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Sample records for acids including proline

  1. Probing the "additive effect" in the proline and proline hydroxamic acid catalyzed asymmetric addition of nitroalkanes to cyclic enones.

    PubMed

    Hanessian, Stephen; Govindan, Subramaniyan; Warrier, Jayakumar S

    2005-11-01

    The effect of chirality and steric bulk of 2,5-disubstituted piperazines as additives in the conjugate addition of 2-nitropropane to cyclohexenone, catalyzed by l-proline, was investigated. Neither chirality nor steric bulk affects the enantioselectivity of addition, which gives 86-93% ee in the presence of achiral and chiral nonracemic 2,5-disubstituted piperazines. Proline hydroxamic acid is shown for the first time to be an effective organocatalyst in the same Michael reaction. PMID:16189834

  2. Enhanced L-lactic acid production in Lactobacillus paracasei by exogenous proline addition based on comparative metabolite profiling analysis.

    PubMed

    Tian, Xiwei; Wang, Yonghong; Chu, Ju; Zhuang, Yingping; Zhang, Siliang

    2016-03-01

    This study investigated cell physiological and metabolic responses of Lactobacillus paracasei to osmotic stresses. Both cellular fatty acid composition and metabolite profiling were responded by increasing unsaturated and epoxy-fatty acid proportions, as well as accumulating some specific intracellular metabolites. Simultaneously, metabolite profiling was adopted to rationally and systematically discover potential osmoprotectants. Consequently, exogenous addition of proline or aspartate was validated to be a feasible and efficacious approach to improve cell growth under hyperosmotic stress in shake flasks. Particularly, with 5-L cultivation system, L-lactic acid concentration increased from 108 to 150 g/L during the following 16-h fermentation in 2 g/L proline addition group, while it only increased from 110 to 140 g/L in no proline addition group. Moreover, glucose consumption rate with proline addition reached 3.49 g/L/h during this phase, 35.8 % higher than that with no proline addition. However, extreme high osmotic pressure would significantly limit the osmoprotection of proline, and the osmolality threshold for L. paracasei was approximately 3600 mOsm/kg. It was suggested that proline principally played a role as a compatible solute accumulated in the cell for hyperosmotic preservation. The strategies of exploiting osmotic protectant with metabolite profiling and enhancing L-lactic acid production by osmoprotectant addition would be potential to provide a new insight for other microorganisms and organic acids production. PMID:26658821

  3. On the influence of low-energy ionizing radiation on the amino acid molecule: proline

    NASA Astrophysics Data System (ADS)

    Tamuliene, Jelena; Romanova, Liudmila; Vukstich, Vasyl; Papp, Alexander; Shkurin, Serhiy; Baliulyte, Laura; Snegursky, Alexander

    2016-06-01

    New data on the electron-impact fragmentation of the amino acid proline molecule are presented as being related to the formation of the ionized products due to the influence of low-energy ionizing radiation on the above molecule. An extensive DFT-theory based on the theoretical approach enabled the main pathways of the proline molecules fragmentation to be elucidated. A series of the produced fragments have been identified. The absolute appearance energies for some of them have been both measured experimentally and calculated theoretically. The data of the experimental studies and theoretical calculations are compared and analyzed. Contribution to the Topical Issue "Low-Energy Interactions related to Atmospheric and Extreme Conditions", edited by S. Ptasinska, M. Smialek-Telega, A. Milosavljevic, B. Sivaraman.

  4. Proline: Mother Nature’s cryoprotectant applied to protein crystallography

    PubMed Central

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-01-01

    l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography. PMID:22868767

  5. Proline: Mother Nature;s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-09-05

    L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

  6. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif.

    PubMed

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-03-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  7. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif

    PubMed Central

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-01-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain 161PPLP164 regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the 161PPLP164 domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  8. Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

    PubMed Central

    Zaprasis, Adrienne; Bleisteiner, Monika; Kerres, Anne; Hoffmann, Tamara

    2014-01-01

    The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. PMID:25344233

  9. LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen.

    PubMed Central

    Schwacke, R; Grallath, S; Breitkreuz, K E; Stransky, E; Stransky, H; Frommer, W B; Rentsch, D

    1999-01-01

    During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes. PMID:10072398

  10. Proline metabolism and transport in plant development.

    PubMed

    Lehmann, Silke; Funck, Dietmar; Szabados, László; Rentsch, Doris

    2010-10-01

    Proline fulfils diverse functions in plants. As amino acid it is a structural component of proteins, but it also plays a role as compatible solute under environmental stress conditions. Proline metabolism involves several subcellular compartments and contributes to the redox balance of the cell. Proline synthesis has been associated with tissues undergoing rapid cell divisions, such as shoot apical meristems, and appears to be involved in floral transition and embryo development. High levels of proline can be found in pollen and seeds, where it serves as compatible solute, protecting cellular structures during dehydration. The proline concentrations of cells, tissues and plant organs are regulated by the interplay of biosynthesis, degradation and intra- as well as intercellular transport processes. Among the proline transport proteins characterized so far, both general amino acid permeases and selective compatible solute transporters were identified, reflecting the versatile role of proline under stress and non-stress situations. The review summarizes our current knowledge on proline metabolism and transport in view of plant development, discussing regulatory aspects such as the influence of metabolites and hormones. Additional information from animals, fungi and bacteria is included, showing similarities and differences to proline metabolism and transport in plants. PMID:20204435

  11. Slow peptide bond formation by proline and other N-alkylamino acids in translation.

    PubMed

    Pavlov, Michael Y; Watts, Richard E; Tan, Zhongping; Cornish, Virginia W; Ehrenberg, Måns; Forster, Anthony C

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis. PMID:19104062

  12. Proline: Mother Nature’s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-08-01

    The amino acid l-proline is shown to be a good cryoprotectant for protein crystals. Four examples are provided; the range of proline used for cryoprotection is 2.0–3.0 M. l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography.

  13. Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

    PubMed Central

    Wang, Kun; Tian, Jian; Turunen, Ossi; Huang, Huoqing; Shi, Pengjun; Hua, Huifang; Wang, Caihong; Wang, Shuanghe

    2014-01-01

    Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability. PMID:24463976

  14. Structural Biology of Proline Catabolism

    PubMed Central

    2009-01-01

    Summary The proline catabolic enzymes proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase catalyze the 4-electron oxidation of proline to glutamate. These enzymes play important roles in cellular redox control, superoxide generation, apoptosis and cancer. In some bacteria, the two enzymes are fused into the bifunctional enzyme, proline utilization A. Here we review the three-dimensional structural information that is currently available for proline catabolic enzymes. Crystal structures have been determined for bacterial monofunctional proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase, as well as the proline dehydrogenase and DNA-binding domains of proline utilization A. Some of the functional insights provided by analyses of these structures are discussed, including substrate recognition, catalytic mechanism, biochemical basis of inherited proline catabolic disorders and DNA recognition by proline utilization A. PMID:18369526

  15. Slow peptide bond formation by proline and other N-alkylamino acids in translation

    PubMed Central

    Pavlov, Michael Y.; Watts, Richard E.; Tan, Zhongping; Cornish, Virginia W.; Ehrenberg, Måns; Forster, Anthony C.

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid (“imino acid”), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3–6 times slower than Phe incorporation from Phe-tRNAPhe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNAPro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the “tRNA abundance” hypothesis. PMID:19104062

  16. Connecting Proline and γ-Aminobutyric Acid in Stressed Plants through Non-Enzymatic Reactions

    PubMed Central

    Signorelli, Santiago; Dans, Pablo D.; Coitiño, E. Laura; Borsani, Omar; Monza, Jorge

    2015-01-01

    The accumulation of proline (Pro) in plants exposed to biotic/abiotic stress is a well-documented and conserved response in most vegetal species. Stress conditions induce the overproduction of reactive oxygen species which can lead to cellular damage. In vitro assays have shown that enzyme inactivation by hydroxyl radicals (·OH) can be avoided in presence of Pro, suggesting that this amino acid could act as an ·OH scavenger. We applied Density Functional Theory coupled with a polarizable continuum model to elucidate how Pro reacts with ·OH. In this work we suggest that Pro reacts favourably with ·OH by H–abstraction on the amine group. This reaction produces the spontaneous decarboxylation of Pro leading to the formation of pyrrolidin-1-yl. In turn, pyrrolidin-1-yl can easily be converted to Δ1-pyrroline, the substrate of the enzyme Δ1-pyrroline dehydrogenase, which produces γ-aminobutyric acid (GABA). GABA and Pro are frequently accumulated in stressed plants and several protective roles have been assigned to these molecules. Thereby we present an alternative non-enzymatic way to synthetize GABA under oxidative stress. Finally this work sheds light on a new beneficial role of Pro accumulation in the maintenance of photosynthetic activity. PMID:25775459

  17. Investigations of the Mechanism of the "Proline Effect" in Tandem Mass Spectrometry Experiments: The "Pipecolic Acid Effect"

    NASA Astrophysics Data System (ADS)

    Raulfs, Mary Disa M.; Breci, Linda; Bernier, Matthew; Hamdy, Omar M.; Janiga, Ashley; Wysocki, Vicki; Poutsma, John C.

    2014-10-01

    The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established "proline effect" through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give yn + ions. In contrast, Pip- and NMA-fragment through a different mechanism, the "pipecolic acid effect," selectively at the amide bond C-terminal to the Pip/NMA residue to give bn + ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the "proline effect' can in part be rationalized by the increased basicity of the prolyl-amide site, the "pipecolic acid effect" cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the "pipecolic acid effect" is stronger than the "proline effect."

  18. Proline Mechanisms of Stress Survival

    PubMed Central

    Liang, Xinwen; Zhang, Lu; Natarajan, Sathish Kumar

    2013-01-01

    Abstract Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response. Antioxid. Redox Signal. 19, 998–1011. PMID:23581681

  19. A novel role for proline in plant floral nectars

    NASA Astrophysics Data System (ADS)

    Carter, Clay; Shafir, Sharoni; Yehonatan, Lia; Palmer, Reid G.; Thornburg, Robert

    2006-02-01

    Plants offer metabolically rich floral nectar to attract visiting pollinators. The composition of nectar includes not only sugars, but also amino acids. We have examined the amino acid content of the nectar of ornamental tobacco and found that it is extremely rich (2 mM) in proline. Because insect pollinators preferentially utilize proline during the initial phases of insect flight and can reportedly taste proline, we determined whether honeybees showed a preference for synthetic nectars rich in proline. We therefore established an insect preference test and found that honeybees indeed prefer nectars rich in the amino acid proline. To determine whether this was a general phenomenon, we also examined the nectars of two insect-pollinated wild perennial species of soybean. These species also showed high levels of proline in their nectars demonstrating that plants often produce proline-rich floral nectar. Because insects such as honeybees prefer proline-rich nectars, we hypothesize that some plants offer proline-rich nectars as a mechanism to attract visiting pollinators.

  20. Water Deficit-Induced Changes in Concentrations in Proline and Some Other Amino Acids in the Phloem Sap of Alfalfa.

    PubMed Central

    Girousse, C.; Bournoville, R.; Bonnemain, J. L.

    1996-01-01

    Changes in amino acid composition of alfalfa (Medicago sativa L.) phloem sap were studies in response to a water deficit. Sap was collected by stylectomy. As the leaf water potential ([psi]) decreased from -0.4 to -2.0 MPa, there was significant increase of the total amino acid concentration, due to that of some amino acids: proline, valine, isoleucine, leucine, glutamic acid, aspartic acid, and threonine. Asparagine concentration, which is the main amino acid assayed in the phloem sap of alfalfa (it accounts for 70% of the total content), did not vary with the plant water status. The other amino acid concentrations remained stable as [psi] varied; in particular, [gamma]-amino butyric acid concentration remained unchanged, whereas it varied in response to wounding. The more striking change in the sieve tubes was the accumulation of proline, which was observed below a [psi] threshold value of about -0.9 MPa (concentration x60 for a decrease of [psi] from -0.9 to -2.0 MPa). The role of such changes in phloem sap amino acid concentration in osmotic adjustment of growing tissues is discussed. PMID:12226278

  1. Glutamine metabolism and function in relation to proline synthesis and the safety of glutamine and proline supplementation.

    PubMed

    Watford, Malcolm

    2008-10-01

    At normal intakes, dietary glutamine and glutamate are metabolized by the small intestine and essentially all glutamine within the body is synthesized de novo through the action of glutamine synthetase. The major sites of net glutamine synthesis are skeletal muscle, lung, and adipose tissue and, under some conditions, the liver. In addition to the small intestine, where glutamine is the major respiratory fuel, other sites of net glutamine utilization include the cells of the immune system, the kidneys, and the liver. The intestine expresses pyrroline 5-carboxylate (P5C) synthase, which means that proline is an end product of intestinal glutamine catabolism. Proline can also be synthesized from ornithine and the exact contribution of the 2 pathways is not certain. Infusion of proline i.v. to increase circulating concentrations is associated with increased proline oxidation and decreased proline synthesis. In contrast, conditions of proline insufficiency, after feeding low-proline diets or in response to high rates of proline catabolism in burn patients, do not result in increased proline synthesis. Glutamine supplementation is widespread and up to 0.57-0.75 g.kg(-1).d(-1) is well tolerated. Similarly, the only study of proline supplementation, in which patients with gyrate atrophy were given 488 mg.kg(-1).d(-1), reported no deleterious side effects. In the absence of controlled trials, it is currently not possible to estimate a safe upper limit for either of these 2 amino acids. PMID:18806115

  2. Synthesis of comb type and semi-interpenetrating networks of acryloyl- L-proline methyl ester and poly (acrylic acid) for Cu (II) immobilization

    NASA Astrophysics Data System (ADS)

    González, Giovanni; Burillo, Guillermina

    2010-08-01

    Graft copolymer hydrogels and semi-interpenetrating networks (s-IPN) of acryloyl- L-proline methyl ester (A-ProOMe) and poly (acrylic acid) (PAAc) were synthesized in methanol solutions, by ionizing radiation (γ rays from a Co 60 source at room temperature). These systems are thermo and pH-sensitive and the pH sensitivity increases from acidic to basic solutions. The Lower Critical Solution Temperature (LCST), due to presence of poly (acryloyl- L-proline methyl ester) (PA-ProOMe) has been found between 18 and 20 °C and an unexpected Upper Critical Solution Temperature (UCST) due to poly acrylic acid (PAAc) has been found between 21 and 22 °C. Preliminary studies on the immobilization of Cu 2+ for both hydrogels were done at several pH values at room temperature. Other techniques employed to characterize the comb type hydrogels and sIPN included scanning electronic microscopy (SEM) and infrared (FTIR-ATR).

  3. Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background The soil bacterium Corynebacterium glutamicum, best known for its glutamate producing ability, is suitable as a producer of a variety of bioproducts. Glutamate is the precursor of the amino acid proline. Proline biosynthesis typically involves three enzymes and a spontaneous cyclisation reaction. Alternatively, proline can be synthesised from ornithine, an intermediate of arginine biosynthesis. The direct conversion of ornithine to proline is catalysed by ornithine cyclodeaminase. An ornithine overproducing platform strain with deletions of argR and argF (ORN1) has been employed for production of derived compounds such as putrescine. By heterologous expression of ocd this platform strain can be engineered further for proline production. Results Plasmid-based expression of ocd encoding the putative ornithine cyclodeaminase of C. glutamicum did not result in detectable proline accumulation in the culture medium. However, plasmid-based expression of ocd from Pseudomonas putida resulted in proline production with yields up to 0.31 ± 0.01 g proline/g glucose. Overexpression of the gene encoding a feedback-alleviated N-acetylglutamate kinase further increased proline production to 0.36 ± 0.01 g/g. In addition, feedback-alleviation of N-acetylglutamate kinase entailed growth-coupled production of proline and reduced the accumulation of by-products in the culture medium. Conclusions The product spectrum of the platform strain C. glutamicum ORN1 was expanded to include the amino acid L-proline. Upon further development of the ornithine overproducing platform strain, industrial production of amino acids of the glutamate family and derived bioproducts such as diamines might become within reach. PMID:23806148

  4. Proline and hydroxyproline metabolism: implications for animal and human nutrition.

    PubMed

    Wu, Guoyao; Bazer, Fuller W; Burghardt, Robert C; Johnson, Gregory A; Kim, Sung Woo; Knabe, Darrell A; Li, Peng; Li, Xilong; McKnight, Jason R; Satterfield, M Carey; Spencer, Thomas E

    2011-04-01

    Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a per-gram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans. PMID:20697752

  5. Proline and hydroxyproline metabolism: implications for animal and human nutrition

    PubMed Central

    Bazer, Fuller W.; Burghardt, Robert C.; Johnson, Gregory A.; Kim, Sung Woo; Knabe, Darrell A.; Li, Peng; Li, Xilong; McKnight, Jason R.; Satterfield, M. Carey; Spencer, Thomas E.

    2013-01-01

    Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a pergram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans. PMID:20697752

  6. Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

    PubMed Central

    Sareddy, Gangadhara R.; Zhang, Quanguang; Wang, Ruimin; Scott, Erin; Zou, Yi; O'Connor, Jason C.; Chen, Yidong; Dong, Yan; Vadlamudi, Ratna K.; Brann, Darrell

    2015-01-01

    17-β estradiol (E2) has been implicated as neuroprotective in a variety of neurodegenerative disorders. However, the underlying mechanism remains unknown. Here, we provide genetic evidence, using forebrain-specific knockout (FBKO) mice, that proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), an estrogen receptor coregulator protein, is essential for the extranuclear signaling and neuroprotective actions of E2 in the hippocampal CA1 region after global cerebral ischemia (GCI). E2-mediated extranuclear signaling (including activation of extracellular signal-regulated kinase and Akt) and antiapoptotic effects [such as attenuation of JNK signaling and increase in phosphorylation of glycogen synthase kinase-3β (GSK3β)] after GCI were compromised in PELP1 FBKO mice. Mechanistic studies revealed that PELP1 interacts with GSK3β, E2 modulates interaction of PELP1 with GSK3β, and PELP1 is a novel substrate for GSK3β. RNA-seq analysis of control and PELP1 FBKO mice after ischemia demonstrated alterations in several genes related to inflammation, metabolism, and survival in PELP1 FBKO mice, as well as a significant reduction in the activation of the Wnt/β-catenin signaling pathway. In addition, PELP1 FBKO studies revealed that PELP1 is required for E2-mediated neuroprotection and for E2-mediated preservation of cognitive function after GCI. Collectively, our data provide the first direct in vivo evidence, to our knowledge, of an essential role for PELP1 in E2-mediated rapid extranuclear signaling, neuroprotection, and cognitive function in the brain. PMID:26627258

  7. Refined regio- and stereoselective hydroxylation of L-pipecolic acid by protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure.

    PubMed

    Koketsu, Kento; Shomura, Yasuhito; Moriwaki, Kei; Hayashi, Mikiro; Mitsuhashi, Satoshi; Hara, Ryotaro; Kino, Kuniki; Higuchi, Yoshiki

    2015-04-17

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe(2+)/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip. PMID:25171735

  8. Fragmentation reactions of deprotonated peptides containing proline. The proline effect.

    PubMed

    Harrison, Alex G; Young, Alex B

    2005-09-01

    The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the ''b(2) ion; further evidence is presented that the ''b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline. PMID:16041740

  9. Proline Modulates the Trypanosoma cruzi Resistance to Reactive Oxygen Species and Drugs through a Novel D, L-Proline Transporter

    PubMed Central

    Sayé, Melisa; Miranda, Mariana R.; di Girolamo, Fabio; de los Milagros Cámara, María; Pereira, Claudio A.

    2014-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC50s for nifurtimox, benznidazole, H2O2 and NO. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease. PMID:24637744

  10. Helix propensities of conformationally restricted amino acids. Non-natural substitutes for helix breaking proline and helix forming alanine.

