to improve ataxic symptoms in patients with sporadic and hereditary forms of ataxia. Here, we investigated the effect of the treatment with Acetyl-DL-leucine on the walking stability of patients with cerebellar ataxia (10x SAOA, 2x MSA-C, 2x ADA, 1x CACNA-1A mutation, 2x SCA 2, 1x SCA 1). Treatment with Acetyl-DL-leucine (500 mg; 3-3-4) significantly improved the coefficient of variation of stride time in 14 out of 18 patients. Moreover, subjective ambulatory scores (FES-I and ABC) and the SARA scores were also improved under treatment. Further prospective studies are necessary to support these class III observational findings.

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

PubMed

Brucet, Marina; Querol-Audí, Jordi; Serra, Maria; Ramirez-Espain, Ximena; Bertlik, Kamila; Ruiz, Lidia; Lloberas, Jorge; Macias, Maria J; Fita, Ignacio; Celada, Antonio

2007-05-11

TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

Physico-Chemical Properties, Aerosolization and Dissolution of Co-Spray Dried Azithromycin Particles with L-Leucine for Inhalation

PubMed Central

Mangal, Sharad; Nie, Haichen; Xu, Rongkun; Guo, Rui; Cavallaro, Alex; Zemlyanov, Dmitry; Zhou, Qi (Tony)

2018-01-01

Purpose Inhalation therapy is popular to treat lower respiratory tract infections. Azithromycin is effective against some bacteria that cause respiratory tract infections; but it has poor water solubility that may limit its efficacy when administrated as inhalation therapy. In this study, dry powder inhaler formulations were developed by co-spray drying azithromycin with L-leucine with a purpose to improve dissolution. Methods The produced powder formulations were characterized regarding particle size, morphology, surface composition and in-vitro aerosolization performance. Effects of L-leucine on the solubility and in-vitro dissolution of azithromycin were also evaluated. Results The spray dried azithromycin alone formulation exhibited a satisfactory aerosol performance with a fine particle fraction (FPF) of 62.5 ± 4.1%. Addition of L-leucine in the formulation resulted in no significant change in particle morphology and FPF, which can be attributed to enrichment of azithromycin on the surfaces of composite particles. Importantly, compared with the spray-dried amorphous azithromycin alone powder, the co-spray dried powder formulations of azithromycin and L-leucine demonstrated a substantially enhanced in-vitro dissolution rate. Such enhanced dissolution of azithromycin could be attributed to the formation of composite system and the acidic microenvironment around azithromycin molecules created by the dissolution of acidic L-leucine in the co-spray dried powder. Fourier transform infrared spectroscopic data showed intermolecular interactions between azithromycin and L-leucine in the co-spray dried formulations. Conclusions We developed the dry powder formulations with satisfactory aerosol performance and enhanced dissolution for a poorly water soluble weak base, azithromycin, by co-spray drying with an amino acid, L-leucine. PMID:29374368

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

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....

Genome-wide analysis of tandem repeats in plants and green algae

Treesearch

Zhixin Zhao; Cheng Guo; Sreeskandarajan Sutharzan; Pei Li; Craig Echt; Jie Zhang; Chun Liang

2014-01-01

Tandem repeats (TRs) extensively exist in the genomes of prokaryotes and eukaryotes. Based on the sequenced genomes and gene annotations of 31 plant and algal species in Phytozome version 8.0 (http://www.phytozome.net/), we examined TRs in a genome-wide scale, characterized their distributions and motif features, and explored their putative biological functions. Among...

Nanoparticles Suitable for BCAA Isolation Can Serve for Use in Magnetic Lipoplex-Based Delivery System for L, I, V, or R-rich Antimicrobial Peptides.

PubMed

Vesely, Radek; Jelinkova, Pavlina; Hegerova, Dagmar; Cernei, Natalia; Kopel, Pavel; Moulick, Amitava; Richtera, Lukas; Heger, Zbynek; Adam, Vojtech; Zitka, Ondrej

2016-03-31

This paper investigates the synthesis of paramagnetic nanoparticles, which are able to bind branched chain amino acids (BCAAs)-leucine, valine, and isoleucine and, thus, serve as a tool for their isolation. Further, by this, we present an approach for encapsulation of nanoparticles into a liposome cavity resulting in a delivery system. Analyses of valine and leucine in entire complex show that 31.3% and 32.6% recoveries are reached for those amino acids. Evaluation of results shows that the success rate of delivery in Escherichia coli ( E. coli ) is higher in the case of BCAAs on nanoparticles entrapped in liposomes (28.7% and 34.7% for valine and leucine, respectively) when compared to nanoparticles with no liposomal envelope (18.3% and 13.7% for valine and leucine, respectively). The nanoparticles with no liposomal envelope exhibit the negative zeta potential (-9.1 ± 0.3 mV); however, their encapsulation results in a shift into positive values (range of 28.9 ± 0.4 to 33.1 ± 0.5 mV). Thus, electrostatic interactions with negatively-charged cell membranes (approx. -50 mV in the case of E. coli ) leads to a better uptake of cargo. Our delivery system was finally tested with the leucine-rich antimicrobial peptide (FALALKALKKALKKLKKALKKAL) and it is shown that hemocompatibility (7.5%) and antimicrobial activity of the entire complex against E. coli , Staphylococcus aureus ( S. aureus ), and methicilin-resistant S. aureus (MRSA) is comparable or better than conventional penicillin antibiotics.

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

USDA-ARS?s Scientific Manuscript database

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

METABOLIC RESPONSES TO DIETARY LEUCINE RESTRICTION INVOLVE REMODELING OF ADIPOSE TISSUE AND ENHANCED HEPATIC INSULIN SIGNALING

PubMed Central

Wanders, Desiree; Stone, Kirsten P.; Dille, Kelly; Simon, Jacob; Pierse, Alicia; Gettys, Thomas W.

2015-01-01

Dietary leucine was incrementally restricted to test whether limiting this essential amino acid (EAA) would fully reproduce the beneficial responses produced by dietary methionine restriction. Restricting leucine by 85% increased energy intake and expenditure within five to seven days of its introduction and reduced overall accumulation of adipose tissue. Leucine restriction (LR) also improved glucose tolerance, increased hepatic release of FGF21 into the blood stream, and enhanced insulin-dependent activation of Akt in liver. However, LR had no effect on hepatic lipid levels and failed to lower lipogenic gene expression in the liver. LR did affect remodeling of white and brown adipose tissue, increasing expression of both thermogenic and lipogenic genes. These findings illustrate that dietary LR reproduces many but not all of the physiological responses of methionine restriction. The primary differences occur in the liver, where methionine and leucine restriction cause opposite effects on tissue lipid levels and expression of lipogenic genes. Together these findings suggest that the sensing systems which detect and respond to dietary restriction of EAAs act through mechanisms that both leucine and methionine are able to engage, and in the case of hepatic lipid metabolism, may be unique to specific EAAs such as methionine. PMID:26643647

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

PubMed

Fossat, Martin J; Dao, Thuy P; Jenkins, Kelly; Dellarole, Mariano; Yang, Yinshan; McCallum, Scott A; Garcia, Angel E; Barrick, Doug; Roumestand, Christian; Royer, Catherine A

2016-12-06

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1 H- 15 N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effect of 8-week leucine supplementation and resistance exercise training on muscle hypertrophy and satellite cell activation in rats.

PubMed

Lim, Chang Hyun; Gil, Ju Hyun; Quan, Helong; Viet, Dang Ha; Kim, Chang Keun

2018-06-01

We investigated the effects of regular leucine intake and/or resistance exercise training on skeletal muscle hypertrophy and satellite cell activity after the administration of different doses of leucine. Ten-week-old Sprague-Dawley rats were assigned to six groups (n = 7 per group): a control group (Con), two groups receiving either 10% (0.135 g/kg.wt) (Leu10) or 50% (0.675 g/kg.wt) (Leu50) leucine supplementation, and three exercise groups receiving 0% (Ex), 10% (Leu10Ex), and 50% (Leu50Ex) leucine supplementation. The rats performed ladder climbing exercises thrice per week for 8 weeks, and received leucine supplements at the same time daily. Muscle phenotypes were assessed by immunohistochemistry. MyoD, myogenin, and IGF1 protein levels were determined by western blot. The Leu50Ex group displayed significantly higher numbers of positive embryonic myosin fibers (0.35 ± 0.08, 250%) and myonuclei (3.29 ± 0.3, 118.7%) than all other groups. And exercise training groups increased the cross-sectional area, the number of satellite cells and protein expression of MyoD, myogenin, and IGF1alpha relative to the Control group (P < 0.05). However, Only leucine supplementation group did not increase skeletal muscle hypertrophy and satellite cell activity, regardless of the dose (P > 0.05). Leucine intake accompanied by regular exercise training may increase satellite cell activation in skeletal muscles, and improve muscle quality more effectively than continuous leucine ingestion alone. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Alu repeats: A source for the genesis of primate microsatellites

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

Arcot, S.S.; Batzer, M.A.; Wang, Zhenyuan

1995-09-01

As a result of their abundance, relatively uniform distribution, and high degree of polymorphism, microsatellites and minisatellites have become valuable tools in genetic mapping, forensic identity testing, and population studies. In recent years, a number of microsatellite repeats have been found to be associated with Alu interspersed repeated DNA elements. The association of an Alu element with a microsatellite repeat could result from the integration of an Alu element within a preexisting microsatellite repeat. Alternatively, Alu elements could have a direct role in the origin of microsatellite repeats. Errors introduced during reverse transcription of the primary transcript derived from anmore » Alu {open_quotes}master{close_quote} gene or the accumulation of random mutations in the middle A-rich regions and oligo(dA)-rich tails of Alu elements after insertion and subsequent expansion and contraction of these sequences could result in the genesis of a microsatellite repeat. We have tested these hypotheses by a direct evolutionary comparison of the sequences of some recent Alu elements that are found only in humans and are absent from nonhuman primates, as well as some older Alu elements that are present at orthologous positions in a number of nonhuman primates. The origin of {open_quotes}young{close_quotes} Alu insertions, absence of sequences that resemble microsatellite repeats at the orthologous loci in chimpanzees, and the gradual expansion of microsatellite repeats in some old Alu repeats at orthologous positions within the genomes of a number of nonhuman primates suggest that Alu elements are a source for the genesis of primate microsatellite repeats. 48 refs., 5 figs., 3 tabs.« less

Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome.

PubMed

Xu, Baoshan; Gogol, Madelaine; Gaudenz, Karin; Gerton, Jennifer L

2016-01-05

Roberts syndrome (RBS) is a human developmental disorder caused by mutations in the cohesin acetyltransferase ESCO2. We previously reported that mTORC1 signaling was depressed and overall translation was reduced in RBS cells and zebrafish models for RBS. Treatment of RBS cells and zebrafish RBS models with L-leucine partially rescued mTOR function and protein synthesis, correlating with increased cell division and improved development. In this study, we use RBS cells to model mTORC1 repression and analyze transcription and translation with ribosome profiling to determine gene-level effects of L-leucine. L-leucine treatment partially rescued translational efficiency of ribosomal subunits, translation initiation factors, snoRNA production, and mitochondrial function in RBS cells, consistent with these processes being mTORC1 controlled. In contrast, other genes are differentially expressed independent of L-leucine treatment, including imprinted genes such as H19 and GTL2, miRNAs regulated by GTL2, HOX genes, and genes in nucleolar associated domains. Our study distinguishes between gene expression changes in RBS cells that are TOR dependent and those that are independent. Some of the TOR independent gene expression changes likely reflect the architectural role of cohesin in chromatin looping and gene expression. This study reveals the dramatic rescue effects of L-leucine stimulation of mTORC1 in RBS cells and supports that normal gene expression and translation requires ESCO2 function.

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

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. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were tr...

Cracking the ANP32 whips: Important functions, unequal requirement, and hints at disease implications

PubMed Central

Reilly, Patrick T; Yu, Yun; Hamiche, Ali; Wang, Lishun

2014-01-01

The acidic (leucine-rich) nuclear phosphoprotein 32 kDa (ANP32) family is composed of small, evolutionarily conserved proteins characterized by an N-terminal leucine-rich repeat domain and a C-terminal low-complexity acidic region. The mammalian family members (ANP32A, ANP32B, and ANP32E) are ascribed physiologically diverse functions including chromatin modification and remodelling, apoptotic caspase modulation, protein phosphatase inhibition, as well as regulation of intracellular transport. In addition to reviewing the widespread literature on the topic, we present a concept of the ANP32s as having a whip-like structure. We also present hypotheses that ANP32C and other intronless sequences should not currently be considered bona fide family members, that their disparate necessity in development may be due to compensatory mechanisms, that their contrasting roles in cancer are likely context-dependent, along with an underlying hypothesis that ANP32s represent an important node of physiological regulation by virtue of their diverse biochemical activities. PMID:25156960

Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance.

PubMed

Saunders, Diane G O; Breen, Susan; Win, Joe; Schornack, Sebastian; Hein, Ingo; Bozkurt, Tolga O; Champouret, Nicolas; Vleeshouwers, Vivianne G A A; Birch, Paul R J; Gilroy, Eleanor M; Kamoun, Sophien

2012-08-01

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

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.

Estimating bacterial production in marine waters from the simultaneous incorporation of thymidine and leucine.

PubMed

Chin-Leo, G; Kirchman, D L

1988-08-01

We examined the simultaneous incorporation of [H]thymidine and [C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 +/- 0.2 [mean +/- standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic acid-insoluble fraction. There were no significant differences in thymidine incorporation between dual- and single-label incubations (dual/ single = 1.03 +/- 0.13). Addition of the two substrates in relatively large amounts (25 nM) did not apparently increase bacterial activity during short incubations (<5 h). With the dual-label method we found that thymidine and leucine incorporation rates covaried over depth profiles of the Chesapeake Bay. Estimates of bacterial production based on thymidine and leucine differed by less than 25%. Although the need for appropriate conversion factors has not been eliminated, the dual-label approach can be used to examine the variation in bacterial production while ensuring that the observed variation in incorporation rates is due to real changes in bacterial production rather than changes in conversion factors or introduction of other artifacts.

Leucine modulates dynamic phosphorylation events in insulin signaling pathway and enhances insulin-dependent glycogen synthesis in human skeletal muscle cells

PubMed Central

2014-01-01

Background Branched-chain amino acids, especially leucine, are known to interact with insulin signaling pathway and glucose metabolism. However, the mechanism by which this is exerted, remain to be clearly defined. In order to examine the effect of leucine on muscle insulin signaling, a set of experiments was carried out to quantitate phosphorylation events along the insulin signaling pathway in human skeletal muscle cell cultures. Cells were exposed to insulin, leucine or both, and phosphorylation events of key insulin signaling molecules were tracked over time so as to monitor time-related responses that characterize the signaling events and could be missed by a single sampling strategy limited to pre/post stimulus events. Results Leucine is shown to increase the magnitude of insulin-dependent phosphorylation of protein kinase B (AKT) at Ser473 and glycogen synthase kinase (GSK3β) at Ser21-9. Glycogen synthesis follows the same pattern of GSK3β, with a significant increase at 100 μM leucine plus insulin stimulus. Moreover, data do not show any statistically significant increase of pGSK3β and glycogen synthesis at higher leucine concentrations. Leucine is also shown to increase the magnitude of insulin-mediated extracellularly regulated kinase (ERK) phosphorylation; however, differently from AKT and GSK3β, ERK shows a transient behavior, with an early peak response, followed by a return to the baseline condition. Conclusions These experiments demonstrate a complementary effect of leucine on insulin signaling in a human skeletal muscle cell culture, promoting insulin-activated GSK3β phosphorylation and glycogen synthesis. PMID:24646332

The ERECTA gene regulates plant transpiration efficiency in Arabidopsis.

PubMed

Masle, Josette; Gilmore, Scott R; Farquhar, Graham D

2005-08-11

Assimilation of carbon by plants incurs water costs. In the many parts of the world where water is in short supply, plant transpiration efficiency, the ratio of carbon fixation to water loss, is critical to plant survival, crop yield and vegetation dynamics. When challenged by variations in their environment, plants often seem to coordinate photosynthesis and transpiration, but significant genetic variation in transpiration efficiency has been identified both between and within species. This has allowed plant breeders to develop effective selection programmes for the improved transpiration efficiency of crops, after it was demonstrated that carbon isotopic discrimination, Delta, of plant matter was a reliable and sensitive marker negatively related to variation in transpiration efficiency. However, little is known of the genetic controls of transpiration efficiency. Here we report the isolation of a gene that regulates transpiration efficiency, ERECTA. We show that ERECTA, a putative leucine-rich repeat receptor-like kinase (LRR-RLK) known for its effects on inflorescence development, is a major contributor to a locus for Delta on Arabidopsis chromosome 2. Mechanisms include, but are not limited to, effects on stomatal density, epidermal cell expansion, mesophyll cell proliferation and cell-cell contact.

Analysis of the DNA sequence of a 15,500 bp fragment near the left telomere of chromosome XV from Saccharomyces cerevisiae reveals a putative sugar transporter, a carboxypeptidase homologue and two new open reading frames.

PubMed

Gamo, F J; Lafuente, M J; Casamayor, A; Ariño, J; Aldea, M; Casas, C; Herrero, E; Gancedo, C

1996-06-15

We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

SdAb heterodimer formation using leucine zippers

NASA Astrophysics Data System (ADS)

Goldman, Ellen R.; Anderson, George P.; Brozozog-Lee, P. Audrey; Zabetakis, Dan

2013-05-01

Single domain antibodies (sdAb) are variable domains cloned from camel, llama, or shark heavy chain only antibodies, and are among the smallest known naturally derived antigen binding fragments. SdAb derived from immunized llamas are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. We hypothesized that the ability to produce heterodimeric sdAb would enable reagents with the robust characteristics of component sdAb, but with dramatically improved overall affinity through increased avidity. Previously we had constructed multimeric sdAb by genetically linking sdAb that bind non-overlapping epitopes on the toxin, ricin. In this work we explored a more flexible approach; the construction of multivalent binding reagents using multimerization domains. We expressed anti-ricin sdAb that recognize different epitopes on the toxin as fusions with differently charged leucine zippers. When the initially produced homodimers are mixed the leucine zipper domains will pair to produce heterodimers. We used fluorescence resonance energy transfer to confirm heterodimer formation. Surface plasmon resonance, circular dichroism, enzyme linked immunosorbent assays, and fluid array assays were used to characterize the multimer constructs, and evaluate their utility in toxin detection.

Reptile Toll-like receptor 5 unveils adaptive evolution of bacterial flagellin recognition.

PubMed

Voogdt, Carlos G P; Bouwman, Lieneke I; Kik, Marja J L; Wagenaar, Jaap A; van Putten, Jos P M

2016-01-07

Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N- and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions.

Reptile Toll-like receptor 5 unveils adaptive evolution of bacterial flagellin recognition

PubMed Central

Voogdt, Carlos G. P.; Bouwman, Lieneke I.; Kik, Marja J. L.; Wagenaar, Jaap A.; van Putten, Jos P. M.

2016-01-01

Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N- and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions. PMID:26738735

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Brown, S. J.; Vergnes, J. P.; Hassell, J. R.; Mann, M. M.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1993-01-01

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides

NASA Astrophysics Data System (ADS)

Zhokhov, Sergey S.; Kovalyov, Sergey V.; Samgina, Tatiana Yu.; Lebedev, Albert T.

2017-08-01

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination.

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

PubMed

Abraham, S; Solomon, W B

2000-09-19

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin

Intragastric administration of leucine or isoleucine lowers the blood glucose response to a mixed-nutrient drink by different mechanisms in healthy, lean volunteers.

PubMed

Ullrich, Sina S; Fitzgerald, Penelope Ce; Schober, Gudrun; Steinert, Robert E; Horowitz, Michael; Feinle-Bisset, Christine

2016-11-01

The branched-chain amino acids leucine and isoleucine lower blood glucose after oral glucose ingestion, and the intraduodenal infusion of leucine decreases energy intake in healthy, lean men. We investigated the effects of the intragastric administration of leucine and isoleucine on the gastric emptying of, and blood glucose responses to, a physiologic mixed-macronutrient drink and subsequent energy intake. In 2 separate studies, 12 healthy, lean subjects received on 3 separate occasions an intragastric infusion of 5 g leucine (leucine-5g) or an intragastric infusion of 10 g leucine (leucine-10g), an intragastric infusion of 5 g isoleucine (isoleucine-5g) or an intragastric infusion of 10 g isoleucine (isoleucine-10g), or a control. Fifteen minutes later, subjects consumed a mixed-nutrient drink (400 kcal, 56 g carbohydrates, 15 g protein, and 12 g fat), and gastric emptying ( 13 C-acetate breath test) and blood glucose, plasma insulin, C-peptide, glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (leucine study only) were measured for 60 min. Immediately afterward, energy intake from a cold, buffet-style meal was assessed. Compared with the control, leucine-10g decreased the blood glucose area under the curve (AUC) (P < 0.05) and tended to reduce peak blood glucose (P = 0.07), whereas effects of leucine-5g were NS. Leucine-10g, but not leucine-5g, increased plasma insulin and C-peptide AUCs (P < 0.01 for both), but neither dose affected glucagon, GLP-1, GIP, cholecystokinin, gastric emptying, or energy intake. Compared with the control, isoleucine-10g reduced the blood glucose AUC and peak blood glucose (P < 0.01), whereas effects of isoleucine-5g were NS. Neither load affected insulin, C-peptide, glucagon, GLP-1, or GIP. Isoleucine-10g, but not isoleucine-5g, slowed gastric emptying (P < 0.05), but gastric emptying was not correlated with the blood glucose AUC. Isoleucine did not affect energy intake

The heptad repeats region is essential for AcMNPV P10 filament formation and not the proline-rich or the C-terminus basic regions.

PubMed

Dong, Chunsheng; Deng, Fei; Li, Dan; Wang, Hualin; Hu, Zhihong

2007-09-01

Baculovirus P10 protein is a small conserved protein and is expressed as bundles of filaments in the host cell during the late phase of virus infection. So far the published results on the domain responsible for filament structural formation have been contradictory. Electron microscopy revealed that the C-terminus basic region was involved in filament structural formation in the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) [van Oers, M.M., Flipsen, J.T., Reusken, C.B., Sliwinsky, E.L., Vlak, J.M., 1993. Functional domains of the p10 protein of Autographa californica nuclear polyhedorsis virus. J. Gen. Virol. 74, 563-574.]. While in the Helicoverpa armigera nucleopolyhedrovirus (HearNPV), the heptad repeats region but not the C-terminus domain was proven to be responsible for filament formation [Dong, C., Li, D., Long, G., Deng, F., Wang, H., Hu, Z., 2005. Identification of functional domains required for HearNPV P10 filament formation. Virology 338, 112-120.]. In this manuscript, fluorescence confocal microscopy was applied to study AcMNPV P10 filament formation. A set of plasmids containing different P10 structural domains fused with a fluorescent protein were constructed and transfected into Sf-9 cells. The data indicated that the heptad repeats region, but not the proline-rich region or the C-terminus basic region, is essential for AcMNPV P10 filament formation. Co-transfection of P10s tagged with different fluorescent revealed that P10s with defective heptad repeats region could not interact with intact heptad repeats region or even full-length P10s to form filament structure. Within the heptad repeats region, deletion of the three amino acids spacing of AcMNPV P10 appeared to have no significant impact on the formation of filament structures, but the content of the heptad repeats region appeared to play a role in the morphology of the filaments.

Genome-Wide Analyses and Functional Classification of Proline Repeat-Rich Proteins: Potential Role of eIF5A in Eukaryotic Evolution

PubMed Central

Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

2014-01-01

The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P) does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly)-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids), only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution. PMID:25364902

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

Serine-rich repeat proteins and pili promote Streptococcus agalactiae colonization of the vaginal tract.

PubMed

Sheen, Tamsin R; Jimenez, Alyssa; Wang, Nai-Yu; Banerjee, Anirban; van Sorge, Nina M; Doran, Kelly S

2011-12-01

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ΔpilA or Δsrr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment.

LGI1 antibody encephalitis and psychosis.

PubMed

Wang, Dahai; Hao, Qinjian; He, Lan; Wang, Qiang

2018-05-01

To describe a case of leucine-rich, glioma inactivated 1 antibody-encephalitis presenting with psychosis. Case report. A young man with leucine-rich, glioma inactivated 1-antibody encephalitis initially presented with acute psychotic symptoms, short-term memory loss and faciobrachial dystonic seizures. Magnetic resonance imaging revealed hippocampal lesions. Electroencephalography revealed frontotemporal slowing of background activity. Increased awareness of leucine-rich, glioma inactivated 1-antibody encephalitis may promote early recognition and treatment.

Estimating Bacterial Production in Marine Waters from the Simultaneous Incorporation of Thymidine and Leucine

PubMed Central

Chin-Leo, Gerardo; Kirchman, David L.

1988-01-01

We examined the simultaneous incorporation of [3H]thymidine and [14C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 ± 0.2 [mean ± standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic acid-insoluble fraction. There were no significant differences in thymidine incorporation between dual- and single-label incubations (dual/ single = 1.03 ± 0.13). Addition of the two substrates in relatively large amounts (25 nM) did not apparently increase bacterial activity during short incubations (<5 h). With the dual-label method we found that thymidine and leucine incorporation rates covaried over depth profiles of the Chesapeake Bay. Estimates of bacterial production based on thymidine and leucine differed by less than 25%. Although the need for appropriate conversion factors has not been eliminated, the dual-label approach can be used to examine the variation in bacterial production while ensuring that the observed variation in incorporation rates is due to real changes in bacterial production rather than changes in conversion factors or introduction of other artifacts. PMID:16347706

Effect of pH and leucine concentration on aerosolization properties of carrier-free formulations of levofloxacin.

PubMed

Barazesh, Ahmadreza; Gilani, Kambiz; Rouini, Mohammadreza; Barghi, Mohammad Ali

2018-06-15

The aim of this study was to evaluate the effect of leucine at different pH values preferred for inhalation on particle characteristics and aerosolization performance of spray dried carrier-free formulations of levofloxacin. A full factorial design was applied to optimize the formulation containing levofloxacin with or without leucine in different pH values and the optimum condition was determined. Particle size and morphology, crystallinity state, electrostatic charge and surface composition of the particles were determined. Aerodynamic properties of the powders were also assessed by an Andersen cascade impactor after aerosolization through an Aerolizer® at an air flow rate of 60 L/min. The pH of initial solution affected various physical properties of the drug containing particles and hence their in vitro deposition. The profound effect of pH was on water content, electrostatic charge and surface composition of the particles. The negative effect of water content on in vitro deposition of the drug was covered by preferred surface accumulation of leucine at pH 6. Optimum formulation which obtained by co-spray drying of the drug with 21.79% leucine at pH 5.98 presented a fine particle fraction equal to 54.38. In conclusion, changing pH of the initial solution influenced the effect of leucine on aerosolization of levofloxacine spray dried particles by modification of their physical properties. Copyright © 2018 Elsevier B.V. All rights reserved.

The Staphylococcus aureus leucine aminopeptidase LAP is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine

PubMed Central

Carroll, Ronan K.; Veillard, Florian; Gagne, Danielle T.; Lindenmuth, Jarrod M.; Poreba, Marcin; Drag, Marcin; Potempa, Jan; Shaw, Lindsey N.

2013-01-01

Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part, due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase - LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria, however, in systemic and localized infection models the pepZ mutant was significantly attenuated in virulence. Recently, a contradictory report has been published, suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition we conduct a biochemical investigation of purified recombinant LAP identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine and that leucine is not the primary target of LAP. PMID:23241672

Repeated burning of eastern tallgrass prairie increases richness and diversity, stabilizing late successional vegetation.