    PubMed

    Alías, Miriam; Ayuso-Tejedor, Sara; Fernández-Recio, Juan; Cativiela, Carlos; Sancho, Javier

    2010-02-21

    Alpha helices are useful scaffolds to build biologically active peptides. The intrinsic stability of an alpha-helix is a key feature that can be successfully designed, and it is governed by the constituting amino acid residues. Their individual contributions to helix stability are given, according to Lifson-Roig theory, by their w parameters, which are known for all proteinogenic amino acids, but not for non-natural ones. On the other hand, non-natural, conformationally-restricted amino acids can be used to impart biochemical stability to peptides intended for in vivo administration. Efficient design of peptides based on these amino acids requires the previous determination of their w parameters. We begin here this task by determining the w parameters of two restricted analogs of alanine: (alpha-methyl)alanine and 1-aminocyclopropanecarboxylic acid. According to their w values (alpha-methyl)alanine is almost as good a helix forming residue as alanine, while 1-aminocyclopropanecarboxylic acid is, similarly to proline, a helix breaker. PMID:20135035

  11. Comparison of inhibitory activity on calcium phosphate precipitation by acidic proline-rich proteins, statherin, and histatin-1.

    PubMed

    Tamaki, N; Tada, T; Morita, M; Watanabe, T

    2002-07-01

    This study quantitatively compares the inhibition of calcium phosphate (CaP) precipitation by the salivary acidic proline-rich proteins (PRPs) statherin and histatin-1. Saliva and CaCl2 in 125 mM imidazole buffer (pH 7.0) were incubated with potassium phosphate and a hydroxyapatite (HAP) suspension, for 30 min at 25 degrees C, then filtered through nitrocellulose. The calcium (Ca) concentration in the filtrate was measured by atomic absorption spectrophotometry, then deducted from that in the initial solution to determine the amount of CaP precipitation after 30 min. The values of the inhibitory activities on CaP precipitation relative to crude parotid saliva were 4.7, 4.9, 6.9, and 65.8 for histatin-1, large PRPs, small PRPs, and statherin, respectively. PMID:12060866

  12. Study of the effects of proline, phenylalanine, and urea foliar application to Tempranillo vineyards on grape amino acid content. Comparison with commercial nitrogen fertilisers.

    PubMed

    Garde-Cerdán, T; López, R; Portu, J; González-Arenzana, L; López-Alfaro, I; Santamaría, P

    2014-11-15

    The aim of this work was to study the influence of foliar application of different nitrogen sources on grape amino acid content. The nitrogen sources applied to Tempranillo grapevines were proline, phenylalanine, urea, and two commercial nitrogen fertilisers, both without and with amino acids in their formulations. All treatments were applied at veraison and one week later. Proline treatment did not affect the must nitrogen composition. However, phenylalanine and urea foliar application enhanced the plants' synthesis of most of the amino acids, producing similar effects. In addition, the spray of commercial nitrogen fertilisers over leaves also induced a rise in grape amino acid concentrations regardless of the presence or absence of amino acids in their formulation. The most effective treatments were phenylalanine and urea followed by nitrogen fertilisers. This finding is of oenological interest for improved must nitrogen composition, ensuring better fermentation kinetics and most likely enhancing wine quality. PMID:24912708

  13. Germination of Candida albicans induced by proline.

    PubMed Central

    Dabrowa, N; Taxer, S S; Howard, D H

    1976-01-01

    Blastospores of Candida albicans germinated in proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glatamate, asparate, and pyruvate families also fostered germinaton but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of L-proline inhibited proline-stimualted germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts. PMID:5375

  14. Abscisic Acid- and Stress-Induced Highly Proline-Rich Glycoproteins Regulate Root Growth in Rice1[W][OPEN

    PubMed Central

    Tseng, I-Chieh; Hong, Chwan-Yang; Yu, Su-May; Ho, Tuan-Hua David

    2013-01-01

    In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX1PX2 motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane. PMID:23886623

  15. Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

    PubMed

    Hamase, K; Inoue, T; Morikawa, A; Konno, R; Zaitsu, K

    2001-11-15

    A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice. PMID:11700980

  16. Survival of Escherichia coli O157:H7 ATCC 43895 in a Model Apple Juice Medium with Different Concentrations of Proline and Caffeic Acid

    PubMed Central

    Reinders, Robert D.; Biesterveld, Steef; Bijker, Peter G. H.

    2001-01-01

    The effects of proline and caffeic acid on the survival of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strain ATCC 43895 in a model apple juice medium were studied. It is hypothesized that the inhibitory effect of caffeic acid may explain why almost all outbreaks of STEC O157:H7 infections linked to apple juice or cider have occurred in October or November. PMID:11375209

  17. The Aspergillus nidulans proline permease as a model for understanding the factors determining substrate binding and specificity of fungal amino acid transporters.

    PubMed

    Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky

    2015-03-01

    Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly(56), Thr(57)), TMS3 (Glu(138)), and TMS6 (Phe(248)), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393

  18. Pre-storage application of oxalic acid alleviates chilling injury in mango fruit by modulating proline metabolism and energy status under chilling stress.

    PubMed

    Li, Peiyan; Zheng, Xiaolin; Liu, Yan; Zhu, Yuyan

    2014-01-01

    Effects of oxalic acid on chilling injury, proline metabolism and energy status in mango fruit were investigated after mango fruit (Mangifera indica L. cv. Zill) were dipped in 5mM oxalic acid solution for 10min at 25°C and then stored at low temperature (10±0.5°C) for 49days thereafter transferred to 25°C for 4days. Pre-storage application of oxalic acid apparently inhibited the development of chilling injury, notably elevated proline accumulation actually associated with increase in Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) activity and decrease in proline dehydrogenase (PDH) activity in the peel and the flesh, without activation of ornithine-δ-aminotransferase (OAT) activity, and maintained high ATP level and energy charge in the flesh during storage. It was suggested that these effects of oxalic acid might collectively contribute to improving chilling tolerance, thereby alleviating chilling injury and maintaining quality of mango fruit in long term cold storage. PMID:24001814

  19. Dye-linked D-amino acid dehydrogenase from the thermophilic bacterium Rhodothermus marinus JCM9785: characteristics and role in trans-4-hydroxy-L-proline catabolism.

    PubMed

    Satomura, Takenori; Ishikura, Masaru; Koyanagi, Takashi; Sakuraba, Haruhiko; Ohshima, Toshihisa; Suye, Shin-ichiro

    2015-05-01

    A gene from the thermophilic Gram-negative bacterium Rhodothermus marinus JCM9785, encoding a dye-linked D-amino acid dehydrogenase homologue, was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was a highly thermostable dye-linked D-amino acid dehydrogenase that retained more than 80% of its activity after incubation for 10 min at up to 70 °C. When enzyme-catalyzed dehydrogenation of several D-amino acids was carried out using 2,6-dichloroindophenol as the electron acceptor, D-phenylalanine was the most preferable substrate among the D-amino acids tested. Immediately upstream of the dye-linked D-amino acid dehydrogenase gene (dadh) was a gene encoding a 4-hydroxyproline 2-epimerase homologue (hypE). That gene was successfully expressed in E. coli, and the gene product exhibited strong 4-hydroxyproline 2-epimerase activity. Reverse transcription PCR and quantitative real-time PCR showed that the six genes containing the dadh and hypE genes were arranged in an operon and were required for catabolism of trans-4-hydroxy-L-proline in R. marinus. This is the first description of a dye-linked D-amino acid dehydrogenase (Dye-DADH) with broad substrate specificity involved in trans-4-hydroxy-L-proline catabolism. PMID:25472442

  20. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny.

    PubMed

    Kavi Kishor, Polavarapu B; Hima Kumari, P; Sunita, M S L; Sreenivasulu, Nese

    2015-01-01

    Proline is a proteogenic amino acid and accumulates both under stress and non-stress conditions as a beneficial solute in plants. Recent discoveries point out that proline plays an important role in plant growth and differentiation across life cycle. It is a key determinant of many cell wall proteins that plays important roles in plant development. The role of extensins, arabinogalactan proteins and hydroxyproline- and proline-rich proteins as important components of cell wall proteins that play pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance is discussed in this review. Molecular insights are also provided here into the plausible roles of proline transporters modulating key events in plant development. In addition, the roles of proline during seed developmental transitions including storage protein synthesis are discussed. PMID:26257754

  1. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny

    PubMed Central

    Kavi Kishor, Polavarapu B.; Hima Kumari, P.; Sunita, M. S. L.; Sreenivasulu, Nese

    2015-01-01

    Proline is a proteogenic amino acid and accumulates both under stress and non-stress conditions as a beneficial solute in plants. Recent discoveries point out that proline plays an important role in plant growth and differentiation across life cycle. It is a key determinant of many cell wall proteins that plays important roles in plant development. The role of extensins, arabinogalactan proteins and hydroxyproline- and proline-rich proteins as important components of cell wall proteins that play pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance is discussed in this review. Molecular insights are also provided here into the plausible roles of proline transporters modulating key events in plant development. In addition, the roles of proline during seed developmental transitions including storage protein synthesis are discussed. PMID:26257754

  2. Effects of Proline and Carbohydrates on the Metabolism of Exogenous Proline by Excised Bean Leaves in the Dark

    PubMed Central

    Stewart, Cecil R.

    1972-01-01

    Proline was metabolized when vacuum infiltrated into starved bean (Phaseolus vulgaris L.) leaves from plants previously in the dark for 48 hours, but an equivalent increase in protein proline was not observed. When 14C-proline was infiltrated into starved leaves, a large percentage of the 14C was recovered in other amino acids, organic acids, and CO2, in addition to that recovered as protein proline. However, extensive oxidation of proline was observed only if enough proline was added to increase substantially the endogenous concentration of proline. Increasing the endogenous concentration did not affect the amount of proline that was incorporated into protein. When added to leaves from plants previously in the light of near saturating intensity for more than 16 hr, very little 14C-proline was oxidized, even when a sufficient amount of proline was added to increase the endogenous concentration. Adding sucrose to starved leaves along with the proline slowed down the oxidation of proline. Thus, it appears that some carbohydrate or intermediate of carbohydrate metabolism may inhibit the oxidation of proline in leaves. Based on kinetics of labeling, the pathway of proline oxidation was by conversion to glutamic acid and subsequent metabolism to intermediates in the Krebs cycle and to CO2. PMID:16658215

  3. Acid catalysis of the formation of the slow-folding species of RNase A: Evidence that the reaction is proline isomerization

    PubMed Central

    Schmid, Franz X.; Baldwin, Robert L.

    1978-01-01

    Unfolded RNase A is known to contain an equilibrium mixture of two forms, a slow-folding form (U1) and a fast-folding form (U2). If U1 is produced after unfolding by the slow cis-trans isomerization of proline residues about X-Pro imide bonds, then the formation of U1 should be catalyzed by strong acids. Therefore, the rate of formation of U1 has been measured at different HClO4 concentrations. After rapid unfolding of the native protein in concentrated HClO4 at 0°, the slow formation of U1 was measured by use of refolding assays. Catalysis of its formation was found at HClO4 concentrations above 5 M. The uncatalyzed reaction follows apparent first-order kinetics but, in the acid-catalyzed range, two reactions are found. The faster reaction produces two-thirds of the slow-folding species and shows acid catalysis above 5 M HClO4. Catalysis of the slower reaction begins at 8 M HClO4. The faster reaction shows a 100-fold increase in rate at 10.6 M HClO4 over the rate of the uncatalyzed reaction of 5 M. The activation enthalpy of the uncatalyzed reaction has been measured in two sets of unfolding conditions: ΔH‡ is 21.5 kcal/mol (1 kcal = 4.2 × 103 J) in 3.3 M HClO4 and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5. Both acid catalysis of the formation of U1 and its high activation enthalpy are consistent with the rate-limiting step being cis-trans isomerization either of X-Pro imide bonds or of peptide bond. The rate of the uncatalyzed reaction is in the range expected for proline isomerization and is 0.1% of that of peptide bond isomerization; thus, the simplest explanation for the formation of U1 is proline isomerization. Earlier data, showing that the kinetic properties of the U1 ⇄ U2 reaction in refolding conditions differ from those of proline isomerization, can be explained if there is kinetic coupling between early steps in the folding of U1 and its conversion to U2. The existence of two acid-catalyzed reactions that are distinguished by the HClO4

  4. Proline Editing: A General and Practical Approach to the Synthesis of Functionally and Structurally Diverse Peptides. Analysis of Steric versus Stereoelectronic Effects of 4-Substituted Prolines on Conformation within Peptides

    PubMed Central

    Pandey, Anil K.; Naduthambi, Devan; Thomas, Krista M.; Zondlo, Neal J.

    2013-01-01

    Functionalized proline residues have diverse applications. Herein we describe a practical approach, proline editing, for the synthesis of peptides with stereospecifically modified proline residues. Peptides are synthesized by standard solid-phase-peptide-synthesis to incorporate Fmoc-Hydroxyproline (4R-Hyp). In an automated manner, the Hyp hydroxyl is protected and the remainder of the peptide synthesized. After peptide synthesis, the Hyp protecting group is orthogonally removed and Hyp selectively modified to generate substituted proline amino acids, with the peptide main chain functioning to “protect” the proline amino and carboxyl groups. In a model tetrapeptide (Ac-TYPN-NH2), 4R-Hyp was stereospecifically converted to 122 different 4-substituted prolyl amino acids, with 4R or 4S stereochemistry, via Mitsunobu, oxidation, reduction, acylation, and substitution reactions. 4-Substituted prolines synthesized via proline editing include incorporated structured amino acid mimetics (Cys, Asp/Glu, Phe, Lys, Arg, pSer/pThr), recognition motifs (biotin, RGD), electron-withdrawing groups to induce stereoelectronic effects (fluoro, nitrobenzoate), handles for heteronuclear NMR (19F:fluoro; pentafluorophenyl or perfluoro-tert-butyl ether; 4,4-difluoro; 77SePh) and other spectroscopies (fluorescence, IR: cyanophenyl ether), leaving groups (sulfonate, halide, NHS, bromoacetate), and other reactive handles (amine, thiol, thioester, ketone, hydroxylamine, maleimide, acrylate, azide, alkene, alkyne, aryl halide, tetrazine, 1,2-aminothiol). Proline editing provides access to these proline derivatives with no solution phase synthesis. All peptides were analyzed by NMR to identify stereoelectronic and steric effects on conformation. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, including azide-alkyne, tetrazinetrans-cyclooctene, oxime, reductive amination, native chemical ligation, Suzuki, Sonogashira, cross-metathesis, and Diels

  5. Hypothesis about the function of membrane-buried proline residues in transport proteins.

    PubMed Central

    Brandl, C J; Deber, C M

    1986-01-01

    In a survey of the bilayer-spanning regions of integral membrane proteins, membrane-buried proline residues were found in nearly all transport proteins examined, whereas membrane-buried regions of nontransport proteins were largely devoid of intramembranous proline residues. When amino acids from the complete sequences of representative sets of transport and nontransport membrane proteins were analyzed for the distribution of proline residues between aqueous vs. membranous domains, proline was shown to be selectively excluded from membranous domains of the nontransport proteins, in accord with expectation from energetic and structural considerations. In contrast, proline residues in transport proteins were evenly distributed between aqueous and membranous domains, consistent with the notion that functional membrane-buried proline residues are selectively included in transport proteins. As cis peptide bonds involving proline arise in proteins and have been implicated in protein dynamic processes, the cis-trans isomerization of an Xaa-Pro peptide bond (Xaa = unspecified amino acid) buried within the membrane--and the resulting redirection of the protein chain--is proposed to provide the reversible conformational change requisite for the regulation (opening/closing) of a transport channel. Parallel to this function, the relatively negative character of the carbonyl groups of Xaa-Pro peptide bonds may promote their participation as intramembranous liganding sites for positive species in proton/cation transport processes. PMID:3456574

  6. Proline dehydrogenase (oxidase) in cancer.

    PubMed

    Liu, Wei; Phang, James M

    2012-01-01

    Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the proline degradative pathway, plays a special role in tumorigenesis and tumor development. Proline metabolism catalyzed by PRODH/POX is closely linked with the tricarboxylic acid (TCA) cycle and urea cycle. The proline cycle formed by the interconversion of proline and Δ(1) -pyrroline-5-carboxylate (P5C) between mitochondria and cytosol interlocks with pentose phosphate pathway. Importantly, by catalyzing proline to P5C, PRODH/POX donates electrons into the electron transport chain to generate ROS or ATP. In earlier studies, we found that PRODH/POX functions as a tumor suppressor to initiate apoptosis, inhibit tumor growth, and block the cell cycle, all by ROS signaling. It also suppresses hypoxia inducible factor signaling by increasing α-ketoglutarate. During tumor progression, PRODH/POX is under the control of various tumor-associated factors, such as tumor suppressor p53, inflammatory factor peroxisome proliferator-activated receptor gamma (PPARγ), onco-miRNA miR-23b*, and oncogenic transcription factor c-MYC. Recent studies revealed the two-sided features of PRODH/POX-mediated regulation. Under metabolic stress such as oxygen and glucose deprivation, PRODH/POX can be induced to serve as a tumor survival factor through ATP production or ROS-induced autophagy. The paradoxical roles of PRODH/POX can be understood considering the temporal and spatial context of the tumor. Further studies will provide additional insights into this protein and on its metabolic effects in tumors, which may lead to new therapeutic strategies. PMID:22886911

  7. Bridging Between Proline Structure, Functions, Metabolism, and Involvement in Organism Physiology.

    PubMed

    Saibi, Walid; Feki, Kaouthar; Yacoubi, Ines; Brini, Faiçal

    2015-08-01

    Much is now known about proline multifunctionality and metabolism; some aspects of its biological functions are still unclear. Here, we discuss some cases in the proline, structure, definition, metabolism, compartmentalization, accumulation, plausible functions and also its implication in homeostasis and organism physiology. Indeed, we report the role of proline in cellular homeostasis, including redox balance and energy status and their implication as biocatalyst for aldolase activity. Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell proliferation or cell death, and trigger specific gene expression, which can be essential for plant recovery from stresses. Although, the regulation and the function of proline accumulation, during abiotic stresses, are not yet completely understood. The engineering of proline metabolism could lead to new opportunities to improve plant tolerance against environmental stresses. This atypical amino acid has a potential role in the toxicity during growth of some microorganism, vegetal, and mammalian species. Furthermore, we note that the purpose through the work is to provide a rich, concise, and mostly cohesive source on proline, considered as a platform and an anchor between several disciplines and biological functions. PMID:26100388

  8. Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

    PubMed Central

    Paes, Lisvane Silva; Suárez Mantilla, Brian; Zimbres, Flávia Menezes; Pral, Elisabeth Mieko Furusho; Diogo de Melo, Patrícia; Tahara, Erich B.; Kowaltowski, Alicia J.; Elias, Maria Carolina; Silber, Ariel Mariano

    2013-01-01

    Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle. PMID:23894476

  9. Solid-Phase Synthesis of Amine/Carboxyl Substituted Prolines and Proline Homologues: Scope and Limitations.

    PubMed

    Zhou, Ziniu; Scott, William L; O'Donnell, Martin J

    2016-01-01

    A solid-phase procedure is used to synthesize racemic peptidomimetics based on the fundamental peptide unit. The peptidomimetics are constructed around proline or proline homologues variably substituted at the amine and carbonyl sites. The procedure expands the diversity of substituted peptidomimetic molecules available to the Distributed Drug Discovery (D3) project. Using a BAL-based solid-phase synthetic sequence the proline or proline homologue subunit is both constructed and incorporated into the peptidomimetic by an α-alkylation, hydrolysis and intramolecular cyclization sequence. Further transformations on solid-phase provide access to a variety of piperazine derivatives representing a class of molecules known to exhibit central nervous system activity. The procedure works well with proline cores, but with larger six- and seven-membered ring homologues the nature of the carboxylic acid acylating the cyclic amine can lead to side reactions and result in poor overall yields. PMID:26999079

  10. Activation of a new proline transport system in Salmonella typhimurium.

    PubMed

    Ekena, K; Liao, M K; Maloy, S

    1990-06-01

    Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes. PMID:2160931

  11. On the Split Personality of Penultimate Proline

    NASA Astrophysics Data System (ADS)

    Glover, Matthew S.; Shi, Liuqing; Fuller, Daniel R.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

    2015-03-01

    The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser1-Pro2-Glu3-Leu4-Pro5-Ser6-Pro7-Gln8-Ala9-Glu10-Lys11. We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis- trans isomerization of Xaa1-Pro2 peptide bonds as Xaa-Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis- and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues.