PubMed

Bowles, Marlin L; Jones, Michael D

2013-03-01

Understanding temporal effects of fire frequency on plant species diversity and vegetation structure is critical for managing tallgrass prairie (TGP), which occupies a mid-continental longitudinal precipitation and productivity gradient. Eastern TGP has contributed little information toward understanding whether vegetation-fire interactions are uniform or change across this biome. We resampled 34 fire-managed mid- and late-successional ungrazed TGP remnants occurring across a dry to wet-mesic moisture gradient in the Chicago region of Illinois, USA. We compared hypotheses that burning acts either as a stabilizing force or causes change in diversity and structure, depending upon fire frequency and successional stage. Based on western TGP, we expected a unimodal species richness distribution across a cover-productivity gradient, variable functional group responses to fire frequency, and a negative relationship between fire frequency and species richness. Species diversity was unimodal across the cover gradient and was more strongly humpbacked in stands with greater fire frequency. In support of a stabilizing hypothesis, temporal similarity of late-successional vegetation had a logarithmic relationship with increasing fire frequency, while richness and evenness remained stable. Temporal similarity within mid-successional stands was not correlated with fire frequency, while richness increased and evenness decreased over time. Functional group responses to fire frequency were variable. Summer forb richness increased under high fire frequency, while C4 grasses, spring forbs, and nitrogen-fixing species decreased with fire exclusion. On mesic and wet-mesic sites, vegetation structure measured by the ratio of woody to graminoid species was negatively correlated with abundance of forbs and with fire frequency. Our findings that species richness responds unimodally to an environmental-productivity gradient, and that fire exclusion increases woody vegetation and leads to loss

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

PubMed

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

2014-09-26

We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. Copyright © 2014 Elsevier Inc. All rights reserved.

The Putative Cerean Exosphere

NASA Astrophysics Data System (ADS)

Schorghofer, Norbert; Byrne, Shane; Landis, Margaret E.; Mazarico, Erwan; Prettyman, Thomas H.; Schmidt, Britney E.; Villarreal, Michaela N.; Castillo-Rogez, Julie; Raymond, Carol A.; Russell, Christopher T.

2017-11-01

The ice-rich crust of dwarf planet 1 Ceres is the source of a tenuous water exosphere, and the behavior of this putative exosphere is investigated with model calculations. Outgassing water molecules seasonally condense around the winter pole in an optically thin layer. This seasonal cap reaches an estimated mass of at least 2× {10}3 {kg}, and the aphelion summer pole may even retain water throughout summer. If this reservoir is suddenly released by a solar energetic particle event, it would form a denser transient water exosphere. Our model calculations also explore species other than H2O. Light exospheric species escape rapidly from Ceres due to its low gravity, and hence their exospheres dissipate soon after their respective source has faded. For example, the theoretical turn-over time in a water exosphere is only 7 hr. A significant fraction of CO2 and SO2 molecules can get trapped and stored in perennially shadowed regions at the current spin axis orientation, but not at the higher spin axis tilt, leaving H2O as the only common volatile expected to accumulate in polar cold traps over long timescales. The D/H fractionation during migration to the cold traps is only about 10%.

Agglomerated novel spray-dried lactose-leucine tailored as a carrier to enhance the aerosolization performance of salbutamol sulfate from DPI formulations.

PubMed

Molina, Carlos; Kaialy, Waseem; Chen, Qiao; Commandeur, Daniel; Nokhodchi, Ali

2017-12-19

Spray-drying allows to modify the physicochemical/mechanical properties of particles along with their morphology. In the present study, L -leucine with varying concentrations (0.1, 0.5, 1, 5, and 10% w/v) were incorporated into lactose monohydrate solution for spray-drying to enhance the aerosolization performance of dry powder inhalers containing spray-dried lactose-leucine and salbutamol sulfate. The prepared spray-dried lactose-leucine carriers were analyzed using laser diffraction (particle size), differential scanning calorimetry (thermal behavior), scanning electron microscopy (morphology), powder X-ray diffraction (crystallinity), Fourier transform infrared spectroscopy (interaction at molecular level), and in vitro aerosolization performance (deposition). The results showed that the efficacy of salbutamol sulfate's aerosolization performance was, in part, due to the introduction of L -leucine in the carrier, prior to being spray-dried, accounting for an increase in the fine particle fraction (FPF) of salbutamol sulfate from spray-dried lactose-leucine (0.5% leucine) in comparison to all other carriers. It was shown that all of the spray-dried carriers were spherical in their morphology with some agglomerates and contained a mixture of amorphous, α-lactose, and β-lactose. It was also interesting to note that spray-dried lactose-leucine particles were agglomerated during the spray-drying process to make coarse particles (volume mean diameter of 79 to 87 μm) suitable as a carrier in DPI formulations.

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.

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

PubMed Central

Moghei, Mahshid; Tavajohi-Fini, Pegah; Beatty, Brendan

2016-01-01

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

Crystal structure of a super leucine zipper, an extended two-stranded super long coiled coil

PubMed Central

Diao, Jiasheng

2010-01-01

Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 Å resolution. The peptide monomer shows a helix trunk with short curved N- and C-termini. In the crystal, two monomers cross in 35° and form an X-shaped dimer, and each X-shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two-stranded, parallel, super long coiled coil rather than a discrete, two-helix coiled coil of the wild-type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild-type leucine zipper, the N-terminus of the mutant has a dramatic conformational change and the C-terminus has one more residue Glu 32 determined. The mutant X-shaped dimer has a large crossing angle of 35° instead of 18° in the wild-type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self-assembling protein fibers. PMID:20027625

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

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

J Diao

2011-12-31

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

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

PubMed

Chen, Jin-Zhong; Wang, Shu; Tang, Rong; Yang, Quan-Sheng; Zhao, Enpeng; Chao, Yaoqiong; Ying, Kang; Xie, Yi; Mao, Yu-Min

2002-09-01

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

Leucine Affects α-Amylase Synthesis through PI3K/Akt-mTOR Signaling Pathways in Pancreatic Acinar Cells of Dairy Calves.

PubMed

Guo, Long; Liang, Ziqi; Zheng, Chen; Liu, Baolong; Yin, Qingyan; Cao, Yangchun; Yao, Junhu

2018-05-23

Dietary nutrient utilization, particularly starch, is potentially limited by digestion in dairy cow small intestine because of shortage of α-amylase. Leucine acts as an effective signal molecular in the mTOR signaling pathway, which regulates a series of biological processes, especially protein synthesis. It has been reported that leucine could affect α-amylase synthesis and secretion in ruminant pancreas, but mechanisms have not been elaborated. In this study, pancreatic acinar (PA) cells were used as a model to determine the cellular signal of leucine influence on α-amylase synthesis. PA cells were isolated from newborn Holstein dairy bull calves and cultured in Dulbecco's modifed Eagle's medium/nutrient mixture F12 liquid media containing four leucine treatments (0, 0.23, 0.45, and 0.90 mM, respectively), following α-amylase activity, zymogen granule, and signal pathway factor expression detection. Rapamycin, a specific inhibitor of mTOR, was also applied to PA cells. Results showed that leucine increased ( p < 0.05) synthesis of α-amylase as well as phosphorylation of PI3K, Akt, mTOR, and S6K1 while reduced ( p < 0.05) GCN2 expression. Inhibition of mTOR signaling downregulated the α-amylase synthesis. In addition, the extracellular leucine dosage significantly influenced intracellular metabolism of isoleucine ( p < 0.05). Overall, leucine regulates α-amylase synthesis through promoting the PI3K/Akt-mTOR pathway and reducing the GCN2 pathway in PA cells of dairy calves. These pathways form the signaling network that controls the protein synthesis and metabolism. It would be of great interest in future studies to explore the function of leucine in ruminant nutrition.

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...

Study on the influence of leucine-rich amelogenin peptide (LRAP) on the remineralization of enamel defects via micro-focus x-ray computed tomography and nanoindentation.

PubMed

Bagheri G, Hossein; Sadr, Alireza; Espigares, Jorge; Hariri, Ilnaz; Nakashima, Syozi; Hamba, Hidenori; Shafiei, Farhad; Moztarzadeh, Fathollah; Tagami, Junji

2015-06-04

Regeneration of severely damaged enamel (e.g. deep demineralized lesions) is currently not possible, because the structural units of enamel crystal construction are removed after its maturation. The aim of this in vitro study was to evaluate the effect of surface impregnation by leucine-rich amelogenin peptide (LRAP) on the remineralization of eroded enamel using micro-focus x-ray computed tomography (µCT). Fifteen bovine enamel blocks were embedded in resin and three zones (sound, demineralization, and remineralization) were defined on each specimen. Lesions were prepared by immersing the samples in demineralization solution for 7 d. The samples were soaked in distilled water or 60 or 120 µg mL(-1) solution of LRAP in water for 30 min. After the surface treatment, specimens were incubated in artificial saliva for either 5 or 10 d at 37 °C. The amount of mineral gain (dΔZ%) and the relative changes in the lesion depth (dLD%), obtained from µCT, were used to evaluate the effect of LRAP on the remineralization of lesions. The effects of LRAP on cross-sectional integrated hardness ΔINH were studied after 10 d using nanoindentation. ANOVA test was used to determine the effect of time and/or LRAP concentration on dΔZ%, dLD% and ΔINH mean values. Tukey's analysis was used for multiple comparison testing (α = 0.05). Analysis of µCT data showed significant effect of time and LRAP concentration on the dΔZ% (p = 0.013, p = 0.003) and the dLD% (p  <  0.001, p = 0.002) mean values. The nanoindentation hardness was significantly improved by 120 µg mL(-1) LRAP (p = 0.02). Also, the peptide treatment affected the mineral distribution throughout the lesion by inhibiting of superficial deposition. This study showed that the treatment of eroded lesions in enamel by LRAP can improve and regulate the pattern of remineralization in vitro.

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

The Biogeography of Putative Microbial Antibiotic Production

PubMed Central

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

2015-01-01

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

Age and sex affect protein metabolism at protein intakes that span the range of adequacy: comparison of leucine kinetics and nitrogen balance data.

PubMed

Conley, Travis B; McCabe, George P; Lim, Eunjung; Yarasheski, Kevin E; Johnson, Craig A; Campbell, Wayne W

2013-04-01

Research suggests that changes in leucine oxidation (leuox) with feeding may reflect adult protein requirements. We evaluated this possibility by assessing the effects of age, sex, and different protein intakes on whole-body leucine kinetics and nitrogen balance. Thirty-four young (n=18, 22-46 years) and old (n=16, 63-81 years) men and women completed three 18-day trials with protein intakes of 0.50, 0.75 and 1.00 g protein·kg body weight(-1)·d(-1). Fasting and fed-state leucine kinetics were quantified on day 12 of each trial using a primed, constant infusion of L-[1-13C]leucine. Protein requirement was estimated using classical nitrogen balance measurements and calculations. Leucine kinetics parameters were influenced by age and sex across all protein intakes. With feeding, leuox increased more in old vs. young adults. Independent of age, fasting and fed-state leuox were lower, and net leucine balance (fasting+fed-state) was higher in women vs. men. Among all subjects and protein intakes, nitrogen balance was correlated with fed-state leuox (r=0.39), fed-state leucine balance (r=0.60), net leucine balance (r=0.49) and the change in leuox from the fasting to fed state (r=0.49) (P<.05 for all results). At the highest protein intake, the change in leuox with feeding was inversely correlated with protein requirement (r=-0.39). These findings indicate that leucine kinetics, especially leuox, reflect nitrogen balance-based estimates of the need for dietary protein and generally support the view that protein requirement is comparable between young and old adults. Copyright © 2013 Elsevier Inc. All rights reserved.

Age and sex affect protein metabolism at protein intakes that span the range of adequacy: comparison of leucine kinetics and nitrogen balance data☆

PubMed Central

Conley, Travis B.; McCabe, George P.; Lim, Eunjung; Yarasheski, Kevin E.; Johnson, Craig A.; Campbell, Wayne W.

2012-01-01

Research suggests that changes in leucine oxidation (leuox) with feeding may reflect adult protein requirements. We evaluated this possibility by assessing the effects of age, sex, and different protein intakes on whole-body leucine kinetics and nitrogen balance. Thirty-four young (n = 18, 22–46 years) and old (n= 16, 63–81 years) men and women completed three 18-day trials with protein intakes of 0.50, 0.75 and 1.00 g protein·kg body weight−1·d−1. Fasting and fed-state leucine kinetics were quantified on day 12 of each trial using a primed, constant infusion of L-[1-13C]leucine. Protein requirement was estimated using classical nitrogen balance measurements and calculations. Leucine kinetics parameters were influenced by age and sex across all protein intakes. With feeding, leuox increased more in old vs. young adults. Independent of age, fasting and fed-state leuox were lower, and net leucine balance (fasting+fed-state) was higher in women vs. men. Among all subjects and protein intakes, nitrogen balance was correlated with fed-state leuox (r=0.39), fed-state leucine balance (r=0.60), net leucine balance (r=0.49) and the change in leuox from the fasting to fed state (r=0.49) (P<.05 for all results). At the highest protein intake, the change in leuox with feeding was inversely correlated with protein requirement (r=−0.39). These findings indicate that leucine kinetics, especially leuox, reflect nitrogen balance-based estimates of the need for dietary protein and generally support the view that protein requirement is comparable between young and old adults. PMID:22841544

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

PubMed

Elhiti, Mohamed; Stasolla, Claudio

2009-02-01

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

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.

Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine.

PubMed

Mazor, Kevin M; Dong, Leiming; Mao, Yuanhui; Swanda, Robert V; Qian, Shu-Bing; Stipanuk, Martha H

2018-05-24

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

Contrasting ability to take up leucine and thymidine among freshwater bacterial groups: implications for bacterial production measurements

PubMed Central

Pérez, María Teresa; Hörtnagl, Paul; Sommaruga, Ruben

2010-01-01

We examined the ability of different freshwater bacterial groups to take up leucine and thymidine in two lakes. Utilization of both substrates by freshwater bacteria was examined at the community level by looking at bulk incorporation rates and at the single-cell level by combining fluorescent in situ hybridization and signal amplification by catalysed reporter deposition with microautoradiography. Our results showed that leucine was taken up by 70–80% of Bacteria-positive cells, whereas only 15–43% of Bacteria-positive cells were able to take up thymidine. When a saturating substrate concentration in combination with a short incubation was used, 80–90% of Betaproteobacteria and 67–79% of Actinobacteria were positive for leucine uptake, whereas thymidine was taken up by < 10% of Betaproteobacteria and by < 1% of the R-BT subgroup that dominated this bacterial group. Bacterial abundance was a good predictor of the relative contribution of bacterial groups to leucine uptake, whereas when thymidine was used Actinobacteria represented the large majority (> 80%) of the cells taking up this substrate. Increasing the substrate concentration to 100 nM did not affect the percentage of R-BT cells taking up leucine (> 90% even at low concentrations), but moderately increased the fraction of thymidine-positive R-BT cells to a maximum of 35% of the hybridized cells. Our results show that even at very high concentrations, thymidine is not taken up by all, otherwise active, bacterial cells. PMID:19725866

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

PubMed

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

2016-04-01

A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members' expression in tomato.