  12. Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein.

    PubMed

    Kraus, Allison

    2016-01-01

    Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity. PMID:26864641

  13. Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12.

    PubMed

    Milner, J L; McClellan, D J; Wood, J M

    1987-07-01

    Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism. PMID:3312483

  14. Proline control of the feeding reaction of Cordylophora.

    PubMed

    FULTON, C

    1963-03-01

    The colonial hydroid Cordylophora is a carnivore whose feeding is induced by substances released from captured prey. An active molecule, probably the only one, has been isolated from a fraction of the laboratory food of Cordylophora, brine shrimp larvae, and identified on paper chromatograms as the imino acid proline. Reagent proline induces the feeding reaction at 10(-5)M. The reaction is specific in that only two alpha-imino acids very closely related to proline were found to possess significant activity: azetidine-2-carboxylic acid and pipecolic acid. The response to proline is inhibited by magnesium ions and enhanced by phosphate. Since previous studies have shown that the feeding reactions of Hydra, Physalia, and Campanularia are controlled by reduced glutathione, the phylogenetic implications of the proline control of feeding in Cordylophora are discussed. The feeding reactions of both Cordylophora and Hydra are also induced by proteases, suggesting similar mechanisms of induction in the two hydroids. PMID:13960251

  15. Exogenous proline relieves growth inhibition caused by NaCl in petunia cells: Metabolism of L-( sup 15 M)-proline followed by sup 15 N NMR

    SciTech Connect

    Heyser, J.W.; Chacon, M.J. )

    1989-04-01

    Exogenous proline stimulated the growth of Petunia hybrida cells on 195 mM NaCl 10-fold as compared with cells grown on 195 mM CaCl medium minus proline. L-({sup 15}N)-proline was fed to cells growing on 0 and 195 mM CaCl, and its metabolism was followed by {sup 15}N NMR analysis of cell extracts. Total proline and amino acids were determined by ninhydrin assay. Proline and primary amino acids were easily resolved in NMR spectra and the amount of {sup 15}N-label which remained in proline was determined. Reduced catabolism of proline in cells grown on NaCl was evident. The role of exogenous proline in conferring increased NaCl tolerance in this nonhalophyte will be discussed.

  16. Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

    PubMed Central

    Burbaum, J. J.; Schimmel, P.

    1992-01-01

    Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304356

  17. Extracellular matrix-mediated control of aortic smooth muscle cell growth and migration by a combination of ascorbic acid, lysine, proline, and catechins.

    PubMed

    Ivanov, Vadim; Ivanova, Svetlana; Roomi, M Waheed; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2007-11-01

    Extracellular matrix (ECM) function and structure are severely compromised at atherosclerotic lesion sites, contributing to initiation and progression of the disease. This study investigated whether ECM biological properties would be beneficially affected by exposure to nutrients essential for collagen synthesis and posttranslational modification. Confluent layers of human aortic smooth muscle cells (SMC) grown on collagen substrate were cultured in the presence of the tested compounds for 7 to 10 days. Pretreated cells were removed from the ECM surface by differential treatment and replaced with secondary innocent SMC cultures. Secondary SMC growth rate and invasiveness were assayed in standard growth medium. ECM protein composition was assayed immunochemically. ECM produced in the presence of ascorbic acid reduced SMC proliferation in a dose-dependent manner. Plant-derived phenolic extracts expressed different degrees of SMC growth inhibition when present during ECM production. A combination of selected nutrients had a greater effect than did individual components. The ECM deposited by SMC in the presence of ascorbate, lysine, proline, and green tea catechins inhibited SMC migration rate up to 70%. The ECM produced under conditions of chronic essential nutrient deficiency can support proatherosclerotic SMC behavior. A combination of selected nutrients can counteract these adverse effects stronger than individual components. PMID:18030064

  18. Proline accumulation is a general response to abiotic stress in the date palm tree (Phoenix dactylifera L.).

    PubMed

    Yaish, M W

    2015-01-01

    Plants exposed to certain abiotic stress conditions tend to produce the amino acid proline, which acts as an active osmolyte, a metal chelator, an antioxidant, and a signaling molecule. There is increasing evidence that proline accumulates in plants due to a wide range of abiotic stress, in particular high soil salinity and drought. Therefore, proline content is often used as a marker-assisted breeding tool aimed at improving drought and salinity tolerance. In this study, it was investigated whether proline accumulation in date palm (Phoenix dactylifera L.) seedlings occurs solely due to high salinity and drought stresses or due to other unspecified abiotic stresses, including salinity and salinity shock, drought, extreme temperatures, and abscisic acid. The free proline assays revealed that this amino acid over-accumulated in the roots and leaves of each stress-treated plant, and was remarkably high when leaves were exposed to suboptimum temperatures and salinity stress. These results indicate that the production of proline is a common response to various abiotic stresses and its differential accumulation cannot be used as a molecular marker in date palm breeding programs aimed at improving drought or salinity tolerance traits in date palms. This conclusion is consistent with the theory that the molecular outcomes of abiotic stresses are often non-specific. PMID:26345930

  19. Role of proline and GABA in sexual reproduction of angiosperms.

    PubMed

    Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

    2015-01-01

    Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

  20. Role of proline and GABA in sexual reproduction of angiosperms

    PubMed Central

    Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

    2015-01-01

    Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

  1. Nine of 16 stereoisomeric polyhydroxylated proline amides are potent β-N-acetylhexosaminidase inhibitors.

    PubMed

    Ayers, Benjamin J; Glawar, Andreas F G; Martínez, R Fernando; Ngo, Nigel; Liu, Zilei; Fleet, George W J; Butters, Terry D; Nash, Robert J; Yu, Chu-Yi; Wormald, Mark R; Nakagawa, Shinpei; Adachi, Isao; Kato, Atsushi; Jenkinson, Sarah F

    2014-04-18

    All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of β-N-acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. β-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors. PMID:24641544

  2. The Proline Regulatory Axis and Cancer

    PubMed Central

    Phang, James Ming; Liu, Wei; Hancock, Chad; Christian, Kyle J.

    2012-01-01

    Studies in metabolism and cancer have characterized changes in core pathways involving glucose and glutamine, emphasizing the provision of substrates for building cell mass. But recent findings suggest that pathways previously considered peripheral may play a critical role providing mechanisms for cell regulation. Several of these mechanisms involve the metabolism of non-essential amino acids, for example, the channeling of glycolytic intermediates into the serine pathway for one-carbon transfers. Historically, we proposed that the proline biosynthetic pathway participated in a metabolic interlock with glucose metabolism. The discovery that proline degradation is activated by p53 directed our attention to the initiation of apoptosis by proline oxidase/dehydrogenase. Now, however, we find that the biosynthetic mechanisms and the metabolic interlock may depend on the pathway from glutamine to proline, and it is markedly activated by the oncogene MYC. These findings add a new dimension to the proline regulatory axis in cancer and present attractive potential targets for cancer treatment. PMID:22737668

  3. Proline metabolism and cancer: emerging links to glutamine and collagen

    PubMed Central

    Phang, James M.; Liu, Wei; Hancock, Chad N.; Fischer, Joseph W.

    2015-01-01

    Purpose of review Proline metabolism impacts a number of regulatory targets in both animals and plants and is especially important in cancer. Glutamine, a related amino acid, is considered second in importance only to glucose as a substrate for tumors. But proline and glutamine are interconvertible and linked in their metabolism. In animals, proline and glutamine have specific regulatory functions and their respective physiologic sources. A comparison of the metabolism of proline and glutamine would help us understand the importance of these two nonessential amino acids in cancer metabolism. Recent findings The regulatory functions of proline metabolism proposed 3 decades ago have found relevance in many areas. For cancer, these functions play a role in apoptosis, autophagy and in response to nutrient and oxygen deprivation. Importantly, proline-derived reactive oxygen species served as a driving signal for reprogramming. This model has been applied by others to metabolic regulation for the insulin-prosurvival axis, induction of adipose triglyceride lipase for lipid metabolism and regulation of embryonic stem cell development. Of special interest, modulatory proteins such as parkinson protein 7 and oral cancer overexpressed 1 interact with pyrroline-5-carboxylate reductase, a critical component of the proline regulatory axis. Although the interconvertibility of proline and glutamine has been long established, recent findings showed that the proto-oncogene, cellular myelocytomatosis oncogene, upregulates glutamine utilization (glutaminase) and routes glutamate to proline biosynthesis (pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductases). Additionally, collagen, which contains large amounts of proline, may be metabolized to serve as a reservoir for proline. This metabolic relationship as well as the new regulatory targets of proline metabolism invites an elucidation of the differential effects of these nonessential amino acids and their production

  4. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  5. The Sodium/Proline Transporter PutP of Helicobacter pylori

    PubMed Central

    Rivera-Ordaz, Araceli; Bracher, Susanne; Sarrach, Sannia; Li, Zheng; Shi, Lei; Quick, Matthias; Hilger, Daniel; Haas, Rainer; Jung, Heinrich

    2013-01-01

    Helicobacter pylori is cause of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. L-proline is a preferred energy source of the microaerophilic bacterium. Previous analyses revealed that HpputP and HpputA, the genes that are predicted to play a central role in proline metabolism as they encode for the proline transporter and proline dehydrogenase, respectively, are essential for stomach colonization. Here, the molecular basis of proline transport in H. pylori by HpPutP was investigated experimentally for the first time. Measuring radiolabeled substrate transport in H. pylori and E. coli heterologously expressing HpputP as well as in proteoliposomes reconstituted with HpPutP, we demonstrate that the observed proline transport in H. pylori is mediated by HpPutP. HpPutP is specific and exhibits a high affinity for L-proline. Notably, L-proline transport is exclusively dependent on Na+ as coupling ion, i.e., Na+/L-proline symport, reminiscent to the properties of PutP of E. coli even though H. pylori lives in a more acidic environment. Homology model-based structural comparisons and substitution analyses identified amino acids crucial for function. HpPutP-catalyzed proline uptake was efficiently inhibited by the known proline analogs 3,4-dehydro-D,L-proline and L-azetidine-2-carboxylic acid. PMID:24358297

  6. Mycorrhizal-mediated lower proline accumulation in Poncirus trifoliata under water deficit derives from the integration of inhibition of proline synthesis with increase of proline degradation.

    PubMed

    Zou, Ying-Ning; Wu, Qiang-Sheng; Huang, Yong-Ming; Ni, Qiu-Dan; He, Xin-Hua

    2013-01-01

    Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵(1)-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation. PMID:24260421

  7. Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

    PubMed Central

    Zou, Ying-Ning; Wu, Qiang-Sheng; Huang, Yong-Ming; Ni, Qiu-Dan; He, Xin-Hua

    2013-01-01

    Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵1-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation. PMID:24260421

  8. Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

    PubMed Central

    Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

    2016-01-01

    Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

  9. Oxidation of Proline and Glutamate by Mitochondria of the Inflorescence of Voodoo Lily (Sauromatum guttatum) 1

    PubMed Central

    Skubatz, Hanna; Meeuse, Bastiaan J. D.; Bendich, Arnold J.

    1989-01-01

    In appendices of Sauromatum guttatum that are developing thermogenicity, mitochondria isolated from successive developmental stages of the inflorescence show an increase in the oxidation rates of proline and glutamate. A similar rise in the oxidation rates of these compounds is observed in mitochondria obtained from the spathe, a nonthermogenic organ of the inflorescence. Changes in oxidative metabolism were also observed in mitochondria isolated from sections of immature appendix treated with salicylic acid (SA) at 0.69 microgram per gram fresh weight indicating that they are induced by SA. At that concentration, however, SA has no effect on oxygen consumption by mitochondria in the presence of glutamate, proline, or malate. Furthermore, oxygen uptake by mitochondria in the presence of proline or glutamate is partially sensitive to salicylhydroxamic acid (SHAM) at concentrations greater than 2 millimolar when in the presence of 1 millimolar KCN. For NADH, succinate, and malate a high capacity of the alternative (cyanide-resistant) pathway is found that is completely sensitive to SHAM at 1.5 to 4 millimolar. The increase in the mitochondrial capacity to oxidize either amino acid is also found in four other Araceae species including both thermogenic and nonthermogenic ones. After anthesis, the rates of proline and glutamate oxidation decline. Images Figure 1 PMID:16667065

  10. Influence of cold stress on contents of soluble sugars, vitamin C and free amino acids including gamma-aminobutyric acid (GABA) in spinach (Spinacia oleracea).

    PubMed

    Yoon, Young-Eun; Kuppusamy, Saranya; Cho, Kye Man; Kim, Pil Joo; Kwack, Yong-Bum; Lee, Yong Bok

    2017-01-15

    The contents of soluble sugars (sucrose, fructose, glucose, maltose and raffinose), vitamin C and free amino acids (34 compounds, essential and non-essential) were quantified in open-field and greenhouse-grown spinaches in response to cold stress using liquid chromatography. In general, greenhouse cultivation produced nutritionally high value spinach in a shorter growing period, where the soluble sugars, vitamin C and total amino acids concentrations, including essential were in larger amounts compared to those grown in open-field scenarios. Further, low temperature exposure of spinach during a shorter growth period resulted in the production of spinach with high sucrose, ascorbate, proline, gamma-aminobutyric acid, valine and leucine content, and these constitute the most important energy/nutrient sources. In conclusion, cultivation of spinach in greenhouse at a low temperature (4-7°C) and exposure for a shorter period (7-21days) before harvest is recommended. This strategy will produce a high quality product that people can eat. PMID:27542466

  11. Thermo- and pH-sensitive gel membranes based on poly-(acryloyl- L-proline methyl ester)- graft-poly(acrylic acid) for selective permeation of metal ions

    NASA Astrophysics Data System (ADS)

    Hasegawa, Shin; Ohashi, Hitoshi; Maekawa, Yasunari; Katakai, Ryoichi; Yoshida, Masaru

    2005-04-01

    Thermo- and pH-responsive gel membranes were synthesized by γ-ray grafting of pH-responsive poly(acrylic acid) (AAc) onto thermo-responsive polymer gel of acryloyl- L-proline methyl ester (A-ProOMe). The gel membranes of poly(A-ProOMe) with 15 mol% graft chains of AAc exhibited both thermo- and pH-responses. Under the condition (pH 6.0, 30°C) in which the thermo-sensitive unit shrinks and the pH-sensitive unit swells, the selective permeation of Li ion over Co and Ni ions can be achieved.

  12. Extraterrestrial Amino Acids in Ureilites Including Almahata Sitta

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Glavin, D. P.; Callahan, M. P.; Dworkin, J. P.

    2011-01-01

    Ureilites are a class of meteorites that lack chondrules (achondrites) but have relatively high carbon abundances, averaging approx.3 wt %. Using highly sensitive liquid chromatography coupled with UV fluorescence and time-of-flight mass spectrometry (LC-FD/ToF-MS), it was recently determined that there are amino acids in. fragment 94 of the Almahata Sitta ureilite[l]. Based on the presence of amino acids that are rare in the Earth's biosphere, as well as the near-racemic enantiomeric ratios of marry of the more common amino acids, it was concluded that most of the detected amino acids were indigenous to the meteorite. Although the composition of the Almahata Sitta ureilite appears to be unlike other recovered ureilites, the discovery of amino acids in this meteorite raises the question of whether other ureilites rnav also contain amino acids. Herein we present the results of LC-FDlTo.F-MS analyses of: a sand sample from the Almahata Sitta strewn held, Almahata Sitta fragments 425 (an ordinary H5 chondrite) and 427 (ureilite), as well as an Antarctic ureilite (Allan lulls, ALHA 77257).

  13. Cluster of genes controlling proline degradation in Salmonella typhimurium.

    PubMed Central

    Ratzkin, B; Roth, J

    1978-01-01

    A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP). PMID:342507

  14. γ-(S)-Trifluoromethyl proline: evaluation as a structural substitute of proline for solid state (19)F-NMR peptide studies.

    PubMed

    Kubyshkin, Vladimir; Afonin, Sergii; Kara, Sezgin; Budisa, Nediljko; Mykhailiuk, Pavel K; Ulrich, Anne S

    2015-03-21

    γ-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with improved yield on a multigram scale. Conformational properties of the amide bond formed by the amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to the corresponding values for intact proline. Therefore, the γ-trifluoromethyl proline was suggested as a structurally low-disturbing proline substitution in peptides for their structural studies by (19)F-NMR. Indeed, the exchange of native proline for γ-trifluoromethyl proline in the peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure. The utility of the amino acid as a selective (19)F-NMR label was demonstrated by observing the re-alignment of the labelled gramicidin S in oriented lipid bilayers. PMID:25703116

  15. Substrate channeling in proline metabolism

    PubMed Central

    Arentson, Benjamin W.; Sanyal, Nikhilesh; Becker, Donald F.

    2012-01-01

    Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is unfavorable at physiological pH. The second intermediate, gamma-glutamyl phosphate, is part of the proline biosynthetic pathway and is extremely labile. Substrate channeling of gamma-glutamyl phosphate is thought to be necessary to protect it from bulk solvent. Because of the unfavorable equilibrium of P5C/GSA and the reactivity of gamma-glutamyl phosphate, substrate channeling likely improves the efficiency of proline metabolism. Here, we outline general strategies for testing substrate channeling and review the evidence for channeling in proline metabolism. PMID:22201749

  16. Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

    PubMed

    Jewell, J B; Kashket, E R

    1991-10-01

    We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered. PMID:1786048

  17. The induction of free proline accumulation by endogenous ABA in Arabidopsis thaliana during drought

    SciTech Connect

    Gottlieb, M.L.; Bray, E.A. )

    1991-05-01

    Endogenous levels of abscisic acid (ABA) and free proline increase in response to drought stress. Exogenous ABA has been shown to induce proline accumulation, suggesting that ABA triggers the amino acid response. To determine if endogenous ABA induces free proline accumulation, increases in ABA and proline during drought stress were compared between wild type (WT), ABA-insensitive (abi) and ABA-deficient (aba) mutants of Arabidopsis thaliana. If elevated levels of endogenous ABA signal the proline response, then the mutants would not be expected to accumulate proline during stress. abi should be unable to respond to increased levels of endogenous ABA, while aba should be unable to accumulate sufficient ABA to elicit a proline response. Drought-stressed three week old shoots of WT, abi, and aba exhibited different patterns of endogenous ABA accumulation, but similar patterns of proline accumulation over 24 hours. Although the patterns of endogenous ABA accumulation differed, maximum levels were similar in WT and abi, but aba produced approximately 25% less. However, free proline accumulated in all three plant lines. abi exhibited a greater, more rapid increase in free proline over that in either WT or aba. aba, however, showed the same pattern and levels of accumulation as that in WT. Since free proline accumulated to at least similar levels in both WT and mutants, regardless of the levels of ABA accumulation, it may be that only a small endogenous ABA accumulation is required for proline accumulation. Alternatively, endogenous ABA may not be the direct signal for the proline response during drought stress.

  18. Involvement of the β3-α3 loop of the Proline Dehydrogenase Domain in Allosteric Regulation of Membrane Association of Proline Utilization A†,‡

    PubMed Central

    Zhu, Weidong; Haile, Ashley M.; Singh, Ranjan K.; Larson, John D.; Smithen, Danielle; Chan, Jie Y.; Tanner, John J.; Becker, Donald F.

    2013-01-01

    Proline utilization A (PutA) from Escherichia coli is a membrane-associated trifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate and moonlights as a transcriptional regulator. As a regulatory protein, PutA represses transcription of the put regulon, which contains the genes encoding PutA and the proline transporter PutP. The binding of proline to the proline dehydrogenase active site and the subsequent reduction of the flavin induces high affinity membrane association of PutA and relieves repression of the put regulon, thereby causing PutA to switch from its regulatory to its enzymatic role. Here, we present evidence suggesting that residues of the β3-α3 loop of the proline dehydrogenase domain (βα)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. Mutation of the conserved residues Asp370 and Glu372 in the β3-α3 loop abrogates the ability of proline to induce functional membrane association. Both in vitro lipid/membrane binding assays and in vivo cell-based assays demonstrate that mutagenesis of Asp370 (D370N/A) or Glu372 (E372A) dramatically impedes PutA functional switching. The crystal structures of the proline dehydrogenase domain mutants PutA86-630D370N and PutA86-630D370A complexed with the proline analog L-tetrahydro-2-furoic acid show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. Rather, these results suggest that the β3-α3 loop may be involved in transmitting the status of the proline dehydrogenase active site and flavin redox state to the distal membrane association domain. PMID:23713611

  19. Catalysis of the Carbonylation of Alcohols to Carboxylic Acids Including Acetic Acid Synthesis from Methanol.

    ERIC Educational Resources Information Center

    Forster, Denis; DeKleva, Thomas W.

    1986-01-01

    Monsanto's highly successful synthesis of acetic acid from methanol and carbon monoxide illustrates use of new starting materials to replace pretroleum-derived ethylene. Outlines the fundamental aspects of the acetic acid process and suggests ways of extending the synthesis to higher carboxylic acids. (JN)

  20. Proline puckering parameters for collagen structure simulations

    SciTech Connect

    Wu, Di

    2015-03-15

    Collagen is made of triple helices rich in proline residues, and hence is influenced by the conformational motions of prolines. Because the backbone motions of prolines are restricted by the helical structures, the only side chain motion—proline puckering—becomes an influential factor that may affect the stability of collagen structures. In molecular simulations, a proper proline puckering population is desired so to yield valid results of the collagen properties. Here we design the proline puckering parameters in order to yield suitable proline puckering populations as demonstrated in the experimental results. We test these parameters in collagen and the proline dipeptide simulations. Compared with the results of the PDB and the quantum calculations, we propose the proline puckering parameters for the selected collagen model simulations.

  1. Proline transport in rat kidney mitochondria.

    PubMed

    Atlante, A; Passarella, S; Pierro, P; Quagliariello, E

    1994-02-15

    Proline transport in rat kidney mitochondria was investigated both by using isotopic techniques and by spectroscopic measurements, in which proline metabolism was essentially allowed to occur. Widely used criteria for demonstrating the occurrence of carrier-mediated transport were successfully applied in both cases. Differences found in the Km and Vmax values, in pH and temperature dependence of proline transport, and in the inhibitor sensitivity demonstrate the existence of two separate translocators for proline in rat kidney mitochondria, i.e., the proline uniporter and the proline/glutamate antiporter. Efflux of glutamate via glutamate/OH- translocator following proline uptake by mitochondria was experimentally ruled out. Discussion is also made of the possible role of such translocators in proline metabolism and in the putative proline/glutamate shuttle. PMID:7906935

  2. Ionization constants of aqueous amino acids at temperatures up to 250°C using hydrothermal pH indicators and UV-visible spectroscopy: Glycine, α-alanine, and proline

    NASA Astrophysics Data System (ADS)

    Clarke, Rodney G. F.; Collins, Christopher M.; Roberts, Jenene C.; Trevani, Liliana N.; Bartholomew, Richard J.; Tremaine, Peter R.

    2005-06-01

    Ionization constants for several simple amino acids have been measured for the first time under hydrothermal conditions, using visible spectroscopy with a high-temperature, high-pressure flow cell and thermally stable colorimetric pH indicators. This method minimizes amino acid decomposition at high temperatures because the data can be collected rapidly with short equilibration times. The first ionization constant for proline and α-alanine, K a,COOH, and the first and second ionization constants for glycine, K a,COOH and K a,NH4+, have been determined at temperatures as high as 250°C. Values for the standard partial molar heat capacity of ionization, Δ rC po, COOH and Δ rC po, NH4+, have been determined from the temperature dependence of ln (K a,COOH) and ln (K a,NH4+). The methodology has been validated by measuring the ionization constant of acetic acid up to 250°C, with results that agree with literature values obtained by potentiometric measurements to within the combined experimental uncertainty. We dedicate this paper to the memory of Dr. Donald Irish (1932-2002) of the University of Waterloo—friend and former supervisor of two of the authors (R.J.B. and P.R.T.).

  3. Localization of two post-proline cleaving peptidases in the midgut of Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in the midgut of Tenebrio molitor larvae with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activit...

  4. Optimization of the catalytic asymmetric addition of nitroalkanes to cyclic enones with trans-4,5-methano-L-proline.

    PubMed

    Hanessian, Stephen; Shao, Zhihui; Warrier, Jayakumar S

    2006-10-12

    [reaction: see text] The conjugate addition of symmetrical 2-nitroalkanes to 2-cycloalkenones catalyzed by trans-4,5-methano-l-proline proceeds with >99% ee and excellent chemical yields. 1-Nitroalkanes afford diastereomeric syn/anti products that can be separated with good individual enantioselectivities. Proline hydroxamic acid and its trans-4,5-methano -l-proline hydroxamic acid are also effective organocatalysts in the addition of 2-nitropropane to 2-cyclohexenone (75% and 81% ee, respectively). PMID:17020303

  5. High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22(Phos) → Phe variant

    PubMed Central

    Iavarone, Federica; D’Alessandro, Alfredo; Tian, Na; Cabras, Tiziana; Messana, Irene; Helmerhorst, Eva J.; Oppenheim, Frank G.; Castagnola, Massimo

    2015-01-01

    During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ~22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22(phos)→Phe. PMID:24771659

  6. Proline as a fuel for insect flight: enhancing carbohydrate oxidation in hymenopterans.

    PubMed

    Teulier, Loïc; Weber, Jean-Michel; Crevier, Julie; Darveau, Charles-A

    2016-07-13

    Bees are thought to be strict users of carbohydrates as metabolic fuel for flight. Many insects, however, have the ability to oxidize the amino acid proline at a high rate, which is a unique feature of this group of animals. The presence of proline in the haemolymph of bees and in the nectar of plants led to the hypothesis that plants may produce proline as a metabolic reward for pollinators. We investigated flight muscle metabolism of hymenopteran species using high-resolution respirometry performed on permeabilized muscle fibres. The muscle fibres of the honeybee, Apis mellifera, do not have a detectable capacity to oxidize proline, as those from the migratory locust, Locusta migratoria, used here as an outgroup representative. The closely related bumblebee, Bombus impatiens, can oxidize proline alone and more than doubles its respiratory capacity when proline is combined with carbohydrate-derived substrates. A distant wasp species, Vespula vulgaris, exhibits the same metabolic phenotype as the bumblebee, suggesting that proline oxidation is common in hymenopterans. Using a combination of mitochondrial substrates and inhibitors, we further show that in B. impatiens, proline oxidation provides reducing equivalents and electrons directly to the electron transport system. Together, these findings demonstrate that some bee and wasp species can greatly enhance the oxidation of carbohydrates using proline as fuel for flight. PMID:27412285

  7. Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline.

    PubMed Central

    Mori, H; Shibasaki, T; Yano, K; Ozaki, A

    1997-01-01

    Proline 3-hydroxylase was purified from Streptomyces sp. strain TH1, and its structural gene was cloned. The purified enzyme hydroxylated free L-proline to cis-3-hydroxy-L-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase (H. Mori, T. Shibasaki, Y. Uosaki, K. Ochiai, and A. Ozaki, Appl. Environ. Microbiol, 62:1903-1907, 1996). The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35 degrees C, respectively. The K(m) values were 0.56 and 0.11 mM for L-proline and 2-oxoglutarate, respectively. The Kcat value of hydroxylation was 3.2 s-1. Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database. A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence. With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR. A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe. The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158. No sequence homolog was found in EMBL, GenBank, and DDBJ databases. ORF 3 was expressed in Escherichia coli DH1. Recombinants showed hydroxylating activity five times higher than that of the original bacterium, Streptomyces sp. strain TH1. It was concluded that the ORF 3 encodes functional proline 3-hydroxylase. PMID:9294421

  8. 21 CFR 582.5650 - Proline.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5650 Proline. (a) Product. Proline (L- and DL-forms). (b) Conditions of use. This substance...

  9. 21 CFR 582.5650 - Proline.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5650 Proline. (a) Product. Proline (L- and DL-forms). (b) Conditions of use. This substance...

  10. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    SciTech Connect

    Nasarabadi, Shanavaz

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  11. Proline accumulation and metabolism-related genes expression profiles in Kosteletzkya virginica seedlings under salt stress

    PubMed Central

    Wang, Hongyan; Tang, Xiaoli; Wang, Honglei; Shao, Hong-Bo

    2015-01-01

    Proline accumulation is a common response to salt stress in many plants. Salt stress also increased proline concentration in roots, stems, and leaves of Kosteletzkya virginica seedling treated with 300 mM NaCl for 24 h and reached 3.75-, 4.76-, and 6.83-fold higher than controls. Further study on proline content in leaves under salt stress showed that proline content increased with increasing NaCl concentrations or time. The proline level peaked at 300 mM NaCl for 24 h and reached more than sixfold higher than control, but at 400 mM NaCl for 24 h proline content fell back slightly along with wilting symptom. To explore the cause behind proline accumulation, we first cloned full length genes related to proline metabolism including KvP5CS1, KvOAT, KvPDH, and KvProT from K. virginica and investigated their expression profiles. The results revealed that the expressions of KvP5CS1 and KvProT were sharply up-regulated by salt stress and the expression of KvOAT showed a slight increase with increasing salt concentrations or time, while the expression of KvPDH was not changed much and slightly decreased before 12 h and then returned to the original level. As the key enzyme genes for proline biosynthesis, the up-regulated expression of KvP5CS1 played a more important role than KvOAT for proline accumulation in leaves under salt stress. The low expression of KvPDH for proline catabolism also made a contribution to proline accumulation before 12 h. PMID:26483809

  12. Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

    NASA Astrophysics Data System (ADS)

    Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L. C.

    2011-12-01

    We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D2O and compare with experimental observations.

  13. The 7-amino-acid site in the proline-rich region of the N-terminal domain of p53 is involved in the interaction with FAK and is critical for p53 functioning.

    PubMed

    Golubovskaya, Vita M; Finch, Richard; Zheng, Min; Kurenova, Elena V; Cance, William G

    2008-04-01

    It is known that p53 alterations are commonly found in tumour cells. Another marker of tumorigenesis is FAK (focal adhesion kinase), a non-receptor kinase that is overexpressed in many types of tumours. Previously we determined that the N-terminal domain of FAK physically interacted with the N-terminal domain of p53. In the present study, using phage display, sitedirected mutagenesis, pulldown and immunoprecipitation assays we localized the site of FAK binding to a 7-amino-acid region(amino acids 65-71) in the N-terminal proline-rich domain of human p53. Mutation of the binding site in p53 reversed the suppressive effect of FAK on p53-mediated transactivation ofp21, BAX (Bcl-2-associated X protein) and Mdm2 (murine double minute 2) promoters. In addition, to functionally test this p53 site, we conjugated p53 peptides [wild-type (containing the wild-type binding site) and mutant (with a mutated 7-aminoacid binding site)] to a TAT peptide sequence to penetrate the cells, and demonstrated that the wild-type p53 peptide disrupted binding of FAK and p53 proteins and significantly inhibited cell viability of HCT116 p53+/+ cells compared with the control mutant peptide and HCT116 p53-/- cells. Furthermore, the TAT-p53 peptide decreased the viability of MCF-7 cells, whereas the mutant peptide did not cause this effect. Normal fibroblast p53+/+ and p53-/- MEF (murine embryonic fibroblast) cells and breast MCF10A cells were not sensitive to p53 peptide. Thus, for the first time, we have identified the binding site of the p53 andFAK interaction and have demonstrated that mutating this site and targeting the site with peptides affects p53 functioning and viability in the cells. PMID:18215142

  14. Proline biosynthesis augments tumor cell growth and aerobic glycolysis: involvement of pyridine nucleotides

    PubMed Central

    Liu, Wei; Hancock, Chad N.; Fischer, Joseph W.; Harman, Meredith; Phang, James M.

    2015-01-01

    The metabolism of the nonessential amino acid proline contributes to tumor metabolic reprogramming. Previously we showed that MYC increases proline biosynthesis (PB) from glutamine. Here we show MYC increases the expression of the enzymes in PB at both protein and mRNA levels. Blockade of PB decreases tumor cell growth and energy production. Addition of Δ1-pyrroline-5-carboxylate (P5C) or proline reverses the effects of P5C synthase knockdown but not P5C reductases knockdown. Importantly, the reversal effect of proline was blocked by concomitant proline dehydrogenase/oxidase (PRODH/POX) knockdown. These findings suggest that the important regulatory contribution of PB to tumor growth derives from metabolic cycling between proline and P5C rather than product proline or intermediate P5C. We further document the critical role of PB in maintaining pyridine nucleotide levels by connecting the proline cycle to glycolysis and to the oxidative arm of the pentose phosphate pathway. These findings establish a novel function of PB in tumorigenesis, linking the reprogramming of glucose, glutamine and pyridine nucleotides, and may provide a novel target for antitumor therapy. PMID:26598224

  15. Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

    PubMed Central

    Verbruggen, N; Hua, X J; May, M; Van Montagu, M

    1996-01-01

    In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis. Images Fig. 3 Fig. 4 PMID:8710950

  16. Proline catalyzed α-aminoxylation reaction in the synthesis of biologically active compounds.

    PubMed

    Kumar, Pradeep; Dwivedi, Namrata

    2013-02-19

    The search for new and efficient ways to synthesize optically pure compounds is an active area of research in organic synthesis. Asymmetric catalysis provides a practical, cost-effective, and efficient method to create a variety of complex natural products containing multiple stereocenters. In recent years, chemists have become more interested in using small organic molecules to catalyze organic reactions. As a result, organocatalysis has emerged both as a promising strategy and as an alternative to catalysis with expensive proteins or toxic metals. One of the most successful and widely studied secondary amine-based organocatalysts is proline. This small molecule can catalyze numerous reactions such as the aldol, Mannich, Michael addition, Robinson annulation, Diels-Alder, α-functionalization, α-amination, and α-aminoxylation reactions. Catalytic and enantioselective α-oxygenation of carbonyl compounds is an important reaction to access a variety of useful building blocks for bioactive molecules. Proline catalyzed α-aminoxylation using nitrosobenzene as oxygen source, followed by in situ reduction, gives enantiomerically pure 1,2-diol. This molecule can then undergo a variety of organic reactions. In addition, proline organocatalysis provides access to an assortment of biologically active natural products including mevinoline (a cholesterol lowering drug), tetrahydrolipstatin (an antiobesity drug), R(+)-α-lipoic acid, and bovidic acid. In this Account, we present an iterative organocatalytic approach to synthesize both syn- and anti-1,3-polyols, both enantio- and stereoselectively. This method is primarily based on proline-catalyzed sequential α-aminoxylation and Horner-Wadsworth-Emmons (HWE) olefination of aldehyde to give a γ-hydroxy ester. In addition, we briefly illustrate the broad application of our recently developed strategy for 1,3-polyols, which serve as valuable, enantiopure building blocks for polyketides and other structurally diverse and

  17. SSR Markers Associated with Proline in Drought Tolerant Wheat Germplasm.

    PubMed

    Iqbal, Muhammad Javid; Maqsood, Yasir; Abdin, Zain Ul; Manzoor, Atif; Hassan, Muhammad; Jamil, Amer

    2016-03-01

    Water stress causes major agricultural loss throughout the world as survival of the crops remained under stress and loss in yield. Plants respond to drought stress by means of different adaptive mechanisms such as accumulation of osmoprotectants to counteract the water stress. Amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities as an osmolyte in response to environmental stresses. Biochemical estimation of proline was done in the drought-affected wheat genotypes by spectrophotometric method. Proline promoted a positive effect as root/shoot ratio was enhanced in wheat germplasm under drought stress. SSR primer pairs (45) were tested for polymorphism among selected wheat genotypes. The dendrogram results have shown the wheat genotype association with the levels of proline during induced drought stress. The relationship between pattern of drought responsive biochemical attributes and DNA markers in the selected wheat genotypes was recognized to select drought tolerant genotypes for sowing in drought affected areas of the country. PMID:26637362

  18. Proline dehydrogenase is essential for proline protection against hydrogen peroxide induced cell death

    PubMed Central

    Natarajan, Sathish Kumar; Zhu, Weidong; Liang, Xinwen; Zhang, Lu; Demers, Andrew J.; Zimmerman, Matthew C.; Simpson, Melanie A.; Becker, Donald F.

    2012-01-01

    Proline metabolism has an underlying role in apoptotic signaling that impacts tumorigenesis. Proline is oxidized to glutamate in the mitochondria with the rate limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53 leading to increased proline oxidation, reactive oxygen species (ROS) formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and retained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress whereas knockdown of Δ1-pyrroline-5-carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the fork head transcription factor class O3a (FoxO3a). The role of PRODH in proline mediated protection was validated in the prostate carcinoma cell line, PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide mediated cell death and that proline/PRODH helps activate Akt in cancer cells. PMID:22796327

  19. On Spontaneously Pulsating Proline-Phenylalanine Peptide Microfibers.

    PubMed

    Godziek, Agnieszka; Maciejowska, Anna; Talik, Ewa; Wrzalik, Roman; Sajewicz, Mieczysław; Kowalska, Teresa

    2016-01-01

    Earlier, we have collected an experimental evidence showing that low molecular weight chiral carboxylic acids (amino acids included) can spontaneously undergo an oscillatory chiral conversion and an oscillatory condensation in abiotic aqueous and non-aqueous liquid systems, stored for certain amount of time under mild external conditions. These earlier findings are summarized in the introductory part of this study. In the second part, a preliminary report is given on spontaneous pulsation of peptide microfibers in the aged proline-phenylalanine (Pro-Phe) solution in 70% aqueous acetonitrile. The experimental evidence originates from a number of advanced analytical techniques. In view of our earlier and present findings, a presumption is made that the mechanism of spontaneous pulsation (formation and decay) of Pro-Phe microfibers is directly related to the oscillatory chiral conversion and oscillatory peptidization. The entity of the discussed results pointing out to spontaneous and uncontrolled instability of peptide structures might be a bad prognostic for employing such structures in nanobiotechnology. PMID:26521956

  20. Proline-based Phosphoramidite Reagents for the Reductive Ligation of S-Nitrosothiols

    PubMed Central

    Xian, Ming

    2015-01-01

    S-Nitrosothiols have many biological implications but are rarely used in organic synthesis. In this work we report the development of proline-based phosphoramidite substrates that can effectively convert S-nitrosothiols to proline-based sulfenamides through a reductive ligation process. A unique property of this method is that the phosphine oxide moiety on the ligation products can be readily removed under acidic conditions. In conjugation with the facile preparation of S-nitrosothiols (RSNO) from the corresponding thiols (RSH), this method provides a new way to prepare proline-based sulfenamides from simple thiol starting materials. PMID:26758493

  1. Quest for Cells Responsible for Age-related Increase of Salivary Glycine and Proline.

    PubMed

    Hino, Shunsuke; Nishiyama, Akira; Matsuta, Tomohiko; Horie, Norio; Shimoyama, Tetsuo; Tanaka, Shoji; Sakagami, Hiroshi

    2016-01-01

    We have previously reported that salivary glycine and proline levels are increased to nearly butanoate level in elderly people. In order to identify the source of glycine and proline, we performed high-performance liquid chromatography analysis of amino acid production to a total of seven oral cells before and after stimulation with inflammation inducers. We found that production of amino acids (per a given number of cells) by normal oral mesenchymal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) was approximately three-fold that of oral squamous cell carcinoma cell lines (HSC-2, HSC-3, HSC-4, Ca9-22), and that production of glycine and especially proline by all these seven cells was much lower than that of glutamine and glutamic acid. Treatment of three oral mesenchymal cells with interleukin (IL)-1β or lipopoly-saccharide (LPS) reproducibly increased the production of glutamic acid and glutamine, but not that of glycine and proline. Glycine and proline only marginally stimulated the IL-8 production by IL-1β-stimulated gingival fibroblast, whereas glycine dose-dependently inhibited the nitric oxide production by lipopolysaccharide-stimulated mouse macrophage-like RAW264.7 cells. These data demonstrated that normal oral mesenchymal cells are not the major source of glycine and proline that accumulates in the saliva of aged people, suggesting the involvement of the deregulation of collagen metabolism during aging. PMID:26912818

  2. Crystal structures and kinetics of monofunctional proline dehydrogenase provide insight into substrate recognition and conformational changes associated with flavin reduction and product release.