PubMed

Zhang, Zhenzhu; Chen, Xiuling; Guan, Xin; Liu, Yang; Chen, Hongyu; Wang, Tingting; Mouekouba, Liana Dalcantara Ongouya; Li, Jingfu; Wang, Aoxue

2014-01-01

Homeodomain-leucine zipper (HD-Zip) proteins are a kind of transcriptional factors that play a vital role in plant growth and development. However, no detailed information of HD-Zip family in tomato has been reported till now. In this study, 51 HD-Zip genes (SlHZ01-51) in this family were identified and categorized into 4 classes by exon-intron and protein structure in tomato (Solanum lycopersicum) genome. The synthetical phylogenetic tree of tomato, Arabidopsis and rice HD-Zip genes were established for an insight into their evolutionary relationships and putative functions. The results showed that the contribution of segmental duplication was larger than that of tandem duplication for expansion and evolution of genes in this family of tomato. The expression profile results under abiotic stress suggested that all SlHZ I genes were responsive to cold stress. This study will provide a clue for the further investigation of functional identification and the role of tomato HD-Zip I subfamily in plant cold stress responses and developmental events.

Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-11-01

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

Identification of critical functional residues of receptor-like kinase ERECTA.

PubMed

Kosentka, Pawel Z; Zhang, Liang; Simon, Yonas A; Satpathy, Binita; Maradiaga, Richard; Mitoubsi, Omar; Shpak, Elena D

2017-03-01

In plants, extracellular signals are primarily sensed by plasma membrane-localized receptor-like kinases (RLKs). ERECTA is a leucine-rich repeat RLK that together with its paralogs ERECTA-like 1 (ERL1) and ERL2 regulates multiple aspects of plant development. ERECTA forms complexes with a range of co-receptors and senses secreted cysteine-rich small proteins from the EPF/EPFL family. Currently the mechanism of the cytoplasmic domain activation and transmission of the signal by ERECTA is unclear. To gain a better understanding we performed a structure-function analysis by introducing altered ERECTA genes into erecta and erecta erl1 erl2 mutants. These experiments indicated that ERECTA's ability to phosphorylate is functionally significant, and that while the cytoplasmic juxtamembrane domain is important for ERECTA function, the C-terminal tail is not. An analysis of multiple putative phosphorylation sites identified four amino acids in the activation segment of the kinase domain as functionally important. Homology of those residues to functionally significant amino acids in multiple other plant RLKs emphasizes similarities in RLK function. Specifically, our data predicts Thr812 as a primary site of phosphor-activation and potential inhibitory phosphorylation of Tyr815 and Tyr820. In addition, our experiments suggest that there are differences in the molecular mechanism of ERECTA function during regulation of stomata development and in elongation of above-ground organs. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Constitutive heterologous overexpression of a TIR-NB-ARC-LRR gene encoding a putative disease resistance protein from wild Chinese Vitis pseudoreticulata in Arabidopsis and tobacco enhances resistance to phytopathogenic fungi and bacteria.

PubMed

Wen, Zhifeng; Yao, Liping; Singer, Stacy D; Muhammad, Hanif; Li, Zhi; Wang, Xiping

2017-03-01

Plants use resistance (R) proteins to detect pathogen effector proteins and activate their innate immune response against the pathogen. The majority of these proteins contain an NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain along with a leucine-rich repeat (LRR), and some also bear a toll interleukin 1 receptor (TIR) domain. In this study, we characterized a gene encoding a TIR-NB-ARC-LRR R protein (VpTNL1) (GenBank accession number KX649890) from wild Chinese grapevine Vitis pseudoreticulata accession "Baihe-35-1", which was identified previously from a transcriptomic analysis of leaves inoculated with powdery mildew (PM; Erysiphe necator (Schw.)). The VpTNL1 transcript was found to be highly induced in V. pseudoreticulata following inoculation with E. necator, as well as treatment with salicylic acid (SA). Sequence analysis demonstrated that the deduced amino acid sequence contained a TIR domain at the N-terminus, along with an NB-ARC and four LRRs domains within the C-terminus. Constitutive expression of VpTNL1 in Arabidopsis thaliana resulted in either a wild-type or dwarf phenotype. Intriguingly, the phenotypically normal transgenic lines displayed enhanced resistance to Arabidopsis PM, Golovinomyces cichoracearum, as well as to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Similarly, constitutive expression of VpTNL1 in Nicotiana tabacum was found to confer enhanced resistance to tobacco PM, Erysiphe cichoacearum DC. Subsequent isolation of the VpTNL1 promoter and deletion analysis indicated that TC-rich repeats and TCA elements likely play an important role in its response to E. necator and SA treatment, respectively. Taken together, these results indicate that VpTNL1 contributes to PM resistance in grapevine and provide an interesting gene target for the future amelioration of grape via breeding and/or biotechnology. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Enteral leucine and protein synthesis in skeletal and cardiac muscle

USDA-ARS?s Scientific Manuscript database

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

Functional insights from the distribution and role of homopeptide repeat-containing proteins

PubMed Central

Faux, Noel G.; Bottomley, Stephen P.; Lesk, Arthur M.; Irving, James A.; Morrison, John R.; de la Banda, Maria Garcia; Whisstock, James C.

2005-01-01

Expansion of “low complex” repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP—involved in important housekeeping processes—retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asparagines, and alanine. Interestingly, while poly-Q repeats are found in vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling. PMID:15805494

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.

Origin of a folded repeat protein from an intrinsically disordered ancestor

PubMed Central

Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

2016-01-01

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin. DOI: http://dx.doi.org/10.7554/eLife.16761.001 PMID:27623012

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.

Serine-Rich Repeat Proteins and Pili Promote Streptococcus agalactiae Colonization of the Vaginal Tract ▿

PubMed Central

Sheen, Tamsin R.; Jimenez, Alyssa; Wang, Nai-Yu; Banerjee, Anirban; van Sorge, Nina M.; Doran, Kelly S.

2011-01-01

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ΔpilA or Δsrr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment. PMID:21984789

Low temperature investigations of dynamic properties in l-leucine - chloranilic acid complex.

PubMed

Hetmańczyk, J; Nowicka-Scheibe, J; Maurin, J K; Pawlukojć, A

2018-07-05

Inelastic neutron scattering (INS) and infra-red (IR) spectroscopy methods were used for determination of dynamic structure of l-leucine - chloranilic acid complex. A theoretical dynamic pattern calculated by the density functional theory (DFT) method for periodic boundary conditions accompanied the experimental ones. Normal modes in the vibrational spectra were defined and described. The characteristic presence of the Hadži's trio enriched by numerous submaxima is observed in the wavenumber range 3200-800 cm -1 . Bands assigned to CH 3 torsion vibrations in the leucine cation were observed at 231 cm -1 and 258 cm -1 in the INS spectrum. Temperature-dependent far-infrared spectra in the temperature range 9 K-290 K were obtained. Vibrational bands were analyzed as a function of temperature. Activation energies for reorientational motion of CH 3 and CH 2 groups were determined by means of the band shape analysis performed for torsional and twisting vibrations of these groups. The estimated energy is equal to E a  = 2.7 ± 0.2 kJ/mol and E a  = 2.17 ± 0.12 kJ/mol for CH 3 and CH 2 groups, respectively. A phase transition at about 130 K in the l-leucine - chloranilic acid complex was observed. Copyright © 2018 Elsevier B.V. All rights reserved.

Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex.

PubMed

Man, Si Ming; Hopkins, Lee J; Nugent, Eileen; Cox, Susan; Glück, Ivo M; Tourlomousis, Panagiotis; Wright, John A; Cicuta, Pietro; Monie, Tom P; Bryant, Clare E

2014-05-20

Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro-IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.

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.

Application of the [3H]Leucine Incorporation Technique for Quantification of Bacterial Secondary Production Associated with Decaying Wetland Plant Litter

PubMed Central

Gillies, Jane E.; Kuehn, Kevin A.; Francoeur, Steven N.; Neely, Robert K.

2006-01-01

The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 μM) required to achieve saturation-level uptake. We assessed the application of the [3H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 μM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [3H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [3H]leucine into protein exhibited a biphasic saturation curve, with lower apparent Km values ranging from 400 nM to 4.2 μM depending on the plant species studied. Upper apparent Km values ranged from 1.3 to 59 μM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent Km values possibly representing bacterial uptake and higher apparent Km values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake. PMID:16957215

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

Effects of intraduodenal infusion of the branched-chain amino acid leucine on ad libitum eating, gut motor and hormone functions, and glycemia in healthy men.

PubMed

Steinert, Robert E; Landrock, Maria F; Ullrich, Sina S; Standfield, Scott; Otto, Bärbel; Horowitz, Michael; Feinle-Bisset, Christine

2015-10-01

Branched-chain amino acids (BCAAs), particularly leucine, act as nutrient signals regulating protein synthesis and degradation as well as glucose metabolism. In addition, leucine has been demonstrated in animal experiments to modulate eating and energy homeostasis. We aimed to characterize the effects of physiologic and supraphysiologic loads of intraduodenal leucine on eating, gut hormone and motor functions, and blood glucose in humans. Twelve lean men were studied on 3 occasions in a randomized, double-blind order. Antropyloroduodenal motility, plasma ghrelin, cholecystokinin, glucagon-like peptide 1, peptide YY, insulin, glucagon, blood glucose, appetite perceptions, and gastrointestinal symptoms were measured during 90-min intraduodenal infusions of leucine at 0.15 kcal/min (total 3.3 g, 13.5 kcal), 0.45 kcal/min (total 9.9 g, 40.5 kcal), or saline (control). Ad libitum eating from a buffet lunch was quantified immediately after the infusions. Leucine at 0.45 kcal/min inhibited eating (energy intake by ∼13%, P < 0.05), increased plasma cholecystokinin, slightly reduced blood glucose and increased plasma insulin, and decreased antral pressures (all P < 0.05). Leucine at 0.15 kcal/min had no effect on food intake, blood glucose, or antral pressures but also slightly increased plasma cholecystokinin (P < 0.05). Neither dose affected plasma ghrelin, glucagon, glucagon-like peptide 1 and peptide YY, or pyloric and duodenal pressures. Plasma leucine concentrations were related to the dose of intraduodenal leucine, with substantial increases during both 0.15 and 0.45 kcal/min. The effects of intraduodenal infusions of free leucine on eating are probably not primarily mediated by changes in gut motor and hormone functions, with perhaps the exception of cholecystokinin. Instead, increased plasma leucine concentrations may be a potential signal mediating the eating-inhibitory effect of leucine. The study was registered as a clinical trial with the Australia and New

Consuming Lower-Protein Nutrition Bars with Added Leucine Elicits Postprandial Changes in Appetite Sensations in Healthy Women.

PubMed

Bolster, Douglas R; Rahn, Maike; Kamil, Alison G; Bristol, Lindsey T; Goltz, Shellen R; Leidy, Heather J; Blaze Mt, Melvin; Nunez, Michael A; Guo, Elizabeth; Wang, Jianquan; Harkness, Laura S

2018-04-20

Higher-protein meals (>25 g protein/meal) have been associated with enhanced satiety but the role of amino acids is unclear. Leucine has been proposed to stimulate satiety in rodents but has not been assessed in humans. We assessed the acute effects of lower-protein nutrition bars, enhanced with a leucine peptide (LP), on postprandial appetite sensations in combination with plasma leucine and peptide YY (PYY) in healthy women. Utilizing a double-blind randomized crossover design, 40 healthy women [28 ± 7.5 y; body mass index (BMI, in kg/m2): 23.5 ± 2.4] consumed the following isocaloric (180 kcal) pre-loads on 3 separate visits: control bar [9 g protein with 0 g added LP (0-g LP)] or treatment bars [11 g protein with 2 g added LP (2-g LP) or 13 g protein with 3 g added LP (3-g LP)]. Pre- and postprandial hunger, desire to eat, prospective food consumption (PFC), fullness, and plasma leucine were assessed every 30 min for 240 min. Plasma PYY was assessed hourly for 240 min (n = 24). Main effects of time (P < 0.0001) and treatment (P < 0.03) were detected for postprandial hunger, desire to eat, PFC, and fullness. Post hoc analyses revealed that the 2-g and 3-g LP bars elicited greater increases in fullness and greater decreases in PFC compared with 0-g LP (all, P < 0.05) with no differences between the 2-g and 3-g LP bars. The 2-g bar elicited greater decreases in hunger and desire to eat compared with the 0-g LP bar (both, P ≤ 0.01), whereas 3-g LP did not. Appetite incremental areas under the curves (iAUCs) and PYY outcomes were not different between bars. A treatment × time interaction was detected for plasma leucine with increases occurring in a leucine-dose-dependent manner (P < 0.0001). Despite the dose-dependent increases in plasma leucine following the consumption of lower-protein bars enhanced with LP, only the 2-g LP bar elicited consistent postprandial changes in select appetite sensations compared with the 0-g LP bar. This study was

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.

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.

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

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.

RNA-binding Protein Trinucleotide repeat-containing 6A Regulates the Formation of Circular RNA 0006916, with Important Functions in Lung Cancer Cells.