    PubMed

    Luo, Min; Arentson, Benjamin W; Srivastava, Dhiraj; Becker, Donald F; Tanner, John J

    2012-12-18

    Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to Δ(1)-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analogue L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by helix α8 and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition. PMID:23151026

  3. Crystal Structures and Kinetics of Monofunctional Proline Dehydrogenase Provide Insight into Substrate Recognition and Conformational Changes Associated With Flavin Reduction and Product Release

    PubMed Central

    Luo, Min; Arentson, Benjamin W.; Srivastava, Dhiraj; Becker, Donald F.; Tanner, John J.

    2012-01-01

    Proline dehydrogenase catalyzes the FAD-dependent oxidation of proline to Δ1- pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analog L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analog binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by the α8 helix and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition. PMID:23151026

  4. Selenazolidine: a selenium containing proline surrogate in peptide science.

    PubMed

    Cordeau, E; Cantel, S; Gagne, D; Lebrun, A; Martinez, J; Subra, G; Enjalbal, C

    2016-09-14

    In the search for new peptide ligands containing selenium in their sequences, we investigated l-4-selenazolidine-carboxylic acid (selenazolidine, Sez) as a proline analog with the chalcogen atom in the γ-position of the ring. In contrast to proteinogenic selenocysteine (Sec) and selenomethionine (SeMet), the incorporation within a peptide sequence of such a non-natural amino acid has never been studied. There is thus a great interest in increasing the possibility of selenium insertion within peptides, especially for sequences that do not possess a sulfur containing amino acid (Cys or Met), by offering other selenated residues suitable for peptide synthesis protocols. Herein, we have evaluated selenazolidine in Boc/Bzl and Fmoc/tBu strategies through the synthesis of a model tripeptide, both in solution and on a solid support. Special attention was paid to the stability of the Sez residue in basic conditions. Thus, generic protocols have been optimized to synthesize Sez-containing peptides, through the use of an Fmoc-Xxx-Sez-OH dipeptide unit. As an example, a new analog of the vasopressin receptor-1A antagonist was prepared, in which Pro was replaced with Sez [3-(4-hydroxyphenyl)-propionyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Sez-Arg-NH2]. Both proline and such pseudo-proline containing peptides exhibited similar pharmacological properties and endopeptidase stabilities indicating that the presence of the selenium atom has minimal functional effects. Taking into account the straightforward handling of Sez as a dipeptide building block in a conventional Fmoc/tBu SPPS strategy, this result suggested a wide range of potential uses of the Sez amino acid in peptide chemistry, for instance as a viable proline surrogate as well as a selenium probe, complementary to Sec and SeMet, for NMR and mass spectrometry analytical purposes. PMID:27506250

  5. STRUCTURE AND KINETICS OF MONOFUNCTIONAL PROLINE DEHYDROGENASE FROM THERMUS THERMOPHILUS

    PubMed Central

    White, Tommi A.; Krishnan, Navasona; Becker, Donald F.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria, but are fused into bifunctional enzymes known as Proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0 Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent exposed compared to PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify 9 conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is −75 mV and the kinetic parameters for proline are Km=27 mM and kcat=13 s−1. 3,4-dehydro-L-proline was found to be an efficient substrate and L-tetrahydro-2-furoic acid is a competitive inhibitor (KI=1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs. PMID:17344208

  6. Stereoselective Synthesis of Quaternary Proline Analogues

    PubMed Central

    Calaza, M. Isabel

    2009-01-01

    This review describes available methods for the diastereoselective and asymmetric synthesis of quaternary prolines. The focus is on the preparation of α-functionalized prolines with the pyrrolidine moiety not embedded in a polycyclic frame. The diverse synthetic approaches are classified according to the bond which is formed to complete the quaternary skeleton. PMID:19655047

  7. Proline transport and osmotic stress response in Escherichia coli K-12.

    PubMed Central

    Grothe, S; Krogsrud, R L; McClellan, D J; Milner, J L; Wood, J M

    1986-01-01

    Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced. PMID:3514577

  8. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate†,‡

    PubMed Central

    Ostrander, Elizabeth L.; Larson, John D.; Schuermann, Jonathan P.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insights into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analog L-tetrahydro-2-furoic acid were determined at resolutions of 1.75 Å, 1.90 Å and 1.85 Å. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline three-fold and decreases the specificity for proline by factors of twenty (Y540S) and fifty (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate. PMID:19140736

  9. Crystal structures of coordination polymers from CaI2 and proline

    PubMed Central

    Lamberts, Kevin; Englert, Ulli

    2015-01-01

    Completing our reports concerning the reaction products from calcium halides and the amino acid proline, two different solids were found for the reaction of l- and dl-proline with CaI2. The enanti­opure amino acid yields the one-dimensional coordination polymer catena-poly[[aqua-μ3-l-proline-tetra-μ2-l-proline-dicalcium] tetra­iodide 1.7-hydrate], {[Ca2(C5H9NO2)5(H2O)]I4·1.7H2O}n, (1), with two independent Ca2+ cations in characteristic seven- and eightfold coordination. Five symmetry-independent zwitterionic l-proline mol­ecules bridge the metal sites into a cationic polymer. Racemic proline forms with Ca2+ cations heterochiral chains of the one-dimensional polymer catena-poly[[di­aquadi-μ2-dl-proline-calcium] diiodide], {[Ca(C5H9NO2)2(H2O)2]I2}n, (2). The centrosymmetric structure is built by one Ca2+ cation that is bridged towards its symmetry equivalents by two zwitterionic proline mol­ecules. In both structures, the iodide ions remain non-coordinating and hydrogen bonds are formed between these counter-anions, the amino groups, coordinating and co-crystallized water mol­ecules. While the overall composition of (1) and (2) is in line with other structures from calcium halides and amino acids, the diversity of the carboxyl­ate coordination geometry is quite surprising. PMID:26090148

  10. Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

    SciTech Connect

    Camilloni, Carlo; Sahakyan, Aleksander B.; Holliday, Michael; Isern, Nancy G.; Zhang, Fengli; Eisenmesser, Elan Z.; Vendruscolo, Michele

    2014-07-15

    Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions1-3. Different families of enzymes, known as peptidyl-prolyl isomerases (PPIases), catalyse this reaction, which involves the interconversion between the cis and trans isomers of the Nterminal amide bond of the amino acid proline2,3. A complete description of the mechanisms by which these enzymes function, however, has remained elusive. Here, we show that cyclophilin A, one of the most common PPIases4, provides a catalytic environment that acts on the substrate through an electrostatic lever mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carboxylic group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. This mechanism resulted from the analysis of an ensemble of conformations populated by cyclophilin A during the enzymatic reaction using a combination of NMR measurements, molecular dynamics simulations and density functional theory calculations. We anticipate that this approach will be helpful in elucidating whether the electrostatic lever mechanism that we describe is common to other PPIases, and more generally to characterise other enzymatic processes.

  11. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana.

    PubMed

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2014-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  12. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    PubMed Central

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2015-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  13. Fatty Acid Composition of Egg Yolk from Chickens Fed a Diet including Marigold (Tagetes erecta L.)

    PubMed Central

    Altuntaş, A.; Aydin, R.

    2014-01-01

    The objective of this study was to determine the effects of diet supplemented with marigold on egg yolk fatty acid composition and egg quality parameters. Sixty hens were assigned into three groups and fed diets supplemented with 0 (control), 10 g kg−1, or 20 g kg−1 marigold for 42 days. Eggs collected at the 6th week of the study were analyzed for fatty acid analysis. Laying performance, egg quality parameters, and feed intake were also evaluated. Yolk color scores in the group fed the 20 g kg−1 marigold-supplemented diet were found greater than control (10.77 versus 9.77). Inclusion of 20 g kg−1 marigold in diet influenced egg weights adversely compared to the control. Diet supplemented with 10 g kg−1 or 20 g kg−1 marigold increased the levels of C16:0 and C18:0 and decreased levels of C16:1 (n-7) and C18:1 (n-9) in the egg yolk. Also, diet including marigold increased total saturated fatty acids (SFA) and decreased monounsaturated fatty acids (MUFA) in the egg yolk. PMID:25587451

  14. Proline transport by brush-border membrane vesicles of lobster antennal glands

    SciTech Connect

    Behnke, R.D.; Wong, R.K.; Huse, S.M.; Reshkin, S.J.; Ahearn, G.A. )

    1990-02-01

    Purified brush-border membrane vesicles (BBMV) of lobster antennal gland labyrinth and bladder were separately formed by a magnesium precipitation technique. L-(3H)proline uptake was stimulated by a transmembrane NaCl gradient (outside (o) greater than inside (i)) to a greater extent in BBMV from labyrinth than those from the bladder. Detailed study of the labyrinth proline-transport processes revealed a specific dependence on NaCl, with negligible stimulatory effects by NaSCN, Na-gluconate, or KCl. A transmembrane proton gradient (o greater than i) was without stimulatory effect on proline transport. A transmembrane potential difference alone, in the presence of equilibrated NaCl and L-(3H)proline, led to net influx of the labeled amino acid, suggesting that the uptake process was electrogenic and capable of bringing about the net transfer of positive charge to the vesicle interior. Although a transmembrane Na gradient alone, in the presence of equilibrated Cl and L-(3H)proline, was able to bring about the net influx of the amino acid, a transmembrane Cl gradient alone under Na- and L-(3H)proline-equilibrated conditions was not, suggesting that only the Na gradient could energize the carrier process through cotransport, while the anion served an essential activating role. Proline influx by these vesicles occurred by the combination of at least one saturable Michaelis-Menten carrier system (apparent Kt = 0.37 mM; apparent JM = 1.19 nmol.mg protein-1.10 s-1) and apparent diffusion (P = 0.33 nmol.mg protein-1.10 s-1.mM-1). Static head analysis of the transport process suggested a cotransport stoichiometry of 2 Na:1 proline with essential activation by Cl ion.

  15. 4-Substituted boro-proline dipeptides: synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activities.

    PubMed

    Wu, Wengen; Liu, Yuxin; Milo, Lawrence J; Shu, Ying; Zhao, Peng; Li, Youhua; Woznica, Iwona; Yu, Gengli; Sanford, David G; Zhou, Yuhong; Poplawski, Sarah E; Connolly, Beth A; Sudmeier, James L; Bachovchin, William W; Lai, Jack H

    2012-09-01

    The boroProline-based dipeptidyl boronic acids were among the first DPP-IV inhibitors identified, and remain the most potent known. We introduced various substitutions at the 4-position of the boroProline ring regioselectively and stereoselectively, and incorporated these aminoboronic acids into a series of 4-substituted boroPro-based dipeptides. Among these dipeptidyl boronic acids, Arg-(4S)-boroHyp (4q) was the most potent inhibitor of DPP-IV, DPP8 and DPP9, while (4S)-Hyp-(4R)-boroHyp (4o) exhibited the most selectivity for DPP-IV over DPP8 and DPP9. PMID:22853995

  16. Proline accumulation protects Saccharomyces cerevisiae cells in stationary phase from ethanol stress by reducing reactive oxygen species levels.

    PubMed

    Takagi, Hiroshi; Taguchi, Junpei; Kaino, Tomohiro

    2016-08-01

    During fermentation processes, Saccharomyces cerevisiae cells are exposed to multiple stresses, including a high concentration of ethanol that represents toxicity through intracellular reactive oxygen species (ROS) generation. We previously reported that proline protected yeast cells from damage caused by various stresses, such as freezing and ethanol. As an anti-oxidant, proline is suggested to scavenge intracellular ROS. In this study, we examined the role of intracellular proline during ethanol treatment in S. cerevisiae strains that accumulate different concentrations of proline. When cultured in YPD medium, there was a significant accumulation of proline in the put1 mutant strain, which is deficient in proline oxidase, in the stationary phase. Expression of the mutant PRO1 gene, which encodes the γ-glutamyl kinase variant (Asp154Asn or Ile150Thr) with desensitization to feedback inhibition by proline in the put1 mutant strain, showed a prominent increase in proline content as compared with that of the wild-type strain. The oxidation level was clearly increased in wild-type cells after exposure to ethanol, indicating that the generation of ROS occurred. Interestingly, proline accumulation significantly reduces the ROS level and increases the survival rate of yeast cells in the stationary phase under ethanol stress conditions. However, there was not a clear correlation between proline content and survival rate in yeast cells. An appropriate level of intracellular proline in yeast might be important for its stress-protective effect. Hence, the engineering of proline metabolism could be promising for breeding stress-tolerant industrial yeast strains. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26833688

  17. Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771?

    PubMed

    Xiong, Z; Farilla, L; Guo, J; McLachlan, S; Rapoport, B

    2001-03-01

    Identification of the thyroid peroxidase (TPO) amino acid residues that comprise the autoantibody immunodominant region is an important goal that has proven difficult because of the conformational nature of the epitopes involved. Recent data suggest that the immunodominant region has been located. Thus, by autoantibody recognition of tryptic fragments of native TPO, as well as of conformational portions of TPO expressed as cell-free translates, the autoantibody immunodominant region appears to include amino acid residues 742-771, near the C terminus of the ectodomain. To evaluate this deduction, we expressed as cell-free translates the full TPO ectodomain, as well as TPO truncated after residues 741 and 771. The epitopic integrity of these molecules was first confirmed by immunoprecipitation by patient sera containing TPO autoantibodies. However, autoantibody recognition could involve a minority of TPO autoantibodies with the individual sera, not fulfilling the strict criteria for immunodominance. In order to obtain definitive data, we performed immunoprecipitations on these TPO variants with four recombinant human monoclonal autoantibodies that define the immunodominant region. All four monoclonal autoantibodies immunoprecipitated TPO 1-741 to the same extent as they did TPO 1-771 and the full TPO ectodomain, indicating that the immunodominant region comprises (at least in large part) amino acid residues upstream of residue 741. PMID:11327613

  18. Amino acid composition, including key derivatives of eccrine sweat: potential biomarkers of certain atopic skin conditions.

    PubMed

    Mark, Harker; Harding, Clive R

    2013-04-01

    The free amino acid (AA) composition of eccrine sweat is different from other biological fluids, for reasons which are not properly understood. We undertook the detailed analysis of the AA composition of freshly isolated pure human eccrine sweat, including some of the key derivatives of AA metabolism, to better understand the key biological mechanisms governing its composition. Eccrine sweat was collected from the axillae of 12 healthy subjects immediately upon formation. Free AA analysis was performed using an automatic AA analyser after ninhydrin derivatization. Pyrrolidine-5-carboxylic acid (PCA) and urocanic acid (UCA) levels were determined using GC/MS. The free AA composition of sweat was dominated by the presence of serine accounting for just over one-fifth of the total free AA composition. Glycine was the next most abundant followed by PCA, alanine, citrulline and threonine, respectively. The data obtained indicate that the AA content of sweat bears a remarkable similarity to the AA composition of the epidermal protein profilaggrin. This protein is the key source of free AAs and their derivatives that form a major part of the natural moisturizing factor (NMF) within the stratum corneum (SC) and plays a major role in maintaining the barrier integrity of human skin. As perturbations in the production of NMF can lead to abnormal barrier function and can arise as a consequence of filaggrin genotype, we propose the quantification of AAs in sweat may serve as a non-invasive diagnostic biomarker for certain atopic skin conditions, that is, atopic dermatitis (AD). PMID:23075272

  19. Two proline porters in Escherichia coli K-12.

    PubMed Central

    Stalmach, M E; Grothe, S; Wood, J M

    1983-01-01

    Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus. PMID:6355059

  20. Proline-induced changes in acetylcholinesterase activity and gene expression in zebrafish brain: reversal by antipsychotic drugs.

    PubMed

    Savio, L E B; Vuaden, F C; Kist, L W; Pereira, T C; Rosemberg, D B; Bogo, M R; Bonan, C D; Wyse, A T S

    2013-10-10

    Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 μM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism. PMID:23867765

  1. Proline Metabolism and Its Implications for Plant-Environment Interaction

    PubMed Central

    Verslues, Paul E.

    2010-01-01

    Proline has long been known to accumulate in plants experiencing water limitation and this has driven studies of proline as a beneficial solute allowing plants to increase cellular osmolarity during water limitation. Proline metabolism also has roles in redox buffering and energy transfer and is involved in plant pathogen interaction and programmed cell death. Some of these unique roles of proline depend on the properties of proline itself, whereas others depend on the “proline cycle” of coordinated proline synthesis in the chloroplast and cytoplasm with proline catabolism in the mitochondria. The regulatory mechanisms controlling proline metabolism, intercellular and intracellular transport and connections of proline to other metabolic pathways are all important to the in vivo functions of proline metabolism. Connections of proline metabolism to the oxidative pentose phosphate pathway and glutamate-glutamine metabolism are of particular interest. The N-acetyl glutamate pathway can also produce ornithine and, potentially, proline but its role and activity are unclear. Use of model systems such as Arabidopsis thaliana to better understand both these long studied and newly emerging functions of proline can help in the design of next-generation experiments testing whether proline metabolism is a promising metabolic engineering target for improving stress resistance of economically important plants. PMID:22303265

  2. Synthesis of Branched Methyl Hydroxy Stearates Including an Ester from Bio-Based Levulinic Acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the synthesis of 5 useful branched methyl alpha-hydroxy oleate esters from commercially available methyl oleate and common organic acids. Of special interest is the synthesis utilizing the natural byproduct, levulinic acid. The other common organic acids used herein were propionic acid, ...

  3. Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli.

    PubMed Central

    Wengender, P A; Miller, K J

    1995-01-01

    The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins. PMID:7887605

  4. Proline porters effect the utilization of proline as nutrient or osmoprotectant for bacteria.

    PubMed

    Wood, J M

    1988-12-01

    Proline is utilized by all organisms as a protein constituent. It may also serve as a source of carbon, energy and nitrogen for growth or as an osmoprotectant. The molecular characteristics of the proline transport systems which mediate the multiple functions of proline in the Gram negative enteric bacteria, Escherichia coli and Salmonella typhimurium, are now becoming apparent. Recent research on those organisms has provided both protocols for the genetic and biochemical characterization of the enzymes mediating proline transport and molecular probes with which the degree of homology among the proline transport systems of archaebacteria, eubacteria and eukaryotes can be assessed. This review has provided a detailed summary of recent research on proline transport in E. coli and S. typhimurium; the properties of other organisms are cited primarily to illustrate the generality of those observations and to show where homologous proline transport systems might be expected to occur. The characteristics of proline transport in eukaryotic microorganisms have recently been reviewed (Horak, 1986). PMID:3072423

  5. Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

    PubMed Central

    Berneman, Armand; Montout, Lory; Goyard, Sophie; Chamond, Nathalie; Cosson, Alain; d’Archivio, Simon; Gouault, Nicolas; Uriac, Philippe; Blondel, Arnaud; Minoprio, Paola

    2013-01-01

    Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles. PMID:23613764

  6. Hypothalamic proline rich polypeptide regulates hematopoiesis.

    PubMed

    Bezirganyan, Kristina B; Davtyan, Tigran K; Galoyan, Armen A

    2010-06-01

    The AGAPEPAEPAQPGVY proline-rich polypeptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects including immunoregulatory, hematopoietic, antimicrobial and anti-neurodegenerative properties. Here we demonstrated that PRP-1 administration influence on redistribution of monocytes, granulocytes and lymphocytes between bone marrow (BM) and peripheral blood and promotes the influx of granulocytes and monocytes/macrophages from BM into peripheral blood and accumulation of immature granulocyte and monocyte in BM and delayed the maturation of T cells in BM. PRP-1 increased colony-forming cell proliferation in rat cells in vivo. In PRP-treated rat BM, the CFU number at day 4, 7 and 14 was considerably increased in comparison with untreated rats BM and no difference was found at day 21 and day 28. We found that PRP-1 enhances erythroid and myeloid colonies formation in human CD34(+) progenitor cell culture in the presence of different growth factors and down-regulates T cells colony formation and specific surface markers expression during induction of human CD34(+) progenitor cells differentiation into T lymphocytes lineage. We suggested that the hypothalamic PRP-1 possibly represents an endogenous peptide whose primary functions are to regulate neuronal survival and differentiation and hematopoiesis within neurosecretory hypothalamus-bone marrow humoral axis. PMID:20020325

  7. Fatty acid composition including cis-9, trans-11 CLA of cooked ground lamb

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little information is available on effect of cooking on beneficial fatty acids such as conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids (PUFA). The objective of this study was to examine impact of cooking on the FA composition of ground lamb of two different muscles. Samples were p...

  8. The formation of ornithine from proline in animal tissues

    PubMed Central

    Smith, A. D.; Benziman, M.; Strecker, H. J.

    1967-01-01

    1. Homogenates of liver or kidney from rat, mouse, dog and guinea pig formed ornithine from proline but not from glutamate. Rat kidney was most active in this reaction and was used for further studies. 2. The overall reaction was found to be catalysed by proline oxidase to yield glutamic γ-semialdehyde, followed by transamination of this product with glutamate as catalysed by ornithine–keto acid aminotransferase. 3. The unfavourable equilibrium of the ornithine–keto acid aminotransferase reaction was overcome chiefly by glutamate dehydrogenase in the tissue, which removed the α-oxoglutarate produced, by reduction with endogenous ammonia and NADH. 4. Aspartate aminotransferase in these preparations also aided in the removal of α-oxoglutarate. In this case the overall reaction was driven also by the rapid decarboxylation of oxaloacetate. 5. No evidence could be found for a pathway of ornithine synthesis involving acylated intermediates as has been observed in some micro-organisms. 6. The rate of ornithine synthesis in homogenates of several rat tissues paralleled the activity of ornithine–keto acid aminotransferase in these tissues, indicating that this enzyme was rate-determining for the synthesis. 7. The possible influence of these reactions on urea synthesis is discussed. PMID:6048797

  9. The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

    PubMed Central

    Zaprasis, Adrienne; Hoffmann, Tamara; Stannek, Lorena; Gunka, Katrin; Commichau, Fabian M.

    2014-01-01

    PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates. PMID:24142252

  10. Assessment of the impact of Aluminum on germination, early growth and free proline content in Lactuca sativa L.

    PubMed

    Silva, Patrícia; Matos, Manuela

    2016-09-01

    Aluminum (Al) toxicity is a major problem in crop production on acid soils. The use of industrial or municipal wastewaters, which may be contaminated with metals, for irrigation in agriculture is common over the world. This action can increase the concentration of these agents in the soil and decrease crops yields. In order to evaluate the toxicological effects of recommended Al levels in irrigation water, under acidic conditions, on lettuce, seeds of two cultivars ("cv Reine de Mai" and "cv White Boston") were exposed to five different Al concentrations (0, 0.05, 0.5, 5 and 20mg/L) and germination percentage, root and shoot lengths were measured. Also, the germination rate and the vigor index were calculated, and the proline content was estimated for all concentrations. Results showed that seed germination was not negatively affected by Al, but the germination rate decreased in both cultivars. For the other factors analyzed, with the exception of 20mg/L concentration for "cv White Boston", Al induced, in general, negative effects including the content of proline that increased in the seeds that were exposed to this metal. The "cv Reine de Mai" was more sensitive for the analyzed concentrations than the other cultivar.. The results indicated that even recommended Al concentrations for irrigation, under acidic conditions, can interfere negatively in seed germination and seedling establishment and possibly with crop production. PMID:27229755

  11. [Rheological properties of human bronchial secretions: demonstration of proline-rich polypeptides and their role (author's transl)].

    PubMed

    Bailleul, V; Richet, C; Hayem, A; Degand, P

    1977-01-17

    Human bronchial secretions were examined for chemical components and rheological properties. Proline-rich polypeptides (PRP) obtained by ultrasonic treatment and by contact with a cationic resin (AG 50WX2) were purified by gel-filtration chromatography and high-voltage electrophoresis. The chemical composition of these components allowed a classification according to their proline, glycine, glutamic acid and lysine contents. Rheological experiments suggest a biological role for the PRP in the fibrillar structure of sputum. PMID:12892

  12. Influence of Fatty Acid Precursors, Including Food Preservatives, on the Growth and Fatty Acid Composition of Listeria monocytogenes at 37 and 10°C ▿

    PubMed Central

    Julotok, Mudcharee; Singh, Atul K.; Gatto, Craig; Wilkinson, Brian J.

    2010-01-01

    Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C4, C5, and C6 branched-chain carboxylic acid, and C3 and C4 straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein. PMID:20048057

  13. Use of prolines for improving growth and other properties of plants and algae

    DOEpatents

    Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

    2004-12-14

    Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

  14. Use of prolines for improving growth and other properties of plants and algae

    DOEpatents

    Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

    2003-04-29

    Increasing the concentration of prolines such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that overexpress glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramnate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

  15. Use of prolines for improving growth and other properties of plants and algae

    DOEpatents

    Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

    2003-07-15

    Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

  16. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

    PubMed

    Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M; Geiger, Otto

    2015-09-01

    Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth. PMID:25711932

  17. Real-time monitoring of matrix acidizing including the effects of diverting agents

    SciTech Connect

    Hill, A.D.; Zhu, D.

    1996-05-01

    Real-time monitoring of the injection rate and pressure during matrix acidizing provides operators with a way to determine the changing skin factor as stimulation proceeds. Current methods are based either on the assumption of steady-state flow in the region around the wellbore affected by acid injection or on computer solution of the transient flow equations describing the unsteady reservoir flow process occurring during acidizing. In this paper, a new method for real-time monitoring of matrix acidizing, the inverse injectivity vs. superposition time function plot, is presented. This new method can be applied with a spreadsheet computer program or a programmable calculator and accounts for the transient flow effects occurring during matrix acidizing at multiple rates and injection pressures. The evolving skin factor during a matrix treatment is readily obtained from the diagnostic plot. Hypothetical examples show how the inverse injectivity plot can be used to assess the efficiency of stimulation and diversion. Comparisons with previously presented field cases show the new method to be a simple and accurate means of monitoring the evolving skin factor during matrix acidizing.

  18. Salt stress encourages proline accumulation by regulating proline biosynthesis and degradation in Jerusalem artichoke plantlets.

    PubMed

    Huang, Zengrong; Zhao, Long; Chen, Dandan; Liang, Mingxiang; Liu, Zhaopu; Shao, Hongbo; Long, Xiaohua

    2013-01-01

    Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation. PMID:23637970

  19. Salt Stress Encourages Proline Accumulation by Regulating Proline Biosynthesis and Degradation in Jerusalem Artichoke Plantlets

    PubMed Central

    Huang, Zengrong; Zhao, Long; Chen, Dandan; Liang, Mingxiang; Liu, Zhaopu; Shao, Hongbo; Long, Xiaohua

    2013-01-01

    Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation. PMID:23637970

  20. Insertion proQ220::Tn5 alters regulation of proline porter II, a transporter of proline and glycine betaine in Escherichia coli.

    PubMed Central

    Milner, J L; Wood, J M

    1989-01-01

    Mutation pro-220::Tn5, which increases the resistance of Escherichia coli to 3,4-dehydroproline (M. E. Stalmach, S. Grothe, and J. M. Wood, J. Bacteriol. 156:481-486, 1983), is not linked to putP, proP, or proU. It was located at 40.4 min on the E. coli chromosomal linkage map, by conjugational and transductional mapping, and is now denoted proQ220::Tn5. Proline porter II was not detectable when proQ220::Tn5 proP+ bacteria were cultivated under optimal conditions or with nutritional stress (amino acid limitation). Toxic proline analog sensitivity and proline porter II activity were partially restored to proQ220::Tn5 proP+ bacteria, but not to a proQ220::Tn5 proP219 strain, by a hyperosmotic shift and by growth under osmotic stress. Elevated expression of a proP::lacZ gene fusion, for bacteria grown under osmotic stress, was not influenced by the proQ220::Tn5 insertion. We propose that the proQ locus encodes a positive regulatory element which elevates proline porter II activity. PMID:2536686

  1. Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N-propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction†,‡

    PubMed Central

    Srivastava, Dhiraj; Zhu, Weidong; Johnson, William H.; Whitman, Christian P.; Becker, Donald F.; Tanner, John J.

    2013-01-01

    Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the ε-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline reduced, membrane-associated form. The 2.15 Å resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane binding domain include major reorganization of the flavin ribityl chain, severe (35 degree) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5), Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the β1-αl loop to complete the active site. PMID:19994913

  2. Kinetic model of water disinfection using peracetic acid including synergistic effects.

    PubMed

    Flores, Marina J; Brandi, Rodolfo J; Cassano, Alberto E; Labas, Marisol D

    2016-01-01

    The disinfection efficiencies of a commercial mixture of peracetic acid against Escherichia coli were studied in laboratory scale experiments. The joint and separate action of two disinfectant agents, hydrogen peroxide and peracetic acid, were evaluated in order to observe synergistic effects. A kinetic model for each component of the mixture and for the commercial mixture was proposed. Through simple mathematical equations, the model describes different stages of attack by disinfectants during the inactivation process. Based on the experiments and the kinetic parameters obtained, it could be established that the efficiency of hydrogen peroxide was much lower than that of peracetic acid alone. However, the contribution of hydrogen peroxide was very important in the commercial mixture. It should be noted that this improvement occurred only after peracetic acid had initiated the attack on the cell. This synergistic effect was successfully explained by the proposed scheme and was verified by experimental results. Besides providing a clearer mechanistic understanding of water disinfection, such models may improve our ability to design reactors. PMID:26819382

  3. Case Studies in Systems Chemistry. Final Report. [Includes Complete Case Study, Carboxylic Acid Equilibria

    ERIC Educational Resources Information Center

    Fleck, George

    This publication was produced as a teaching tool for college chemistry. The book is a text for a computer-based unit on the chemistry of acid-base titrations, and is designed for use with FORTRAN or BASIC computer systems, and with a programmable electronic calculator, in a variety of educational settings. The text attempts to present computer…

  4. Tumour-specific proline vulnerability uncovered by differential ribosome codon reading.

    PubMed

    Loayza-Puch, Fabricio; Rooijers, Koos; Buil, Levi C M; Zijlstra, Jelle; Oude Vrielink, Joachim F; Lopes, Rui; Ugalde, Alejandro Pineiro; van Breugel, Pieter; Hofland, Ingrid; Wesseling, Jelle; van Tellingen, Olaf; Bex, Axel; Agami, Reuven

    2016-02-25

    Tumour growth and metabolic adaptation may restrict the availability of certain amino acids for protein synthesis. It has recently been shown that certain types of cancer cells depend on glycine, glutamine, leucine and serine metabolism to proliferate and survive. In addition, successful therapies using L-asparaginase-induced asparagine deprivation have been developed for acute lymphoblastic leukaemia. However, a tailored detection system for measuring restrictive amino acids in each tumour is currently not available. Here we harness ribosome profiling for sensing restrictive amino acids, and develop diricore, a procedure for differential ribosome measurements of codon reading. We first demonstrate the functionality and constraints of diricore using metabolic inhibitors and nutrient deprivation assays. Notably, treatment with L-asparaginase elicited both specific diricore signals at asparagine codons and high levels of asparagine synthetase (ASNS). We then applied diricore to kidney cancer and discover signals indicating restrictive proline. As for asparagine, this observation was linked to high levels of PYCR1, a key enzyme in proline production, suggesting a compensatory mechanism allowing tumour expansion. Indeed, PYCR1 is induced by shortage of proline precursors, and its suppression attenuated kidney cancer cell proliferation when proline was limiting. High PYCR1 is frequently observed in invasive breast carcinoma. In an in vivo model system of this tumour, we also uncover signals indicating restrictive proline. We further show that CRISPR-mediated knockout of PYCR1 impedes tumorigenic growth in this system. Thus, diricore has the potential to reveal unknown amino acid deficiencies, vulnerabilities that can be used to target key metabolic pathways for cancer treatment. PMID:26878238

  5. Effects of glycine and proline on the calcium activation properties of skinned muscle fibre segments from crayfish and rat.

    PubMed

    Powney, E L; West, J M; Stephenson, D G; Dooley, P C

    2003-01-01

    The effects of the polar amino acid glycine (20 mmol l(-1)) and the non-polar amino acid proline (20 mmol l(-1)) on Ca(2+)-activated contraction have been examined in four types of striated muscle fibres. Single fibres dissected from the claw muscle of a crustacean (long- and short-sarcomere) and the hindlimb muscles of the rat (slow-twitch from soleus and fast-twitch from extensor digitorum longus) were activated in matched solutions that either contained the amino acid ('test') or not ('control'). The steady-state force produced in these solutions was used to determine the relation between force production and pCa (-log10[Ca2+]). The results show that in the concentrations used, glycine and proline had only small effects on the maximum Ca(2+)-activated force, pCa corresponding to 10, 50 and 90% maximum force (pCa10, pCa50, pCa90, respectively) or on the slope of the force-pCa curves in the four different fibre types. The relative lack of effects of glycine and proline on contractile activation would confer a distinct physiological advantage to force production of muscle of Cherax, where the concentrations of glycine and proline vary considerably. Finally, the results show that glycine and proline may be useful to balance control solutions when the effects of other amino acids or zwitterions on contractile activation are examined. PMID:14677649

  6. Eggshell composition of squamate reptiles: relationship between eggshell permeability and amino acid distribution.

    PubMed

    Sexton, Owen J; Bramble, Judith E; Heisler, I Lorraine; Phillips, Christopher A; Cox, David L

    2005-10-01

    Most snakes and lizards produce eggs with flexible shells that interact with the environment to maintain water balance. Geckos produce rigid eggshells that are independent of an external source of water and can be oviposited in more open, dryer locations. In this study, we analyzed and compared the amino acid composition of 24 lizard species, six snake species, and four outgroups (including avian and reptilian elastin and chicken eggshell). Rigid Gecko eggshells had significantly lower levels of seven of the 17 amino acids evaluated. Multivariate analysis showed that proline was the most important amino acid in distinguishing between these two groups of eggshells, occurring at significantly higher levels in flexible eggshells. High levels of proline have also been observed in the eggshells of other species. Proline and other amino acids are associated with the alleviation of water and salt stress in plants. PMID:16195850

  7. PROLINE IS REQUIRED FOR THE STIMULATION OF DNA SYNTHESIS IN HEPATOCYTE CULTURES BY EGF (EPIDERMAL GROWTH FACTOR)

    EPA Science Inventory

    Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo and in vitro (4,9). The authors report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has th...

  8. The puckering free-energy surface of proline

    NASA Astrophysics Data System (ADS)

    Wu, Di

    2013-03-01

    Proline has two preferred puckering states, which are often characterized by the pseudorotation phase angle and amplitude. Although proline's five endocyclic torsion angles can be utilized to calculate the phase angle and amplitude, it is not clear if there is any direct correlation between each torsion angle and the proline-puckering pathway. Here we have designed five proline puckering pathways utilizing each torsion angle χj (j = 1˜5) as the reaction coordinate. By examining the free-energy surfaces of the five puckering pathways, we find they can be categorized into two groups. The χ2 pathway (χ2 is about the Cβ—Cγ bond) is especially meaningful in describing proline puckering: it changes linearly with the puckering amplitude and symmetrically with the phase angle. Our results show that this conclusion applies to both trans and cis proline conformations. We have also analyzed the correlations of proline puckering and its backbone torsion angles ϕ and ψ. We show proline has preferred puckering states at the specific regions of ϕ, ψ angles. Interestingly, the shapes of ψ-χ2 free-energy surfaces are similar among the trans proline in water, cis proline in water and cis proline in the gas phase, but they differ substantially from that of the trans proline in the gas phase. Our calculations are conducted using molecular simulations; we also verify our results using the proline conformations selected from the Protein Data Bank. In addition, we have compared our results with those calculated by the quantum mechanical methods.

  9. Binding of a proline-independent hydrophobic motif by the Candida albicans Rvs167-3 SH3 domain.

    PubMed

    Gkourtsa, Areti; van den Burg, Janny; Avula, Teja; Hochstenbach, Frans; Distel, Ben

    2016-09-01

    Src-homology 3 (SH3) domains are small protein-protein interaction modules. While most SH3 domains bind to proline-x-x-proline (PxxP) containing motifs in their binding partners, some SH3 domains recognize motifs other than proline-based sequences. Recently, we showed that the SH3 domain of Candida albicans Rvs167-3 binds peptides enriched in hydrophobic residues and containing a single proline residue (RΦxΦxΦP, where x is any amino acid and Φ is a hydrophobic residue). Here, we demonstrate that the proline in this motif is not required for Rvs167-3 SH3 recognition. Through mutagenesis studies we show that binding of the peptide ligand involves the conserved tryptophan in the canonical PxxP binding pocket as well as residues in the extended n-Src loop of Rvs167-3 SH3. Our studies establish a novel, proline-independent, binding sequence for Rvs167-3 SH3 (RΦxΦxΦ) that is comprised of a positively charged residue (arginine) and three hydrophobic residues. PMID:27393996

  10. Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase.