PubMed

Dai, Xin; Zhang, Nan; Cheng, Ying; Yang, Ti; Chen, Yingnan; Liu, Zhenzhong; Wang, Zhishan; Yang, Chengfeng; Jiang, Yiguo

2018-05-03

Circular RNAs (circRNAs) are widespread and diverse endogenous RNAs distinct from traditional linear RNAs, which may regulate gene expression in eukaryotes. However, the function of human circRNAs, including their potential role in lung cancer, remains largely unknown. We screened the circRNA circ0006916, which was evidently down-regulated in 16HBE-T cells (anti-benzopyrene-trans-7, 8-dihydrodiol-9, 10-epoxide-transformed human bronchial epithelial cells), and in A549 and H460 cell lines. Silencing of circ0006916, but not its parental gene homer scaffolding protein 1 (HOMER1), promoted cell proliferation via speeding up the cell cycle process rather than by inhibiting apoptosis; conversely, overexpression of circ0006916 had the opposite effect. Luciferase screening assay indicated that circ0006916 bound to miR-522-3p and inhibited pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) activity. We also explored the effect of the RNA-binding protein trinucleotide repeat-containing 6A (TNRC6A) on circ0006916 production. Circ0006916 expression was decreased after silencing TNRC6A. TNRC6A bound to the intron regions around the circRNA-forming exons of circ0006916, as shown by RNA immunoprecipitation assay combined with sequencing analysis. The association of circ0006916 with TNRC6A was further verified by RNA pull-down assays. We then constructed a carrier and confirmed that TNRC6A binding to the flanked intron region of circ0006916 was necessary for generation of circ0006916. These results demonstrate that TNRC6A regulates the biogenesis of the circRNA circ0006916, which has a regulatory role in cell growth.

The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation

PubMed Central

Aubee, Joseph I.; Olu, Morenike

2016-01-01

The translation of rpoS (σS), the general stress/stationary phase sigma factor, is tightly regulated at the post-transcriptional level by several factors via mechanisms that are not clearly defined. One of these factors is MiaA, the enzyme necessary for the first step in the N6-isopentyl-2-thiomethyladenosinemethyladenosine 37 (ms2i6A37) tRNA modification. We tested the hypothesis that an elevated UUX-Leucine/total leucine codon ratio can be used to identify transcripts whose translation would be sensitive to loss of the MiaA-dependent modification. We identified iraP as another candidate MiaA-sensitive gene, based on the UUX-Leucine/total leucine codon ratio. An iraP-lacZ fusion was significantly decreased in the absence of MiaA, consistent with our predictive model. To determine the role of MiaA in UUX-Leucine decoding in rpoS and iraP, we measured β-galactosidase-specific activity of miaA− rpoS and iraP translational fusions upon overexpression of leucine tRNAs. We observed suppression of the MiaA effect on rpoS, and not iraP, via overexpression of tRNALeuX but not tRNALeuZ. We also tested the hypothesis that the MiaA requirement for rpoS and iraP translation is due to decoding of UUX-Leucine codons within the rpoS and iraP transcripts, respectively. We observed a partial suppression of the MiaA requirement for rpoS and iraP translational fusions containing one or both UUX-Leucine codons removed. Taken together, this suggests that MiaA is necessary for rpoS and iraP translation through proper decoding of UUX-Leucine codons and that rpoS and iraP mRNAs are both modification tunable transcripts (MoTTs) via the presence of the modification. PMID:26979278

Prolonged leucine infusion differentially affects tissue protein synthesis in neonatal pigs

USDA-ARS?s Scientific Manuscript database

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

The effect of a high protein diet on leucine and alanine turnover in acid maltase deficiency.

PubMed Central

Umpleby, A M; Trend, P S; Chubb, D; Conaglen, J V; Williams, C D; Hesp, R; Scobie, I N; Wiles, C M; Spencer, G; Sönksen, P H

1989-01-01

Leucine and alanine production rate was measured in 5 patients with acid maltase deficiency in the postabsorptive state, following 6 months on a normal diet with placebo and 6 months on an isocaloric high protein diet (16-22% protein). Whole body leucine production rate, a measure of protein degradation, expressed in terms of lean body mass was significantly greater than in five control subjects. Following the high protein diet, leucine production rate was decreased in four of the five patients but this was not statistically significant. Alanine production rate expressed in terms of lean body mass was significantly greater than in control subjects. After the high protein diet, alanine production rate and concentration were significantly decreased (p less than 0.05). There were no significant improvements in any of the clinically relevant variables measured in these patients. It is possible that a larger increase in protein intake over a longer time period may have a clinical effect. PMID:2507747

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

Identification of positive selection in disease response genes within members of the Poaceae.

PubMed

Rech, Gabriel E; Vargas, Walter A; Sukno, Serenella A; Thon, Michael R

2012-12-01

Millions of years of coevolution between plants and pathogens can leave footprints on their genomes and genes involved on this interaction are expected to show patterns of positive selection in which novel, beneficial alleles are rapidly fixed within the population. Using information about upregulated genes in maize during Colletotrichum graminicola infection and resources available in the Phytozome database, we looked for evidence of positive selection in the Poaceae lineage, acting on protein coding sequences related with plant defense. We found six genes with evidence of positive selection and another eight with sites showing episodic selection. Some of them have already been described as evolving under positive selection, but others are reported here for the first time including genes encoding isocitrate lyase, dehydrogenases, a multidrug transporter, a protein containing a putative leucine-rich repeat and other proteins with unknown functions. Mapping positively selected residues onto the predicted 3-D structure of proteins showed that most of them are located on the surface, where proteins are in contact with other molecules. We present here a set of Poaceae genes that are likely to be involved in plant defense mechanisms and have evidence of positive selection. These genes are excellent candidates for future functional validation.

Report on the development of putative functional SSR and SNP markers in passion fruits.

PubMed

da Costa, Zirlane Portugal; Munhoz, Carla de Freitas; Vieira, Maria Lucia Carneiro

2017-09-06

Passionflowers Passiflora edulis and Passiflora alata are diploid, outcrossing and understudied fruit bearing species. In Brazil, passion fruit cultivation began relatively recently and has earned the country an outstanding position as the world's top producer of passion fruit. The fruit's main economic value lies in the production of juice, an essential exotic ingredient in juice blends. Currently, crop improvement strategies, including those for underexploited tropical species, tend to incorporate molecular genetic approaches. In this study, we examined a set of P. edulis transcripts expressed in response to infection by Xanthomonas axonopodis, (the passion fruit's main bacterial pathogen that attacks the vines), aiming at the development of putative functional markers, i.e. SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). A total of 210 microsatellites were found in 998 sequences, and trinucleotide repeats were found to be the most frequent (31.4%). Of the sequences selected for designing primers, 80.9% could be used to develop SSR markers, and 60.6% SNP markers for P. alata. SNPs were all biallelic and found within 15 gene fragments of P. alata. Overall, gene fragments generated 10,003 bp. SNP frequency was estimated as one SNP every 294 bp. Polymorphism rates revealed by SSR and SNP loci were 29.4 and 53.6%, respectively. Passiflora edulis transcripts were useful for the development of putative functional markers for P. alata, suggesting a certain level of sequence conservation between these cultivated species. The markers developed herein could be used for genetic mapping purposes and also in diversity studies.

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis

PubMed Central

Müller, Pari-Sima; Schulz, Ramona; Maretto, Silvia; Costello, Ita; Srinivas, Shankar; Bikoff, Elizabeth; Robertson, Elizabeth

2011-01-01

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium. PMID:21350012

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

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.

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

Effect of dietary nutrients on ileal endogenous losses of threonine, cysteine, methionine, lysine, leucine and protein in broiler chicks.

PubMed

Cerrate, S; Vignale, S K; Ekmay, R; England, J; Coon, C

2018-04-01

An isotope dose technique was utilized (i) to determine endogenous amino acid (AA) and protein losses and (ii) to propose adjusted values for AA requirements. The endogenous flow rate was calculated from the pool of enrichment in plasma AA, assuming similitude to enrichment of endogenous AA. In experiment 1, chicks were orally administered D4-lysine at 2% of estimated lysine intake from 16 to 24 days to find the isotopic steady state of the atom percent excess (APE) of lysine for plasma and jejunal and ileal digesta. The APE of D4-lysine in plasma, jejunal digesta and ileal digesta reached the isotopic steady state at 5.5, 3.4 and 2.0 days, respectively, by using the broken-line model. It was assumed that the isotopic steady state at 5 days identified for D4-lysine is also representative for the 15N-labeled AA. In experiment 2, chicks were fed diets from 1 to 21 days with increasing levels of fat (6%, 8%, 12%, 13% extract ether), protein (26%, 28.5%, 31% CP) or fiber (14%, 16%, 18% NDF) by adding poultry fat, soybean meal, blended animal protein or barley. Chicks were orally administered 15N-threonine, 15N-cysteine, 15N-methionine, 15N-lysine and 15N-leucine at 2% of estimated daily intake for 5 days from 17 to 21 days of age. Dietary nutrients influenced endogenous losses (EL), where dietary fat stimulated EL of lysine (P=0.06), leucine and protein (P=0.07); dietary protein enhanced EL of leucine and protein; and finally the dietary fiber increased EL of leucine. Dietary nutrients also affected apparent ileal digestibility (AID). Dietary fat increased AID of cysteine but decreased AID of lysine. Dietary protein reduced AID of protein, threonine, lysine and leucine, and similarly dietary fiber decreased AID of protein, threonine, methionine, lysine and leucine. In contrast, dietary fat or protein did not affect real ileal digestibility (RID) of protein and AA except threonine and leucine. The dietary fiber reduced the RID of protein, threonine and leucine. This

l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

PubMed Central

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E.; Gerton, Jennifer L.

2015-01-01

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

PubMed

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

2015-03-15

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. © The Author 2014. Published by Oxford University Press.

Diverse roles of ERECTA family genes in plant development.

PubMed

Shpak, Elena D

2013-12-01

Multiple receptor-like kinases (RLKs) enable intercellular communication that coordinates growth and development of plant tissues. ERECTA family receptors (ERfs) are an ancient family of leucine-rich repeat RLKs that in Arabidopsis consists of three genes: ERECTA, ERL1, and ERL2. ERfs sense secreted cysteine-rich peptides from the EPF/EPFL family and transmit the signal through a MAP kinase cascade. This review discusses the functions of ERfs in stomata development, in regulation of longitudinal growth of aboveground organs, during reproductive development, and in the shoot apical meristem. In addition the role of ERECTA in plant responses to biotic and abiotic factors is examined. Elena D. Shpak (Corresponding author). © 2013 Institute of Botany, Chinese Academy of Sciences.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the

Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene.

PubMed Central

Koch, K S; Gleiberman, A S; Aoki, T; Leffert, H L; Feren, A; Jones, A L; Fodor, E J

1995-01-01

An unusual S1-nuclease sensitive microsatellite (STMS) has been found in the single copy, rat polymeric immunoglobulin receptor gene (PIGR) terminal exon. In Fisher rats, elements within or beyond the STMS are expressed variably in the 3' untranslated regions (3'UTRs) of two 'Groups' of PIGR-encoded hepatic mRNAs (pIg-R) during liver regeneration. STMS elements include neighboring constant regions (a 60-bp d[GA]-rich tract with a chi-like octamer, followed by 15 tandem d[GGA] repeats) that merge directly with 36 or 39 tandem d[GAA] repeats (Fisher or Wistar strains, respectively) interrupted by d[AA] between their 5th-6th repeat units. The Wistar STMS is flanked upstream by two regions of nearly contiguous d[CA] or d[CT] repeats in the 3' end of intron 8; and downstream, by a 283 bp 'unit' containing several inversions at its 5' end, and two polyadenylation signals at its 3' end. The 283 nt unit is expressed in Group 1 pIg-R mRNAs; but it is absent in the Group 2 family so that their GAA repeats merge with their poly A tails. In contrast to genomic sequence, GGA triplet repeats are amplified (n > or = 24-26), whereas GAA triplet repeats are truncated variably (n < or = 9-37) and expressed uninterruptedly in both mRNA Groups. These results suggest that 3' end processing of the rat PIGR gene may involve misalignment, slippage and premature termination of RNA polymerase II. The function of this unusual processing and possible roles of chi-like octamers in quiescent or extrahepatic tissues are discussed. Images PMID:7739889

Effects of dietary leucine and phenylalanine on pancreas development, enzyme activity, and relative gene expression in milk-fed Holstein dairy calves.

PubMed

Cao, Y C; Yang, X J; Guo, L; Zheng, C; Wang, D D; Cai, C J; Liu, S M; Yao, J H

2018-05-01

This study aimed to investigate the effect of dietary supplementation with leucine and phenylalanine on pancreas development, enzyme activity, and related gene expression in male Holstein calves. Twenty male Holstein calves [1 d of age, 38 ± 3 kg of body weight (BW)] were randomly assigned to 1 of the following 4 treatment groups with 5 calves in each group: control, leucine supplementation (1.435 g/L of milk), phenylalanine supplementation (0.725 g/L of milk), and leucine and phenylalanine (1.435 + 0.725 g/L of milk). The diets were made isonitrogenous with the inclusion of alanine in each respective treatment. The feeding trial lasted for 8 wk, including 1 wk for adaption and 7 wk for the feeding experiment. Leucine tended to increase the concentration of total pancreatic protein (mg/kg of BW). Phenylalanine increased the concentrations of plasma insulin, cholecystokinin, and pancreatic DNA (mg/g) and the expression of trypsin gene but decreased the pancreatic protein:DNA ratio and tended to decrease the pancreas weight (g/kg of BW). No differences were observed in total pancreatic DNA (mg/pancreas and mg/kg of BW), pancreatic protein (mg/pancreas), or activities of α-amylase, trypsin, and lipase. The relative expression levels of the genes encoding α-amylase and lipase did not differ among the 4 groups. The supplementation of both leucine and phenylalanine showed an interaction on the pancreas weight (g and g/kg of BW) and a tendency of an interaction on the pancreatic protein concentration (mg/g of pancreas and mg/kg of BW) and the plasma glucose concentration. Leucine tended to increase the size of the pancreatic cells, whereas phenylalanine tended to increase the number of pancreatic cells. However, neither AA affected the activities of the pancreatic enzymes of the calves. These results indicate that leucine and phenylalanine supplementation in milk-fed Holstein calves differentially affect pancreatic growth and development. Copyright © 2018 American

Electrophoretically mediated microanalysis of leucine aminopeptidase using two-photon excited fluorescence detection on a microchip.

PubMed

Zugel, S A; Burke, B J; Regnier, F E; Lytle, F E

2000-11-15

Two-photon excited fluorescence detection was performed on a microfabricated electrophoresis chip. A calibration curve of the fluorescent tag beta-naphthylamine was performed, resulting in a sensitivity of 2.5 x 10(9) counts M(-1) corresponding to a detection limit of 60 nM. Additionally, leucine aminopeptidase was assayed on the chip using electrophoretically mediated microanalysis. The differential electroosmotic mobilities of the enzyme and substrate, L-leucine beta-naphthylamide, allowed for efficient mixing in an open channel, resulting in the detection of a 30 nM enzyme solution under constant potential. A zero potential incubation for 1 min yielded a calculated detection limit of 4 nM enzyme.

Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean.

PubMed

Burt, Andrew J; William, H Manilal; Perry, Gregory; Khanal, Raja; Pauls, K Peter; Kelly, James D; Navabi, Alireza

2015-01-01

Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co-4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co-4 is localized. Three SCAR markers with known linkage to Co-4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK-4 loci found in previous studies. It is possible that the Co-4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases.

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

Investigating intermolecular forces associated with thrombus initiation using optical tweezers

NASA Astrophysics Data System (ADS)

Arya, Maneesh; Lopez, Jose A.; Romo, Gabriel M.; Dong, Jing-Fei; McIntire, Larry V.; Moake, Joel L.; Anvari, Bahman

2002-05-01

Thrombus formation occurs when a platelet membrane receptor, glycoprotein (GP) Ib-IX-V complex, binds to its ligand, von Willebrand factor (vWf), in the subendothelium or plasma. To determine which GP Ib-IX-V amino acid sequences are critical for bond formation, we have used optical tweezers to measure forces involved in the binding of vWf to GP Ib-IX-V variants. Inasmuch as GP Ib(alpha) subunit is the primary component in human GP Ib-IX-V complex that binds to vWf, and that canine GP Ib(alpha) , on the other hand, does not bind to human vWf, we progressively replaced human GP Ib(alpha) amino acid sequences with canine GP Ib(alpha) sequences to determine the sequences essential for vWf/GP Ib(alpha) binding. After measuring the adhesive forces between optically trapped, vWf-coated beads and GP Ib(alpha) variants expressed on mammalian cells, we determined that leucine- rich repeat 2 of GP Ib(alpha) was necessary for vWf/GP Ib-IX- V bond formation. We also found that deletion of the N- terminal flanking sequence and leucine-rich repeat 1 reduced adhesion strength to vWf but did not abolish binding. While divalent cations are known to influence binding of vWf, addition of 1mM CaCl2 had no effect on measured vWf/GP Ib(alpha) bond strengths.

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis

PubMed Central

Anderson, Ryan G.; Cherkis, Karen A.; Law, Terry F.; Liu, Qingli L.; Machius, Mischa; Nimchuk, Zachary L.; Yang, Li; Chung, Eui-Hwan; El Kasmi, Farid; Hyunh, Michael; Sondek, John E.; Dangl, Jeffery L.

2017-01-01

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll–interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR–TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that “truncated” NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system. PMID:28137883

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

ERIC Educational Resources Information Center

Meyer, Scott C.

2015-01-01

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

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

In silico analysis of the polygalacturonase inhibiting protein 1 from apple, Malus domestica.

PubMed

Matsaunyane, Lerato Bt; Oelofse, Dean; Dubery, Ian A

2015-03-11

The Malus domestica polygalacturonase inhibiting protein 1 (MdPGIP1) gene, encoding the M. domestica polygalacturonase inhibiting protein 1 (MdPGIP1), was isolated from the Granny Smith apple cultivar (GenBank accession no. DQ185063). The gene was used to transform tobacco and potato for enhanced resistance against fungal diseases. Analysis of the MdPGIP1 nucleotide sequence revealed that the gene comprises 993 nucleotides that encode a 330 amino acid polypeptide. In silico characterization of the MdPGIP1 polypeptide revealed domains typical of PGIP proteins, which include a 24 amino acid putative signal peptide, a potential cleavage site [Alanine-Leucine-Serine (ALS)] for the signal peptide, a 238 amino acid leucine-rich repeat (LRR) domain, a 46 amino acid N-terminal domain and a 22 amino acid C-terminal domain. The hydropathic evaluation of MdPGIP1 indicated a repetitive hydrophobic motif in the LRR domain and a hydrophilic surface area consistent with a globular protein. The typical consensus glycosylation sequence of Asn-X-Ser/Thr was identified in MdPGIP1, indicating potential N-linked glycosylation of MdPGIP1. The molecular mass of non-glycosylated MdPGIP1 was calculated as 36.615 kDa and the theoretical isoelectric point as 6.98. Furthermore, the secondary and tertiary structure of MdPGIP1 was modelled, and revealed that MdPGIP1 is a curved and elongated molecule that contains sheet B1, sheet B2 and 310-helices on its LRR domain. The overall properties of the MdPGIP1 protein is similar to that of the prototypical Phaseolus vulgaris PGIP 2 (PvPGIP2), and the detected differences supported its use in biotechnological applications as an inhibitor of targeted fungal polygalacturonases (PGs).

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

PubMed Central

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

2015-01-01

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

A Rich-Club Organization in Brain Ischemia Protein Interaction Network

PubMed Central

Alawieh, Ali; Sabra, Zahraa; Sabra, Mohammed; Tomlinson, Stephen; Zaraket, Fadi A.

2015-01-01

Ischemic stroke involves multiple pathophysiological mechanisms with complex interactions. Efforts to decipher those mechanisms and understand the evolution of cerebral injury is key for developing successful interventions. In an innovative approach, we use literature mining, natural language processing and systems biology tools to construct, annotate and curate a brain ischemia interactome. The curated interactome includes proteins that are deregulated after cerebral ischemia in human and experimental stroke. Network analysis of the interactome revealed a rich-club organization indicating the presence of a densely interconnected hub structure of prominent contributors to disease pathogenesis. Functional annotation of the interactome uncovered prominent pathways and highlighted the critical role of the complement and coagulation cascade in the initiation and amplification of injury starting by activation of the rich-club. We performed an in-silico screen for putative interventions that have pleiotropic effects on rich-club components and we identified estrogen as a prominent candidate. Our findings show that complex network analysis of disease related interactomes may lead to a better understanding of pathogenic mechanisms and provide cost-effective and mechanism-based discovery of candidate therapeutics. PMID:26310627

Identification of a "glycine-loop"-like coiled structure in the 34 AA Pro,Gly,Met repeat domain of the biomineral-associated protein, PM27.

PubMed

Wustman, Brandon A; Santos, Rudolpho; Zhang, Bo; Evans, John Spencer

2002-12-05

Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain. Copyright 2002 Wiley Periodicals, Inc.

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.

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

PubMed Central

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

2012-01-01

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

Identification of the centromeric repeat in the threespine stickleback fish (Gasterosteus aculeatus).

PubMed

Cech, Jennifer N; Peichel, Catherine L

2015-12-01

Centromere sequences exist as gaps in many genome assemblies due to their repetitive nature. Here we take an unbiased approach utilizing centromere protein A (CENP-A) chomatin immunoprecipitation followed by high-throughput sequencing to identify the centromeric repeat sequence in the threespine stickleback fish (Gasterosteus aculeatus). A 186-bp, AT-rich repeat was validated as centromeric using both fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on interphase nuclei and metaphase spreads. This repeat hybridizes strongly to the centromere on all chromosomes, with the exception of weak hybridization to the Y chromosome. Together, our work provides the first validated sequence information for the threespine stickleback centromere.

The 15N-leucine single-injection method allows for determining endogenous losses and true digestibility of amino acids in cecectomized roosters

PubMed Central

Hu, Rujiu; Li, Jing; Soomro, Rab Nawaz; Wang, Fei; Feng, Yan; Yang, Xiaojun

2017-01-01

This study was conducted to assess the influence of dietary protein content in poultry when using the 15N-leucine single-injection method to determine endogenous amino acid losses (EAALs) in poultry. Forty-eight cecectomized roosters (2.39 ± 0.23 kg) were randomly allocated to eight dietary treatments containing protein levels of 0, 3%, 6%, 9%, 12%, 15%, 18% and 21%. Each bird was precisely fed an experimental diet of 25 g/kg of body weight. After feeding, all roosters were subcutaneously injected with a 15N-leucine solution at a dose of 20 mg/kg of body weight. Blood was sampled 23 h after the injection, and excreta samples were continuously collected during the course of the 48-h experiment. The ratio of 15N-enrichment of leucine in crude mucin to free leucine in plasma ranged from 0.664 to 0.763 and remained relatively consistent (P > 0.05) across all treatments. The amino acid (AA) profiles of total endogenous AAs, except isoleucine, alanine, aspartic acid, cysteine, proline and serine, were not influenced (P > 0.05) by dietary protein contents. The predominant endogenous AAs in the excreta were glutamic acid, aspartic acid, threonine, serine and proline. The order of the relative proportions of these predominant AAs also remained relatively constant (P > 0.05). The endogenous losses of total AAs determined with the 15N-leucine single-injection method increased curvilinearly with the dietary protein contents. The true digestibility of most AAs and total AAs was independent of their respective dietary protein levels. Collectively, the 15N-leucine single-injection method is appropriate for determining EAALs and the true digestibility of AAs in poultry fed varying levels of protein-containing ingredients. PMID:29166671

The 15N-leucine single-injection method allows for determining endogenous losses and true digestibility of amino acids in cecectomized roosters.

PubMed

Hu, Rujiu; Li, Jing; Soomro, Rab Nawaz; Wang, Fei; Feng, Yan; Yang, Xiaojun; Yao, Junhu

2017-01-01

This study was conducted to assess the influence of dietary protein content in poultry when using the 15N-leucine single-injection method to determine endogenous amino acid losses (EAALs) in poultry. Forty-eight cecectomized roosters (2.39 ± 0.23 kg) were randomly allocated to eight dietary treatments containing protein levels of 0, 3%, 6%, 9%, 12%, 15%, 18% and 21%. Each bird was precisely fed an experimental diet of 25 g/kg of body weight. After feeding, all roosters were subcutaneously injected with a 15N-leucine solution at a dose of 20 mg/kg of body weight. Blood was sampled 23 h after the injection, and excreta samples were continuously collected during the course of the 48-h experiment. The ratio of 15N-enrichment of leucine in crude mucin to free leucine in plasma ranged from 0.664 to 0.763 and remained relatively consistent (P > 0.05) across all treatments. The amino acid (AA) profiles of total endogenous AAs, except isoleucine, alanine, aspartic acid, cysteine, proline and serine, were not influenced (P > 0.05) by dietary protein contents. The predominant endogenous AAs in the excreta were glutamic acid, aspartic acid, threonine, serine and proline. The order of the relative proportions of these predominant AAs also remained relatively constant (P > 0.05). The endogenous losses of total AAs determined with the 15N-leucine single-injection method increased curvilinearly with the dietary protein contents. The true digestibility of most AAs and total AAs was independent of their respective dietary protein levels. Collectively, the 15N-leucine single-injection method is appropriate for determining EAALs and the true digestibility of AAs in poultry fed varying levels of protein-containing ingredients.

Empirical Leucine-to-Carbon Conversion Factors for Estimating Heterotrophic Bacterial Production: Seasonality and Predictability in a Temperate Coastal Ecosystem▿

PubMed Central

Calvo-Díaz, Alejandra; Morán, Xosé Anxelu G.

2009-01-01

Leucine-to-carbon conversion factors (CFs) are needed for converting substrate incorporation into biomass production of heterotrophic bacteria. During 2006 we performed 20 dilution experiments for determining the spatiotemporal variability of empirical CFs in temperate Atlantic coastal waters. Values (0.49 to 1.92 kg C mol Leu−1) showed maxima in autumn to early winter and minima in summer. Spatially averaged CFs were significantly negatively correlated with in situ leucine incorporation rates (r = −0.91) and positively correlated with phosphate concentrations (r = 0.76). These relationships, together with a strong positive covariation between cell-specific leucine incorporation rates and carbon contents (r = 0.85), were interpreted as a strategy to maximize survival through protein synthesis and low growth rates under nutrient limitation (low CFs) until favorable conditions stimulate cell division relative to protein synthesis (high CFs). A multiple regression with in situ leucine incorporation rates and cellular carbon contents explained 96% of CF variance in our ecosystem, suggesting their potential prediction from more easily measurable routine variables. The use of the theoretical CF of 1.55 kg C mol Leu−1 would have resulted in a serious overestimation (73%) of annual bacterial production rates. Our results emphasize the need for considering the temporal scale in CFs for bacterial production studies. PMID:19304821

Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach.

PubMed

Paal, Jürgen; Henselewski, Heike; Muth, Jost; Meksem, Khalid; Menéndez, Cristina M; Salamini, Francesco; Ballvora, Agim; Gebhardt, Christiane

2004-04-01

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.

Involvement of the Neutral Amino Acid Transporter SLC6A15 and Leucine in Obesity-Related Phenotypes

PubMed Central

Drgonova, Jana; Jacobsson, Josefin A.; Han, Joan C.; Yanovski, Jack A.; Fredriksson, Robert; Marcus, Claude; Schiöth, Helgi B.; Uhl, George R.

2013-01-01

Brain pathways, including those in hypothalamus and nucleus of the solitary tract, influence food intake, nutrient preferences, metabolism and development of obesity in ways that often differ between males and females. Branched chain amino acids, including leucine, can suppress food intake, alter metabolism and change vulnerability to obesity. The SLC6A15 (v7-3) gene encodes a sodium-dependent transporter of leucine and other branched chain amino acids that is expressed by neurons in hypothalamus and nucleus of the solitary tract. We now report that SLC6A15 knockout attenuates leucine's abilities to reduce both: a) intake of normal chow and b) weight gain produced by access to a high fat diet in gender-selective fashions. We identify SNPs in the human SLC6A15 that are associated with body mass index and insulin resistance in males. These observations in mice and humans support a novel, gender-selective role for brain amino acid compartmentalization mediated by SLC6A15 in diet and obesity-associated phenotypes. PMID:24023709

Arginine supplementation modulates pig plasma lipids, but not hepatic fatty acids, depending on dietary protein level with or without leucine.