    PubMed

    Hasegawa, T; Yokogawa, T

    2000-01-01

    A major proline tRNA was purified from bulk Escherichia coli A19 tRNA by affinity chromatography with a biotinylated DNA probe. Its nucleotide sequence including modified nucleotides was determined by the post-labelling technique. In order to study the recognition sites of this proline tRNA for prolyl-tRNA synthetase, various mutant transcripts were prepared using an in vitro transcription system with T7 RNA polymerase. Based on the results of in vitro kinetic analyses of mutant transcripts, it was concluded that the second and third letters, G35 and G36, of the anticodon, G37 of the anticodon loop, the discriminator base A73, G72 of the acceptor stem, G49 and U17A that existed in the corner of an L-shaped structure are the recognition sites of proline tRNA for prolyl-tRNA synthetase. PMID:12903242

  11. Longitudinal distributions of dicarboxylic acids, ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids in the marine aerosols from the central Pacific including equatorial upwelling

    NASA Astrophysics Data System (ADS)

    Hoque, Mir Md. Mozammal; Kawamura, Kimitaka

    2016-03-01

    Remote marine aerosol samples (total suspended particles) were collected during a cruise in the central Pacific from Japan to Mexico (1°59'N-35°N and 171°54'E-90°58'W). The aerosol samples were analyzed for dicarboxylic acids (C2-C11), ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids as well as organic and elemental carbon, water-soluble organic carbon, and total nitrogen (WSTN). During the study, diacids were the most abundant compound class followed by fatty acids, ω-oxoacids, and α-dicarbonyls. Molecular compositions of diacids showed a predominance of oxalic (C2) acid followed by malonic (C3) and succinic (C4) acids. Oxalic acid comprises 74% of total diacids. This result suggests that photochemical production of oxalic acid is significant over the central Pacific. Spatial distributions of diacids, ω-oxoacids, pyruvic acid, α-dicarbonyls, and fatty acids together with total carbon and WSTN showed higher abundances in the eastern equatorial Pacific where the upwelling of high-nutrient waters followed by high biological productivity is common, indicating that their in situ production is important in the warmer central Pacific through photochemical oxidation from their gaseous and particulate precursors. This study demonstrates that there is a strong linkage in biogeochemical cycles of carbon in the sea-air interface via ocean upwelling, phytoplankton productivity, sea-to-air emissions of organic matter, and formation of secondary organic aerosols in the eastern equatorial Pacific.

  12. L-proline: a highly effective cryoprotectant for mouse oocyte vitrification

    PubMed Central

    Zhang, Lu; Xue, Xu; Yan, Jie; Yan, Li-Ying; Jin, Xiao-Hu; Zhu, Xiao-Hui; He, Zhi-Zhu; Liu, Jing; Li, Rong; Qiao, Jie

    2016-01-01

    Recent studies have shown that L-proline is a natural osmoprotectant and an antioxidant to protect cells from injuries such as that caused by freezing and thawing in many species including plant, ram sperm and human endothelial cells. Nevertheless, this nontoxic cryoprotectant has not yet been applied to mammalian oocyte vitrification. In this study we evaluated the efficiency and safety of the new cryoprotectant in oocyte vitrification. The results indicated that L-proline improves the survival rate of vitrified oocytes, protects mitochondrial functions and could be applied as a new cryoprotectant in mouse oocyte vitrification. PMID:27412080

  13. L-proline: a highly effective cryoprotectant for mouse oocyte vitrification.

    PubMed

    Zhang, Lu; Xue, Xu; Yan, Jie; Yan, Li-Ying; Jin, Xiao-Hu; Zhu, Xiao-Hui; He, Zhi-Zhu; Liu, Jing; Li, Rong; Qiao, Jie

    2016-01-01

    Recent studies have shown that L-proline is a natural osmoprotectant and an antioxidant to protect cells from injuries such as that caused by freezing and thawing in many species including plant, ram sperm and human endothelial cells. Nevertheless, this nontoxic cryoprotectant has not yet been applied to mammalian oocyte vitrification. In this study we evaluated the efficiency and safety of the new cryoprotectant in oocyte vitrification. The results indicated that L-proline improves the survival rate of vitrified oocytes, protects mitochondrial functions and could be applied as a new cryoprotectant in mouse oocyte vitrification. PMID:27412080

  14. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

    PubMed

    Van de Wouwer, Dorien; Vanholme, Ruben; Decou, Raphaël; Goeminne, Geert; Audenaert, Dominique; Nguyen, Long; Höfer, René; Pesquet, Edouard; Vanholme, Bartel; Boerjan, Wout

    2016-09-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  15. Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation

    PubMed Central

    Zhang, Lu; Xue, Xu; Yan, Jie; Yan, Li-Ying; Jin, Xiao-Hu; Zhu, Xiao-Hui; He, Zhi-Zhu; Liu, Jing; Li, Rong; Qiao, Jie

    2016-01-01

    Background: L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods: The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results: L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions: It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification. PMID:27503023

  16. Design of cyclic peptides featuring proline predominantly in the cis conformation under physiological conditions.

    PubMed

    Malešević, Miroslav; Schumann, Michael; Jahreis, Günther; Fischer, Gunter; Lücke, Christian

    2012-09-24

    Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue β-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space. PMID:22969011

  17. Molecular cloning and expression analysis of the gene encoding proline dehydrogenase from Jatropha curcas L.

    PubMed

    Wang, Haibo; Ao, Pingxing; Yang, Shuanglong; Zou, Zhurong; Wang, Shasha; Gong, Ming

    2015-03-01

    Proline dehydrogenase (ProDH) (EC 1.5.99.8) is a key enzyme in the catabolism of proline. The enzyme JcProDH and its complementary DNA (cDNA) were isolated from Jatropha curcas L., an important woody oil plant used as a raw material for biodiesels. It has been classified as a member of the Pro_dh superfamily based on multiple sequence alignment, phylogenetic characterization, and its role in proline catabolism. Its cDNA is 1674 bp in length with a complete open reading frame of 1485 bp, which encodes a polypeptide chain of 494 amino acids with a predicted molecular mass of 54 kD and a pI of 8.27. Phylogenetic analysis indicated that JcProDH showed high similarity with ProDH from other plants. Reverse transcription PCR (RT-PCR) analysis revealed that JcProDH was especially abundant in the seeds and flowers but scarcely present in the stems, roots, and leaves. In addition, the expression of JcProDH increased in leaves experiencing environmental stress such as cold (5 °C), heat (42 °C), salt (300 mM), and drought (30 % PEG6000). The JcProDH protein was successfully expressed in the yeast strain INVSc1 and showed high enzyme activity in proline catabolism. This result confirmed that the JcProDH gene negatively participated in the stress response. PMID:25502926

  18. Engineering of yeast Put4 permease and its application to lager yeast for efficient proline assimilation.

    PubMed

    Omura, Fumihiko; Fujita, Atsushi; Miyajima, Keiji; Fukui, Nobuyuki

    2005-06-01

    The Saccharomyces cerevisiae Put4 permease is significant for the transport of proline, alanine, and glycine. Put4p downregulation is counteracted by npi1 mutation that affects the cellular ubiquitination function. Here we describe mutant Put4 permeases, in which up to nine lysine residues in the cytoplasmic N-terminal domain have been replaced by arginine. The steady-state protein level of the mutant permease Put4-20p (Lys9, Lys34, Lys35, Lys60, Lys68, Lys71, Lys93, Lys105, Lys107 --> Arg) was largely higher compared to that of the wild-type Put4p, indicating that the N-terminal lysines can undergo ubiquitination and the subsequent degradation steps. Proline is the only amino acid that yeast assimilates with difficulty under standard brewing conditions. A lager yeast strain provided with Put4-20p was able to assimilate proline efficiently during beer fermentations. These results suggest possible industrial applications of the mutant Put4 permeases in improved fermentation systems for beer and other alcoholic beverages based on proline-rich fermentable sources. PMID:15973048

  19. Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

    PubMed Central

    Chao, H.; Davies, P. L.; Sykes, B. D.; Sönnichsen, F. D.

    1993-01-01

    To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity. PMID:8401227

  20. Glutamine synthetase in the phloem plays a major role in controlling proline production

    PubMed Central

    Brugiere, N; Dubois, F; Limami, AM; Lelandais, M; Roux, Y; Sangwan, RS; Hirel, B

    1999-01-01

    To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress. PMID:10521528

  1. Screening of Bread Wheat Genotypes for Drought Tolerance Using Phenotypic and Proline Analyses

    PubMed Central

    Mwadzingeni, Learnmore; Shimelis, Hussein; Tesfay, Samson; Tsilo, Toi J.

    2016-01-01

    Drought stress is one of the leading constraints to wheat (Triticum aestivum L.) production globally. Breeding for drought tolerance using novel genetic resources is an important mitigation strategy. This study aimed to determine the level of drought tolerance among diverse bread wheat genotypes using agronomic traits and proline analyses and to establish correlation of proline content and agronomic traits under drought-stress conditions in order to select promising wheat lines for breeding. Ninety-six diverse genotypes including 88 lines from the International Maize and Wheat Improvement Center (CIMMYT)'s heat and drought nurseries, and eight local checks were evaluated under greenhouse and field conditions during 2014/15 and 2015/16 making four testing environments. The following phenotypic traits were collected after stress imposed during the heading to anthesis period: the number of days to heading (DTH), days to maturity (DTM), productive tiller number (TN), plant height (PH), spike length (SL), spikelet per spike (SPS), kernels per spike (KPS), thousand kernel weight (TKW) and grain yield (GY) and proline content (PC). Analysis of variance, Pearson's correlation coefficient, principal component and stress tolerance index were calculated. Genotypes with high yield performance under stressed and optimum conditions maintained high values for yield components. Proline content significantly increased under stress, but weakly correlated with agronomic traits under both optimal and water limited conditions. The positive correlation observed between grain yield and proline content under-drought stress conditions provides evidence that proline accumulation might ultimately be considered as a tool for effective selection of drought tolerant genotypes. The study selected 12 genotypes with high grain yields under drought stressed conditions and favorable adaptive traits useful for breeding. PMID:27610116

  2. Screening of Bread Wheat Genotypes for Drought Tolerance Using Phenotypic and Proline Analyses.

    PubMed

    Mwadzingeni, Learnmore; Shimelis, Hussein; Tesfay, Samson; Tsilo, Toi J

    2016-01-01

    Drought stress is one of the leading constraints to wheat (Triticum aestivum L.) production globally. Breeding for drought tolerance using novel genetic resources is an important mitigation strategy. This study aimed to determine the level of drought tolerance among diverse bread wheat genotypes using agronomic traits and proline analyses and to establish correlation of proline content and agronomic traits under drought-stress conditions in order to select promising wheat lines for breeding. Ninety-six diverse genotypes including 88 lines from the International Maize and Wheat Improvement Center (CIMMYT)'s heat and drought nurseries, and eight local checks were evaluated under greenhouse and field conditions during 2014/15 and 2015/16 making four testing environments. The following phenotypic traits were collected after stress imposed during the heading to anthesis period: the number of days to heading (DTH), days to maturity (DTM), productive tiller number (TN), plant height (PH), spike length (SL), spikelet per spike (SPS), kernels per spike (KPS), thousand kernel weight (TKW) and grain yield (GY) and proline content (PC). Analysis of variance, Pearson's correlation coefficient, principal component and stress tolerance index were calculated. Genotypes with high yield performance under stressed and optimum conditions maintained high values for yield components. Proline content significantly increased under stress, but weakly correlated with agronomic traits under both optimal and water limited conditions. The positive correlation observed between grain yield and proline content under-drought stress conditions provides evidence that proline accumulation might ultimately be considered as a tool for effective selection of drought tolerant genotypes. The study selected 12 genotypes with high grain yields under drought stressed conditions and favorable adaptive traits useful for breeding. PMID:27610116

  3. Metabolism of 3H- and 14C-labeled glutamate, proline, and alanine in normal and adrenalectomized rats using different sites of tracer administration and sampling.

    PubMed

    Said, H M; Chenoweth, M; Dunn, A

    1989-08-01

    Alanine, glutamate and proline labeled with 14C and 3H were infused into fasted normal and adrenalectomized rats. Alanine was administered by the A-V mode (arterial administration-venous sampling), and glutamate and proline by both the A-V and V-A (venous administration-arterial sampling) modes. The kinetics of 14C alanine and 14C glutamate differed markedly from those of the tritium-labeled compounds, but there was little difference in the kinetics of 3H and 14C proline. The replacement rate calculated from the A-V mode for glutamate was about half that obtained in the V-A mode, but there was little difference with proline. The masses of the amino acids (total content of amino acids in the body) were calculated from the washout curves of the tritium-labeled compounds after the infusion of tracer was terminated. The masses for the normal rats were 407 mumol/kg for alanine, 578 mumol/kg for glutamate and 296 mumol/kg for proline. The so-called distribution spaces calculated conventionally from total masses and the amino acid concentrations in plasma are much greater than the volume of the body, reflecting the fact that amino acid concentrations in tissues greatly exceed those in plasma. Adrenalectomy markedly affected the kinetics of the three amino acids, and their replacement rates were greatly reduced. The proline and glutamate masses were reduced by at least one half, while that of alanine was unchanged. Adrenalectomy markedly reduced the conversion of proline to glutamate. The hydrocortisone regimen used in this study restored the metabolism of alanine and glutamate to normal, but had no effect on that of proline. PMID:2569659

  4. Proline transport in Leishmania donovani amastigotes: dependence on pH gradients and membrane potential.

    PubMed

    Glaser, T A; Mukkada, A J

    1992-03-01

    Amastigotes of Leishmania donovani develop and multiply within the acidic phagolysosomes of mammalian macrophages. Isolated amastigotes are acidophilic; they catabolize substrates and synthesize macromolecules optimally at pH 5.5. Substrate transport in amastigotes has not been characterized. Here we show that amastigotes exhibit an uphill transport of proline (active transport) with an acid pH optimum (pH 5.5). It is dependent upon metabolic energy and is driven by proton motive force. Agents which selectively disturb the component forces of proton motive force, such as carbonyl cyanide chlorophenylhydrazone, nigericin and valinomycin, inhibit proline transport. Transport is sensitive to dicyclohexylcarbodiimide and insensitive to ouabain, demonstrating the involvement of a proton ATPase in the maintenance of proton motive force. It is suggested that the plasma membrane pH gradient probably makes the greatest contribution to proton motive force that drives substrate transport in the amastigote stage. PMID:1533014

  5. Role of proline and pyrroline-5-carboxylate metabolism in plant defense against invading pathogens.

    PubMed

    Qamar, Aarzoo; Mysore, Kirankumar S; Senthil-Kumar, Muthappa

    2015-01-01

    Pyrroline-5-carboxylate (P5C) is an intermediate product of both proline biosynthesis and catabolism. Recent evidences indicate that proline-P5C metabolism is tightly regulated in plants, especially during pathogen infection and abiotic stress. However, role of P5C and its metabolism in plants has not yet been fully understood. Studies indicate that P5C synthesized in mitochondria has a role in both resistance (R)-gene-mediated and non-host resistance against invading pathogens. Proline dehydrogenase and delta-ornithine amino transferase-encoding genes, both involved in P5C synthesis in mitochondria are implicated in defense response of Nicotiana benthamiana and Arabidopsis thaliana against bacterial pathogens. Such defense response is proposed to involve salicylic acid-dependent pathway, reactive oxygen species (ROS) and hypersensitive response (HR)-associated cell death. Recently HR, a form of programmed cell death (PCD), has been proposed to be induced by changes in mitochondrial P5C synthesis or the increase in P5C levels per se in plants inoculated with either a host pathogen carrying suitable avirulent (Avr) gene or a non-host pathogen. Consistently, A. thaliana mutant plants deficient in P5C catabolism showed HR like cell death when grown in external P5C or proline supplemented medium. Similarly, yeast and plant cells under oxidative stress were shown to increase ROS production and PCD due to increase in P5C levels. Similar mechanism has also been reported as one of the triggers for apoptosis in mammalian cells. This review critically analyzes results from various studies and enumerates the pathways for regulation of P5C levels in the plant cell, especially in mitochondria, during pathogen infection. Further, mechanisms regulating P5C- mediated defense responses, namely HR are outlined. This review also provides new insights into the differential role of proline-P5C metabolism in plants exposed to pathogen infection. PMID:26217357

  6. Comparative Aspects of Tissue Glutamine and Proline Metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cellular metabolism of glutamine and proline are closely interrelated since they can be interconverted with glutamate and ornithine via the mitochondrial pathway involving pyrolline-5-carboxylate (P5C). In adults, glutamine and proline are converted via P5C to citrulline in the gut, then citrul...

  7. Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.

    PubMed Central

    Straub, P F; Reynolds, P H; Althomsons, S; Mett, V; Zhu, Y; Shearer, G; Kohl, D H

    1996-01-01

    We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses. PMID:8572700

  8. Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions.

    PubMed

    Albert, Benjamin; Le Cahérec, Françoise; Niogret, Marie-Françoise; Faes, Pascal; Avice, Jean-Christophe; Leport, Laurent; Bouchereau, Alain

    2012-08-01

    Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25-85% of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape. PMID:22526495

  9. Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution.

    PubMed

    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

  10. 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

  11. Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa.

    PubMed

    Trimeche, A; Yvon, J M; Vidament, M; Palmer, E; Magistrini, M

    1999-07-01

    The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were evaluated after thawing by computer automated sperm analyzer. The percentage of motile spermatozoa (faster than 30 microns/sec) was assessed. In addition, the velocity of the average path (VAP), the straight line velocity (VSL), the curvilinear velocity (VCL) and the amplitude of the lateral head displacement (ALH) were also measured. In Experiment 1, only glutamine (40 mM) significantly improved sperm motility (56.0% +/- 3.0 vs 49.7% +/- 1.6; P < 0.05) compared with the control extender, while velocities were unaffected at concentrations of 40 to 120 mM. However, at 160 mM, a significant decrease in motility and velocity was observed for all amino acids. In Experiment 2, motility in glutamine (range 41.1% +/- 3.8%; 42.4% +/- 3.6) and proline (43.0% +/- 3.7; 45.6% +/- 3.8) extenders compared with the control (34.7% +/- 1.6) was improved significantly (P < 0.05). Sperm velocity was improved at concentrations higher than 40 mM glutamine and 50 mM proline. PMID:10734416

  12. α-Quaternary Proline Derivatives by Intramolecular Diastereoselective Arylation of N-Carboxamido Proline Ester Enolates.

    PubMed

    Maury, Julien; Clayden, Jonathan

    2015-11-01

    Pyrrolidine-2-carboxylate esters substituted in the 3-, 4- or 5-positions were converted to their N'-aryl urea derivatives. Deprotonation at the 2-position to form a potassium enolate led to migration of the N'-aryl substituent to the 2 position of the pyrrolidine ring, followed by cyclization of the resulting urea to give bicyclic α-aryl hydantoin derivatives of substituted prolines. Depending on the substitution pattern of the starting material, high diastereoselectivity was observed in the aryl migration, allowing formation of the products in enantiomerically enriched form, despite the intermediacy of a planar enolate. The hydrolysis of the bicyclic hydantoins under basic conditions gave a range of enantiopure and enantioenriched quaternary α-aryl proline derivatives. PMID:26421443

  13. Role of oral nitrate in the nitrosation of ( UC)proline by conventional microflora and germ-free rats

    SciTech Connect

    Mallett, A.K.; Rowland, I.R.; Walters, D.G.; Gangolli, S.D.; Cottrell, R.C.; Massey, R.C.

    1985-11-01

    The urinary excretion of N-nitroso-L-(U- UC)proline by conventional microflora and germ free rats was used to assess the role of the gut bacteria and oral nitrate in the endogenous formation of N-nitroso compounds. The formation of nitrosoproline was qualitatively similar in conventional and germfree rats suggesting no involvement of the intestinal flora in this reaction. Furthermore, nitrosamino acid production was similar following the administration of nitrate and (U- UC)proline or (U- UC)proline alone, demonstrating no involvement of exogenous nitrate under the conditions of the experiment. Dietary contamination with nitrate/nitrite was negligible. The results are consistent with the suggestion that nitrate/nitrite reserves in the body are important in the formation of nitrosoproline in vivo.