PubMed

Madeira, Marta Sofia Morgado Dos Santos; Rolo, Eva Sofia Alves; Pires, Virgínia Maria Rico; Alfaia, Cristina Maria Riscado Pereira Mateus; Coelho, Diogo Francisco Maurício; Lopes, Paula Alexandra Antunes Brás; Martins, Susana Isabel Vargas; Pinto, Rui Manuel Amaro; Prates, José António Mestre

2017-05-30

In the present study, the effect of arginine and leucine supplementation, and dietary protein level, were investigated in commercial crossbred pigs to clarify their individual or combined impact on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid sensitive factors. The experiment was conducted on fifty-four entire male pigs (Duroc × Pietrain × Large White × Landrace crossbred) from 59 to 92 kg of live weight. Each pig was randomly assigned to one of six experimental treatments (n = 9). The treatments followed a 2 × 3 factorial arrangement, providing two levels of arginine supplementation (0 vs. 1%) and three levels of basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet with 2% of leucine, RPDL). Significant interactions between arginine supplementation and protein level were observed across plasma lipids. While dietary arginine increased total lipids, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and triacylglycerols in NPD, the inverse effect was observed in RPD. Overall, dietary treatments had a minor impact on hepatic fatty acid composition. RPD increased 18:1c9 fatty acid while the combination of leucine and RPD reduced 18:0 fatty acid. Arginine supplementation increased the gene expression of FABP1, which contributes for triacylglycerols synthesis without affecting hepatic fatty acids content. RPD, with or without leucine addition, upregulated the lipogenic gene CEBPA but downregulated the fat oxidation gene LPIN1. Arginine supplementation was responsible for a modulated effect on plasma lipids, which is dependent on dietary protein level. It consistently increased lipaemia in NPD, while reducing the correspondent metabolites in RPD. In contrast, arginine had no major impact, neither on hepatic fatty acids content nor on fatty acid composition. Likewise, leucine supplementation of RPD, regardless the presence of arginine, promoted no changes on total fatty acids in

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

Deuterium incorporation experiments from (3R)- and (3S)-[3-2H]leucine into characteristic isoprenoidal lipid-core of halophilic archaea suggests the involvement of isovaleryl-CoA dehydrogenase.

PubMed

Yamauchi, Noriaki; Tanoue, Ryo

2017-11-01

The stereochemical reaction course for the two C-3 hydrogens of leucine to produce a characteristic isoprenoidal lipid in halophilic archaea was observed using incubation experiments with whole cell Halobacterium salinarum. Deuterium-labeled (3R)- and (3S)-[3- 2 H]leucine were freshly prepared as substrates from 2,3-epoxy-4-methyl-1-pentanol. Incorporation of deuterium from (3S)-[3- 2 H]leucine and loss of deuterium from (3R)-[3- 2 H]leucine in the lipid-core of H. salinarum was observed. Taken together with the results of our previous report, involving the incubation of chiral-labeled [5- 2 H]leucine, these results strongly suggested an involvement of isovaleryl-CoA dehydrogenase in leucine conversion to isoprenoid lipid in halophilic archaea. The stereochemical course of the reaction (anti-elimination) might have been the same as that previously reported for mammalian enzyme reactions. Thus, these results suggested that branched amino acids were metabolized to mevalonate in archaea in a manner similar to other organisms.

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

EPA Science Inventory

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

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.

Are the TTAGG and TTAGGG telomeric repeats phylogenetically conserved in aculeate Hymenoptera?

NASA Astrophysics Data System (ADS)

Menezes, Rodolpho S. T.; Bardella, Vanessa B.; Cabral-de-Mello, Diogo C.; Lucena, Daercio A. A.; Almeida, Eduardo A. B.

2017-10-01

Despite the (TTAGG)n telomeric repeat supposed being the ancestral DNA motif of telomeres in insects, it was repeatedly lost within some insect orders. Notably, parasitoid hymenopterans and the social wasp Metapolybia decorata (Gribodo) lack the (TTAGG)n sequence, but in other representatives of Hymenoptera, this motif was noticed, such as different ant species and the honeybee. These findings raise the question of whether the insect telomeric repeat is or not phylogenetically predominant in Hymenoptera. Thus, we evaluated the occurrence of both the (TTAGG)n sequence and the vertebrate telomere sequence (TTAGGG)n using dot-blotting hybridization in 25 aculeate species of Hymenoptera. Our results revealed the absence of (TTAGG)n sequence in all tested species, elevating the number of hymenopteran families lacking this telomeric sequence to 13 out of the 15 tested families so far. The (TTAGGG)n was not observed in any tested species. Based on our data and compiled information, we suggest that the (TTAGG)n sequence was putatively lost in the ancestor of Apocrita with at least two subsequent independent regains (in Formicidae and Apidae).

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

USDA-ARS?s Scientific Manuscript database

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

Repeat region of Brugia malayi sheath protein (Shp-1) carries Dominant B epitopes recognized in filarial endemic population.

PubMed

Jawaharlal, Jeya Prita Parasurama; Madhumathi, Jayaprakasam; Prince, Rajaiah Prabhu; Kaliraj, Perumal

2014-09-01

Transmission of lymphatic filariasis is mediated through microfilariae (L1 stage of the parasite) which is encased in an eggshell called sheath. The sheath protein Shp-1 stabilizes the structure due to the unique repeat region with Met-Pro-Pro-Gln-Gly sequences. Microfilarial proteins could be used as transmission blocking vaccines. Since the repeat region of Shp-1 was predicted to carry putative B epitopes, this region was used to analyze its reactivity with clinical samples towards construction of peptide vaccine. In silico analysis of Shp-1 showed the presence of B epitopes in the region 49-107. The polypeptide epitopic region Shp-149-107 was cloned and expressed in Escherichia coli. Antibody reactivity of the Shp-149-107 construct was evaluated in filarial endemic population by ELISA. Putatively immune endemic normals (EN) showed significantly high reactivity (P < 0.05) when compared to all the other categories. Antibody reactivity of Shp-1 repeat region was similar to that of whole protein proving that this region carries B epitopes responsible for its humoral response in humans. Thus this can be employed for inducing anti-microfilarial immunity in the infected population that may lead to reduction in transmission intensity and also it could be used along with other epitopes from different stages of the parasite in order to manage the disease effectively.

Homez, a homeobox leucine zipper gene specific to the vertebrate lineage.

PubMed

Bayarsaihan, Dashzeveg; Enkhmandakh, Badam; Makeyev, Aleksandr; Greally, John M; Leckman, James F; Ruddle, Frank H

2003-09-02

This work describes a vertebrate homeobox gene, designated Homez (homeodomain leucine zipper-encoding gene), that encodes a protein with an unusual structural organization. There are several regions within Homez, including three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously expressed in human and murine tissues, although the expression pattern is more restricted during mouse development. Genomic analysis revealed that human and mouse genes are located at 14q11.2 and 14C, respectively, and are composed of two exons. The zebrafish and pufferfish homologs share high similarity to mammalian sequences, particularly within the homeodomain sequences. Based on homology of homeodomains and on the similarity in overall protein structure, we delineate Homez and members of ZHX family of zinc finger homeodomain factors as a subset within the superfamily of homeobox-containing proteins. The type and composition of homeodomains in the Homez subfamily are vertebrate-specific. Phylogenetic analysis indicates that Homez lineage was separated from related genes >400 million years ago before separation of ray- and lobe-finned fishes. We apply a duplication-degeneration-complementation model to explain how this family of genes has evolved.

Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics

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

Lichtenstein, A.H.; Cohn, J.S.; Hachey, D.L.

1990-09-01

The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of (5,5,5,-2H3)leucine, (4,4,4,-2H3)valine, and (6,6-2H2,1,2-13C2)lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine)more » mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine), and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.« less

Leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent regeneration.

PubMed

Takayama, Kazuya; Muto, Akihiko; Kikuchi, Yutaka

2018-05-29

In animal regeneration, control of position-dependent cell proliferation is crucial for the complete restoration of patterned appendages in terms of both, shape and size. However, detailed mechanisms of this process are largely unknown. In this study, we identified leucine/glutamine and v-ATPase/lysosomal acidification, via mechanistic target of rapamycin complex 1 (mTORC1) activation, as effectors of amputation plane-dependent zebrafish caudal fin regeneration. mTORC1 activation, which functions in cell proliferation, was regulated by lysosomal acidification possibly via v-ATPase activity at 3 h post amputation (hpa). Inhibition of lysosomal acidification resulted in reduced growth factor-related gene expression and suppression of blastema formation at 24 and 48 hpa, respectively. Along the proximal-distal axis, position-dependent lysosomal acidification and mTORC1 activation were observed from 3 hpa. We also report that Slc7a5 (L-type amino acid transporter), whose gene expression is position-dependent, is necessary for mTORC1 activation upstream of lysosomal acidification during fin regeneration. Furthermore, treatment with leucine and glutamine, for both proximal and distal fin stumps, led to an up-regulation in cell proliferation via mTORC1 activation, indicating that leucine/glutamine signaling possesses the ability to change the position-dependent regeneration. Our findings reveal that leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent zebrafish fin regeneration.

The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

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

Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidencemore » towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.« less

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

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

Pelletier, V.; Marks, L.; Wagner, D.A.

1991-08-01

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

Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex.

PubMed

Onischenko, Evgeny; Tang, Jeffrey H; Andersen, Kasper R; Knockenhauer, Kevin E; Vallotton, Pascal; Derrer, Carina P; Kralt, Annemarie; Mugler, Christopher F; Chan, Leon Y; Schwartz, Thomas U; Weis, Karsten

2017-11-02

Nuclear pore complexes (NPCs) are ∼100 MDa transport channels assembled from multiple copies of ∼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

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

Leucine, Not Total Protein, Content of a Supplement Is the Primary Determinant of Muscle Protein Anabolic Responses in Healthy Older Women.

PubMed

Devries, Michaela C; McGlory, Chris; Bolster, Douglas R; Kamil, Alison; Rahn, Maike; Harkness, Laura; Baker, Steven K; Phillips, Stuart M

2018-06-13

Older adults show a blunted muscle protein synthesis (MPS) response to postprandial hyperaminoacidemia relative to younger adults. Evidence suggests that this anabolic resistance can be overcome by consuming greater quantities of leucine. The purpose of this trial was to determine whether the addition of leucine to a smaller dose (10 g) of milk proteins would, when compared with a larger dose (25 g) of whey protein isolate (WPI), result in similar increases in acute (hourly) and integrated (daily) myofibrillar protein synthesis (myoPS). Healthy older (mean ± SD age: 69 ± 1 y) women (n = 11/group) were randomly assigned with the use of a single-blind, parallel-group design to twice-daily consumption of either WPI [25 g WPI (3 g l-leucine)] or leucine (LEU; 10 g milk protein with 3 g total l-leucine) for 6 d. Participants performed unilateral resistance exercise to allow assessment of the impact of the supplement alone and with resistance exercise. We determined acute (13C6-phenylanine) and integrated [using deuterated water (D2O)] rates of myoPS in the fasting (acute), basal (integrated), nonexercised, and exercised states. Acute myoPS increased in both legs in response to LEU (fed: 45%; fed+exercise: 71%; P < 0.001) and WPI (fed: 29%; fed+exercise: 47%; P < 0.001) compared with fasting; the increase was greater with LEU than with WPI in the exercised leg (46%; P = 0.04) but not in the rested leg (P = 0.07). The acute myoPS response was greater in the exercised leg than in the rested leg for both WPI (63%) and LEU (58%) (P < 0.001). Integrated myoPS increased with WPI and LEU in the exercised leg (both 9%; P < 0.001) during supplementation, and with WPI (3%; P = 0.02) but not LEU (2%, P = 0.1) in the rested leg compared with the basal state. A lower-protein (10 compared with 25 g/dose), leucine-matched beverage induced similar increases in acute and integrated myoPS in healthy older women. Lower-protein supplements with added leucine may

A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.

PubMed

Kogi, M; Fukushige, S; Lefevre, C; Hadano, S; Ikeda, J E

1997-06-01

In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.

Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in Pseudomonas aeruginosa

PubMed Central

Förster-Fromme, Karin; Höschle, Birgit; Mack, Christina; Bott, Michael; Armbruster, Wolfgang; Jendrossek, Dieter

2006-01-01

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (atuABCDEFGH and liuRABCDE) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens. This result concurred with the finding that P. fluorescens, but not P. putida, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR, was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster. PMID:16820476

Medium-Chain Triglycerides in Combination with Leucine and Vitamin D Increase Muscle Strength and Function in Frail Elderly Adults in a Randomized Controlled Trial.

PubMed

Abe, Sakiko; Ezaki, Osamu; Suzuki, Motohisa

2016-05-01

Sarcopenia, the loss of skeletal muscle mass, strength, and function, is common in elderly individuals but difficult to treat. A combination of nutrients was investigated to treat sarcopenia in very frail elderly adults. We enrolled 38 elderly nursing home residents (11 men and 27 women with a mean ± SD age of 86.6 ± 4.8 y) in a 3-mo randomized, controlled, single-blind, parallel group trial. The participants were randomly allocated to 3 groups. The first group received a daily l-leucine (1.2 g) and cholecalciferol (20 μg)-enriched supplement with 6 g medium-chain triglycerides (TGs) (MCTs) (LD + MCT); the second group received the same leucine and cholecalciferol-enriched supplement with 6 g long-chain TGs (LD + LCT); and the third group did not receive any supplements (control). The supplement and oils were taken at dinner, and changes in muscle mass, strength, and function were monitored. The increase in body weight in the LD + MCT (1.1 ± 1.0 kg) and LD + LCT (0.8 ± 1.1 kg) groups was greater than that in the control group (-0.5 ± 0.9 kg) (P < 0.05). After 3 mo, participants in the LD + MCT group had a 13.1% increase in right-hand grip strength (1.2 ± 1.0 kg, P < 0.01), a 12.5% increase in walking speed (0.078 ± 0.080 m/s, P < 0.05), a 68.2% increase in a 10-s leg open-and-close test performance (2.31 ± 1.68 n/10 s, P < 0.001), and a 28.2% increase in peak expiratory flow (53 ± 59 L/min, P < 0.01). No significant improvements in muscle mass, strength, or function were observed in the LD + LCT or control groups. The combined supplementation of MCTs (6 g), leucine-rich amino acids, and cholecalciferol at dinner may improve muscle strength and function in frail elderly individuals. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as UMIN000017567. © 2016 American Society for Nutrition.

Leucine partially protects muscle mass and function during bed rest in middle-aged adults1,2

PubMed Central

English, Kirk L; Mettler, Joni A; Ellison, Jennifer B; Mamerow, Madonna M; Arentson-Lantz, Emily; Pattarini, James M; Ploutz-Snyder, Robert; Sheffield-Moore, Melinda; Paddon-Jones, Douglas

2016-01-01

Background: Physical inactivity triggers a rapid loss of muscle mass and function in older adults. Middle-aged adults show few phenotypic signs of aging yet may be more susceptible to inactivity than younger adults. Objective: The aim was to determine whether leucine, a stimulator of translation initiation and skeletal muscle protein synthesis (MPS), can protect skeletal muscle health during bed rest. Design: We used a randomized, double-blind, placebo-controlled trial to assess changes in skeletal MPS, cellular signaling, body composition, and skeletal muscle function in middle-aged adults (n = 19; age ± SEM: 52 ± 1 y) in response to leucine supplementation (LEU group: 0.06 g ∙ kg−1 ∙ meal−1) or an alanine control (CON group) during 14 d of bed rest. Results: Bed rest decreased postabsorptive MPS by 30% ± 9% (CON group) and by 10% ± 10% (LEU group) (main effect for time, P < 0.05), but no differences between groups with respect to pre-post changes (group × time interactions) were detected for MPS or cell signaling. Leucine protected knee extensor peak torque (CON compared with LEU group: −15% ± 2% and −7% ± 3%; group × time interaction, P < 0.05) and endurance (CON compared with LEU: −14% ± 3% and −2% ± 4%; group × time interaction, P < 0.05), prevented an increase in body fat percentage (group × time interaction, P < 0.05), and reduced whole-body lean mass loss after 7 d (CON compared with LEU: −1.5 ± 0.3 and −0.8 ± 0.3 kg; group × time interaction, P < 0.05) but not 14 d (CON compared with LEU: −1.5 ± 0.3 and −1.0 ± 0.3 kg) of bed rest. Leucine also maintained muscle quality (peak torque/kg leg lean mass) after 14 d of bed-rest inactivity (CON compared with LEU: −9% ± 2% and +1% ± 3%; group × time interaction, P < 0.05). Conclusions: Bed rest has a profoundly negative effect on muscle metabolism, mass, and function in middle-aged adults. Leucine supplementation may partially protect muscle health during relatively

Molecular characterization and origin of novel bipartite cold-regulated ice recrystallization inhibition proteins from cereals.

PubMed

Tremblay, Karine; Ouellet, François; Fournier, Julie; Danyluk, Jean; Sarhan, Fathey

2005-06-01

To understand the molecular basis of freezing tolerance in plants, several low temperature-responsive genes have been identified from wheat. Among these are two genes named TaIRI-1 and TaIRI-2 (Triticum aestivum ice recrystallization inhibition) that are up-regulated during cold acclimation in freezing-tolerant species. Phytohormones involved in pathogen defense pathways (jasmonic acid and ethylene) induce the expression of one of the two genes. The encoded proteins are novel in that they have a bipartite structure that has never been reported for antifreeze proteins. Their N-terminal part shows similarity with the leucine-rich repeat-containing regions present in the receptor domain of receptor-like protein kinases, and their C-terminus is homologous to the ice-binding domain of some antifreeze proteins. The recombinant TaIRI-1 protein inhibits the growth of ice crystals, confirming its function as an ice recrystallization inhibition protein. The TaIRI genes were found only in the species belonging to the Pooideae subfamily of cereals. Comparative genomic analysis suggested that molecular evolutionary events took place in the genome of freezing-tolerant cereals to give rise to these genes with putative novel functions. These apparent adaptive DNA rearrangement events could be part of the molecular mechanisms that ensure the survival of hardy cereals in the harsh freezing environments.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

PubMed Central

Guaitoli, Giambattista; Raimondi, Francesco; Gilsbach, Bernd K.; Gómez-Llorente, Yacob; Deyaert, Egon; Renzi, Fabiana; Li, Xianting; Schaffner, Adam; Jagtap, Pravin Kumar Ankush; Boldt, Karsten; von Zweydorf, Felix; Gotthardt, Katja; Lorimer, Donald D.; Yue, Zhenyu; Burgin, Alex; Janjic, Nebojsa; Sattler, Michael; Versées, Wim; Ueffing, Marius; Ubarretxena-Belandia, Iban; Kortholt, Arjan; Gloeckner, Christian Johannes

2016-01-01

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. PMID:27357661

The immunotranscriptome of the Caribbean reef-building coral Pseudodiploria strigosa.

PubMed

Ocampo, Iván D; Zárate-Potes, Alejandra; Pizarro, Valeria; Rojas, Cristian A; Vera, Nelson E; Cadavid, Luis F

2015-09-01

The viability of coral reefs worldwide has been seriously compromised in the last few decades due in part to the emergence of coral diseases of infectious nature. Despite important efforts to understand the etiology and the contribution of environmental factors associated to coral diseases, the mechanisms of immune response in corals are just beginning to be studied systematically. In this study, we analyzed the set of conserved immune response genes of the Caribbean reef-building coral Pseudodiploria strigosa by Illumina-based transcriptome sequencing and annotation of healthy colonies challenged with whole live Gram-positive and Gram-negative bacteria. Searching the annotated transcriptome with immune-related terms yielded a total of 2782 transcripts predicted to encode conserved immune-related proteins that were classified into three modules: (a) the immune recognition module, containing a wide diversity of putative pattern recognition receptors including leucine-rich repeat-containing proteins, immunoglobulin superfamily receptors, representatives of various lectin families, and scavenger receptors; (b) the intracellular signaling module, containing components from the Toll-like receptor, transforming growth factor, MAPK, and apoptosis signaling pathways; and (3) the effector module, including the C3 and factor B complement components, a variety of proteases and protease inhibitors, and the melanization-inducing phenoloxidase. P. strigosa displays a highly variable and diverse immune recognition repertoire that has likely contributed to its resilience to coral diseases.

mRNA export: threading the needle

PubMed Central

Gaouar, Ouassila; Germain, Hugo

2013-01-01

After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress. PMID:23526740

Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation*

PubMed Central

Dixon, Emma V.; Claridge, Jolyon K.; Harvey, David J.; Baruah, Kavitha; Yu, Xiaojie; Vesiljevic, Snezana; Mattick, Susan; Pritchard, Laura K.; Krishna, Benjamin; Scanlan, Christopher N.; Schnell, Jason R.; Higgins, Matthew K.; Zitzmann, Nicole; Crispin, Max

2014-01-01

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans. PMID:24668806

Molecular characteristics of insect vitellogenins and vitellogenin receptors.

PubMed

Sappington, T W; Raikhel, A S

1998-01-01

shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.

Compartmental Modeling and Dosimetry of in Vivo Metabolic Studies of Leucine and Three Secretory Proteins in Humans Using Radioactive Tracers

NASA Astrophysics Data System (ADS)

Venkatakrishnan, Vaidehi

1995-01-01

Physical and mathematical models provide a systematic means of looking at biological systems. Radioactive tracer kinetic studies open a unique window to study complex tracee systems such as protein metabolism in humans. This research deals with compartmental modeling of tracer kinetic data on leucine and apolipoprotein metabolism obtained using an endogenous tritiated leucine tracer administered as a bolus, and application of compartmental modeling techniques for dosimetric evaluation of metabolic studies of radioiodinated apolipoproteins. Dr. Waldo R. Fisher, Department of Medicine, was the coordinating research supervisor and the work was carried out in his laboratory. A compartmental model for leucine kinetics in humans has been developed that emphasizes its recycling pathways which were examined over two weeks. This model builds on a previously published model of Cobelli et al, that analyzed leucine kinetic data up to only eight hours. The proposed model includes different routes for re-entry of leucine from protein breakdown into plasma accounting for proteins which turn over at different rates. This new model successfully incorporates published models of three secretory proteins: albumin, apoA-I, and VLDL apoB, in toto thus increasing its validity and utility. The published model of apoA-I, based on an exogenous radioiodinated tracer, was examined with data obtained using an endogenous leucine tracer using compartmental techniques. The analysis concludes that the major portion of apoA-I enters plasma by a fast pathway but the major fraction of apoA-I in plasma resides with a second slow pathway; further the study is suggestive of a precursor-product relationship between the two plasma apoA-I pools. The possible relevance of the latter suggestion to the aberrant kinetics of apoA-I in Tangier disease is discussed. The analysis of apoA-II data resulted in similar conclusions. A methodology for evaluating the dosimetry of radioiodinated apolipoproteins by

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

Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean

PubMed Central

Burt, Andrew J.; William, H. Manilal; Perry, Gregory; Khanal, Raja; Pauls, K. Peter; Kelly, James D.; Navabi, Alireza

2015-01-01

Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co–4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co–4 is localized. Three SCAR markers with known linkage to Co–4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK–4 loci found in previous studies. It is possible that the Co–4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases. PMID:26431031

NIK1-mediated translation suppression functions as a plant antiviral immunity mechanism.

PubMed

Zorzatto, Cristiane; Machado, João Paulo B; Lopes, Kênia V G; Nascimento, Kelly J T; Pereira, Welison A; Brustolini, Otávio J B; Reis, Pedro A B; Calil, Iara P; Deguchi, Michihito; Sachetto-Martins, Gilberto; Gouveia, Bianca C; Loriato, Virgílio A P; Silva, Marcos A C; Silva, Fabyano F; Santos, Anésia A; Chory, Joanne; Fontes, Elizabeth P B

2015-04-30

Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus-plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defence mechanism similar to that employed in non-viral infections. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses. Here we demonstrate in Arabidopsis that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a newly identified MYB-like protein, L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), to downregulate translational machinery genes fully. LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral messenger RNA association with polysome fractions and enhanced tolerance to begomovirus. By contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

NLRP3 Upregulation in Retinal Pigment Epithelium in Age-Related Macular Degeneration.

PubMed

Wang, Yujuan; Hanus, Jakub W; Abu-Asab, Mones S; Shen, Defen; Ogilvy, Alexander; Ou, Jingxing; Chu, Xi K; Shi, Guangpu; Li, Wei; Wang, Shusheng; Chan, Chi-Chao

2016-01-08

Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1β) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.

Comparative Genomics of Non-TNL Disease Resistance Genes from Six Plant Species.

PubMed

Nepal, Madhav P; Andersen, Ethan J; Neupane, Surendra; Benson, Benjamin V

2017-09-30

Disease resistance genes (R genes), as part of the plant defense system, have coevolved with corresponding pathogen molecules. The main objectives of this project were to identify non-Toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (nTNL) genes and elucidate their evolutionary divergence across six plant genomes. Using reference sequences from Arabidopsis , we investigated nTNL orthologs in the genomes of common bean, Medicago , soybean, poplar, and rice. We used Hidden Markov Models for sequence identification, performed model-based phylogenetic analyses, visualized chromosomal positioning, inferred gene clustering, and assessed gene expression profiles. We analyzed 908 nTNL R genes in the genomes of the six plant species, and classified them into 12 subgroups based on the presence of coiled-coil (CC), nucleotide binding site (NBS), leucine rich repeat (LRR), resistance to Powdery mildew 8 (RPW8), and BED type zinc finger domains. Traditionally classified CC-NBS-LRR (CNL) genes were nested into four clades (CNL A-D) often with abundant, well-supported homogeneous subclades of Type-II R genes. CNL-D members were absent in rice, indicating a unique R gene retention pattern in the rice genome. Genomes from Arabidopsis , common bean, poplar and soybean had one chromosome without any CNL R genes. Medicago and Arabidopsis had the highest and lowest number of gene clusters, respectively. Gene expression analyses suggested unique patterns of expression for each of the CNL clades. Differential gene expression patterns of the nTNL genes were often found to correlate with number of introns and GC content, suggesting structural and functional divergence.

Comparative Genomics of Non-TNL Disease Resistance Genes from Six Plant Species

PubMed Central

Andersen, Ethan J.; Neupane, Surendra; Benson, Benjamin V.

2017-01-01

Disease resistance genes (R genes), as part of the plant defense system, have coevolved with corresponding pathogen molecules. The main objectives of this project were to identify non-Toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (nTNL) genes and elucidate their evolutionary divergence across six plant genomes. Using reference sequences from Arabidopsis, we investigated nTNL orthologs in the genomes of common bean, Medicago, soybean, poplar, and rice. We used Hidden Markov Models for sequence identification, performed model-based phylogenetic analyses, visualized chromosomal positioning, inferred gene clustering, and assessed gene expression profiles. We analyzed 908 nTNL R genes in the genomes of the six plant species, and classified them into 12 subgroups based on the presence of coiled-coil (CC), nucleotide binding site (NBS), leucine rich repeat (LRR), resistance to Powdery mildew 8 (RPW8), and BED type zinc finger domains. Traditionally classified CC-NBS-LRR (CNL) genes were nested into four clades (CNL A-D) often with abundant, well-supported homogeneous subclades of Type-II R genes. CNL-D members were absent in rice, indicating a unique R gene retention pattern in the rice genome. Genomes from Arabidopsis, common bean, poplar and soybean had one chromosome without any CNL R genes. Medicago and Arabidopsis had the highest and lowest number of gene clusters, respectively. Gene expression analyses suggested unique patterns of expression for each of the CNL clades. Differential gene expression patterns of the nTNL genes were often found to correlate with number of introns and GC content, suggesting structural and functional divergence. PMID:28973974

NLRP3 Upregulation in Retinal Pigment Epithelium in Age-Related Macular Degeneration

PubMed Central

Wang, Yujuan; Hanus, Jakub W.; Abu-Asab, Mones S.; Shen, Defen; Ogilvy, Alexander; Ou, Jingxing; Chu, Xi K.; Shi, Guangpu; Li, Wei; Wang, Shusheng; Chan, Chi-Chao

2016-01-01

Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1β) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis. PMID:26760997

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.