  14. Synthesis, Surface Active Properties and Cytotoxicity of Sodium N-Acyl Prolines.

    PubMed

    Sreenu, Madhumanchi; Narayana Prasad, Rachapudi Badari; Sujitha, Pombala; Kumar, Chityal Ganesh

    2015-01-01

    Sodium N-acyl prolines (NaNAPro) were synthesized using mixture of fatty acids obtained from coconut, palm, karanja, Sterculia foetida and high oleic sunflower oils via Schotten-Baumann reaction in 58-75% yields to study the synergetic effect of mixture of hydrophobic fatty acyl functionalities like saturation, unsaturation and cyclopropene fatty acids with different chain lengths and aliphatic hetero cyclic proline head group on their surface and cytotoxicity activities. The products were characterized by chromatographic and spectral techniques. The synthesized products were evaluated for their surface active properties such as surface tension, wetting power, foaming characteristics, emulsion stability, calcium tolerance, critical micelle concentration (CMC) and thermodynamic properties. The results revealed that all the products exhibited superior surface active properties like CMC, calcium tolerance and emulsion stability as compared to the standard surfactant, sodium lauryl sulphate (SLS). In addition, palm, Sterculia foetida and high oleic sunflower fatty N-acyl prolines exhibited promising cytotoxicity against different tumor cell lines. PMID:26521810

  15. Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

    PubMed Central

    Robinson, R; Kauffman, D L; Waye, M M; Blum, M; Bennick, A; Keller, P J

    1989-01-01

    Human submandibular/sublingual saliva contains one non-glycosylated basic proline-rich protein whereas parotid saliva contains multiple such components. The submandibular protein has a primary structure identical with the C-terminal segment [TZ] of the human parotid acidic proline-rich proteins that contain 150 amino acid residues (Mr 16,000). Northern-blot analyses of human parotid and submandibular glands revealed that mRNAs containing the HaeIII repeat sequence typical for acidic proline-rich proteins are expressed in both of these salivary glands whereas mRNAs for non-glycosylated basic proline-rich proteins containing a typical BstN1 repeat sequence are expressed in the parotid but not in the submandibular gland. Products of translation in vitro of mRNAs from human parotid and submandibular glands were also examined. Two immunoprecipitable bands with Mr 29,000 and 28,000 were obtained by translation of both parotid and submandibular mRNA. In the presence of microsomal membranes these proteins gave rise to proteins electrophoretically identical with the secreted acidic proline-rich proteins of Mr 16,000. These proteins were cleaved by kallikrein, giving rise to proteins with electrophoretic mobilities identical with those of a smaller acidic proline-rich protein with Mr 11,000 and peptide TZ. Additional immunoprecipitable bands with Mr ranging from 35,000 to 46,000 were seen when parotid mRNA was used for translation in vitro, and are believed to be precursors of the basic proline-rich proteins encoded by the BstN1 repeat type mRNA. Neither these bands nor a separate precursor for the basic non-glycosylated proline-rich protein was detected when submandibular mRNA was used for translation in vitro. It is suggested that the non-glycosylated basic proline-rich protein present in human submandibular saliva arises by cleavage of acidic proline-rich proteins. Images Fig. 1. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2688632

  16. Proline synthesis in barley under iron deficiency and salinity.

    PubMed

    Arias-Baldrich, Cirenia; Bosch, Nadja; Begines, Digna; Feria, Ana B; Monreal, José A; García-Mauriño, Sofía

    2015-07-01

    This work investigates proline synthesis in six barley varieties subjected to iron deficiency, salinity or both stresses. The highest growth under Fe sufficiency corresponded to Belgrano and Shakira. A moderate augment of leaf phosphoenolpyruvate carboxylase (PEPC) activity was observed in all six varieties in response to Fe deficiency, consistently in leaves and sporadically in roots. All six varieties accumulated proline under Fe deficiency, to a higher extent in leaves than in roots. The decrease of Fe supply from 100 μM NaFe(III)-EDTA to 0.5 μM NaFe(III)-EDTA reduced growth and photosynthetic pigments similarly in the six barley varieties. On the contrary, differences between varieties could be observed with respect to increased or, conversely, decreased proline content as a function of the amount of NaFe(III)-EDTA supplied. These two opposite types were represented by Belgrano (higher proline under Fe deficiency) and Shakira (higher proline under Fe sufficiency). Time-course experiments suggested that leaf PEPC activity was not directly responsible for supplying C for proline synthesis under Fe deficiency. High proline levels in the leaves of Fe-deficient Belgrano plants in salinity were associated to a better performance of this variety under these combined stresses. PMID:26125122

  17. Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Δ1-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria

    PubMed Central

    Watanabe, Seiya; Tanimoto, Yoshiaki; Yamauchi, Seiji; Tozawa, Yuzuru; Sawayama, Shigeki; Watanabe, Yasuo

    2014-01-01

    trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685–6695; J Biol Chem (2012) 287, 32674–32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ1-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ1-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism. PMID:24649405

  18. Adjacent proline residues in the inhibitory domain of the Oct-2 transcription factor play distinct functional roles.

    PubMed Central

    Liu, Y Z; Lee, I K; Locke, I; Dawson, S J; Latchman, D S

    1998-01-01

    A 40 amino acid region of Oct-2 from amino acids 142 to 181 functions as an active repressor domain capable of inhibiting both basal activity and activation of promoters containing a TATA box, but not of those that contain an initiator element. Based on our observation that the equivalent region of the closely related Oct-1 factor does not act as an inhibitory domain, we have mutated specific residues in the Oct-2 domain in an attempt to probe their importance in repressor domain function. Although mutations of several residues have no or minimal effect, mutation of proline 175 to arginine abolishes the ability to inhibit both basal and activated transcription. In contrast, mutation of proline 174 to arginine confers upon the domain the ability to repress activation of an initiator-containing promoter by acidic activation domains, and also suppresses the effect of the proline 175 mutation. Hence, adjacent proline residues play key roles in the functioning of the inhibitory domain and in limiting its specificity to TATA-box-containing promoters. PMID:9580701

  19. Proline-glutamate chimera's side chain conformation directs the type of β-hairpin structure.

    PubMed

    Maity, Jyotirmoy; Gerling, Ulla I M; Vukelić, Stella; Schäfer, Andreas; Koksch, Beate

    2014-01-01

    Our aim was to study the impact of two proline chimeras, containing a glutamic acid side chain in cis- or trans-configuration, on secondary structure formation. We further investigated to what extent the configuration of the side chain contributes to the overall peptide conformation. We used a 10 residue peptide (IYSNPDGTWT) that forms a β-hairpin in water. The turn-forming proline was substituted with either a cis- or trans-proline-glutamic acid chimera, resulting in the peptides IYSNPcis -E DGTWT (P1_Pcis-E) and IYSNP(trans-E)DGTWT (P1_Ptrans-E). We studied the conformation of the modified peptides by circular dichroism (CD) and NMR-spectroscopy, and SEC/static light scattering (SLS) analysis. NMR analysis reveals that the modified peptides maintain the β-hairpin conformation in aqueous solution. At 5 °C and pH 4.3, the peptide (P1_Pcis-E) was found to adopt two coexisting β-hairpin conformations (2:2 β-hairpin, and 3:5 β-hairpin). In contrast to that, the peptide (P1_Ptrans-E) adopts a 2:2 β-hairpin that exists in equilibrium with a 4:4 β-hairpin conformation. The adoption of ordered β-hairpin structures for both modified peptides could be confirmed by CD spectroscopy, while SEC/SLS analysis showed a monomeric oligomerization state for all three investigated peptides. With the combination of several NMR methods, we were able to elucidate that even small alterations in the side chain conformation of the proline-glutamate chimera (cis or trans) can significantly influence the conformation of the adopted β-hairpin. PMID:24221353

  20. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-11-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding. PMID:7479887

  1. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed Central

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479887

  2. Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12.

    PubMed Central

    Cayley, S; Lewis, B A; Record, M T

    1992-01-01

    K+) increased, with increasing external osmolarity. We predicted that the observed changes in nK+ and Vcyto would have large and approximately compensating effects on key protein-nucleic acid interactions of gene expression, and we proposed that Vf was the fundamental determinant of growth rate in osmotically stressed cells. The properties of cells cultured in the presence of betaine and proline appear completely consistent with our previous work and proposals. Accumulation of betaine and, to a lesser extent, proline shifts the set of linked physiological parameters (nK+, Vcyto, growth rate) to those characteristic of growth at lower osmolarity in the absence of osmoprotectants. Models for the thermodynamic basis and physiological consequences of the effect of osmoprotectants on Vcyto and Vf are discussed. PMID:1537801

  3. Impact of hedonic evaluation on consumers' preferences for beef attributes including its enrichment with n-3 and CLA fatty acids.

    PubMed

    Baba, Yasmina; Kallas, Zein; Costa-Font, Montserrat; Gil, José María; Realini, Carolina E

    2016-01-01

    The impact of hedonic evaluation on consumers' preferences for beef attributes was evaluated (origin, animal diet, fat content, color, price) including its enrichment with omega-3 (n-3) and conjugated linoleic acid (CLA) fatty acids. One group of consumers (n=325) received information about n-3 and CLA, while the other group (n=322) received no information. Consumers conducted a Discrete Choice Experiment (DCE), using the recently developed Generalized Multinomial Logit model; followed by a blind hedonic evaluation of beef samples, which were identified after tasting, and finally repeated the DCE. Results showed that hedonic evaluation had a significant impact on consumers' preferences, which were similar after tasting for all consumers, with less emphasis on the fat content, color, and origin attributes and greater emphasis on animal diet. Preference for n-3 enriched beef increased, while preference for CLA enriched beef was still not significant after tasting. The information provided had a significant effect on consumers' beef preferences, but no significant impact on beef liking scores. PMID:26331961

  4. Another Role of Proline: Stabilization Interactions in Proteins and Protein Complexes Concerning Proline and Tryptophane

    SciTech Connect

    Biedermannova, Lada; Riley, Kevin E.; Berka, Karel; Hobza, Pavel; Vondrasek, Jiri

    2008-09-11

    Proline–tryptophan complexes derived from experimental structures are investigated by quantum chemical procedures known to properly describe the London dispersion energy. We study two geometrical arrangements: the “L-shaped”, stabilized by an H-bond, and the “stacked-like”, where the two residues are in parallel orientation without any H-bond. Interestingly, the interaction energies in both cases are comparable and very large (~7 kcal mol⁻¹). The strength of stabilization in the stacked arrangement is rather surprising considering the fact that only one partner has an aromatic character. The interaction energy decomposition using the SAPT method further demonstrates the very important role of dispersion energy in such arrangement. To elucidate the structural features responsible for this unexpectedly large stabilization we examined the role of the nitrogen heteroatom and the importance of the cyclicity of the proline residue. We show that the electrostatic interaction due to the presence of the dipole, caused by the nitrogen heteroatom, contributes largely to the strength of the interaction. Nevertheless, the cyclic arrangement of proline, which allows for the largest amount of dispersive contact with the aromatic partner, also has a notable-effect. Geometry optimizations carried out for the “stackedlike” complexes show that the arrangements derived from protein structure are close to their gas phase optimum geometry, suggesting that the environment has only a minor effect on the geometry of the interaction. We conclude that the strength of proline non-covalent interactions, combined with this residue’s rigidity, might be the explanation for its prominent role in protein stabilization and recognition processes.

  5. Predictors of the short-term effect of isoleucine-proline-proline/valine-proline-proline lactotripeptides from casein on office and ambulatory blood pressure in subjects with pharmacologically untreated high-normal blood pressure or first-degree hypertension.

    PubMed

    Cicero, A F G; Rosticci, M; Ferroni, A; Bacchelli, S; Veronesi, M; Strocchi, E; Borghi, C

    2012-01-01

    Our aim was to evaluate the predictors of Isoleucine-Proline-Proline/Valine-Proline-Proline (IPP-VPP) lactotripeptides (LTPs) antihypertensive effect in the context of a short-term large double-blind randomized clinical trial involving 164 pharmacologically untreated subjects in primary prevention for cardiovascular disease. When compared with the baseline, office systolic blood pressure (SBP) (-3.42 mm Hg, P < .001) and diastolic blood pressure (DBP) (-2.35 mm Hg, P < .001) significantly decreased, in the LTP-treated patients only. No significant change in predictors during the study of ambulatory blood pressure measurement (ABPM) parameters was observed. A short-term supplementation with LTPs significantly improves the office SBP and DBP, especially in male subjects. The main predictor of LTP antihypertensive effect was the baseline BP. PMID:22574612

  6. Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein.

    PubMed Central

    Gajardo, R; Vende, P; Poncet, D; Cohen, J

    1997-01-01

    Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7. PMID:9032355

  7. Cis–Trans Isomerizations of Proline Residues are Key to Bradykinin Conformations

    PubMed Central

    Pierson, Nicholas A.; Chen, Liuxi; Russell, David H.; Clemmer, David E.

    2013-01-01

    A recent ion mobility – mass spectrometry (IM–MS) study of the nonapeptide bradykinin (BK, amino acid sequence Arg1–Pro2–Pro3–Gly4–Phe5–Ser6–Pro7–Phe8–Arg9) found evidence for 10 populations of conformations that depend upon the solution composition [J. Am. Chem. Soc. 2011, 133, 13810]. Here, the role of the three proline residues (Pro2, Pro3, and Pro7) in establishing these conformations is investigated using a series of seven analogue peptides in which combinations of alanine residues are substituted for prolines. IM–MS distributions of the analogue peptides, when compared to the distribution for bradykinin, indicate the multiple structures are associated with different combinations of cis and trans forms of the three proline residues. These data are used to assign the structures to different peptide populations that are observed under various solution conditions. The assignments also show the connectivity between structures when collisional activation is used to convert one state into another. PMID:23373819

  8. On the evolutionary significance of the size and planarity of the proline ring

    NASA Astrophysics Data System (ADS)

    Behre, Jörn; Voigt, Roland; Althöfer, Ingo; Schuster, Stefan

    2012-10-01

    Proline is a proteinogenic amino acid in which the side chain forms a ring, the pyrrolidine ring. This is a five-membered ring made up of four carbons and one nitrogen. Here, we study the evolutionary significance of this ring size. It is shown that the size of the pyrrolidine ring has the advantage of being nearly planar and strain-free, based on a general mathematical assertion saying that the angular sum of a polygon is maximum if it is planar and convex. We also provide a sketch of the proof to this assertion. The optimality of the ring size of proline can be derived from a triangle inequality for angles. Quasi-planarity is physiologically significant because it allows an easier and evolutionarily old type of fit into binding grooves of proteins with which proline-rich proteins interact. Finally, we present a comparison with other planar, nearly planar and non-planar biomolecules such as neurotransmitters, hormones and toxins, involving, for example, aromatic rings, cyclopentanone and 1,3-dioxole.

  9. Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

    PubMed

    Nguyen, Phuong Q T; Luu, Thuy T; Bai, Yang; Nguyen, Giang K T; Pervushin, Konstantin; Tam, James P

    2015-04-24

    Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family. PMID:25832441

  10. Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport.

    PubMed Central

    Shelden, M C; Loughlin, P; Tierney, M L; Howitt, S M

    2001-01-01

    The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane. PMID:11368789

  11. Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress.

    PubMed

    Zouari, Mohamed; Ben Ahmed, Chedlia; Elloumi, Nada; Bellassoued, Khaled; Delmail, David; Labrousse, Pascal; Ben Abdallah, Ferjani; Ben Rouina, Bechir

    2016-06-01

    Proline plays an important role in plant response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effectiveness of exogenous proline (10 and 20 mM) in alleviating cadmium induced inhibitory effects in young olive plants (Olea europaea L. cv. Chemlali) exposed to two Cd levels (10 and 30 mg CdCl2 kg(-1) soil). The Cd treatment induced substantial accumulation of Cd in both root and leaf tissues and a decrease in gas exchange, photosynthetic pigments contents, uptake of essential elements (Ca, Mg and K) and plant biomass. Furthermore, an elevation of antioxidant enzymes activities (superoxide dismutase, catalase, glutathione peroxydase) and proline content in association with relatively high amounts of hydrogen peroxide, thiobarbituric acid reactive substances and electrolyte leakage were observed. Interestingly, the application of exogenous proline alleviated the oxidative damage induced by Cd accumulation. In fact, Cd-stressed olive plants treated with proline showed an increase of antioxidant enzymes activities, photosynthetic activity, nutritional status, plant growth and oil content of olive fruit. Generally, it seems that proline supplementation alleviated the deleterious effects of young olive plants exposed to Cd stress. PMID:26946284

  12. Raw and fungal-treated olive-mill wastewater effects on selected parameters of lettuce (Lactuca sativa L.) growth--the role of proline.

    PubMed

    Ouzounidou, Georgia; Ntougias, Spyridon; Asfi, Maria; Gaitis, Fragiskos; Zervakis, Georgios I

    2012-01-01

    Olive-mill wastewater (OMW) constitutes a major agricultural waste stream for which disposal is associated with significant environmental repercussions. No data are available on the effects of biotreated OMW and of the protective role of exogenously provided proline on plant physiology. In the present study, OMW was administered, either raw or previously treated by the white-rot fungus Pleurotus ostreatus, with or without proline amendment, to lettuce plants growing in sterilized sand. Biotreated OMW and proline addition resulted in significant moderation of OMW adverse effects on plant biomass production and ascorbic acid content, while their synergistic action alleviated the severe negative impact on net photosynthetic rate, water use efficiency and photosynthetic activity (Fv/Fo) invoked by the effluent. Moreover, biotreated OMW supplemented with proline, moderated the decrease in chlorophylls exerted by raw OMW, but it did not contribute at restoring carotenoids content. Restoration of plant transpiration was complete when biotreated OMW was used (with or without proline); proline alone mitigated the negative impact of OMW on photosynthetic efficiency (Fv/Fm and Fv'/Fm'). It seems that key photosynthetic parameters could be exploited as suitable evaluators of wastewater-induced plant toxicity, while plant fertigation with biotreated and/or supplemented OMW could be an interesting prospect in valorizing this effluent. PMID:22560036

  13. Quantifying Additive Interactions of the Osmolyte Proline with Individual Functional Groups of Proteins: Comparisons with Urea and Glycine Betaine, Interpretation of m-Values

    PubMed Central

    Diehl, Roger C.; Guinn, Emily J.; Capp, Michael W.; Tsodikov, Oleg V.; Record, M. Thomas

    2013-01-01

    To quantify interactions of the osmolyte L-proline with protein functional groups and predict its effects on protein processes, we use vapor pressure osmometry to determine chemical potential derivatives dµ2/dm3 = µ23 quantifying preferential interactions of proline (component 3) with 21 solutes (component 2) selected to display different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, and/or amide or cationic N surface. Solubility data yield µ23 values for 4 less-soluble solutes. Values of µ23 are dissected using an ASA-based analysis to test the hypothesis of additivity and obtain α-values (proline interaction potentials) for these eight surface types and three inorganic ions. Values of µ23 predicted from these α-values agree with experiment, demonstrating additivity. Molecular interpretation of α-values using the solute partitioning model yields partition coefficients (Kp) quantifying the local accumulation or exclusion of proline in the hydration water of each functional group. Interactions of proline with native protein surface and effects of proline on protein unfolding are predicted from α-values and ASA information and compared with experimental data, with results for glycine betaine and urea, and with predictions from transfer free energy analysis. We conclude that proline stabilizes proteins because of its unfavorable interactions with (exclusion from) amide oxygens and aliphatic hydrocarbon surface exposed in unfolding, and that proline is an effective in vivo osmolyte because of the osmolality increase resulting from its unfavorable interactions with anionic (carboxylate and phosphate) and amide oxygens and aliphatic hydrocarbon groups on the surface of cytoplasmic proteins and nucleic acids. PMID:23909383

  14. Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

    PubMed

    Cabassa-Hourton, Cécile; Schertl, Peter; Bordenave-Jacquemin, Marianne; Saadallah, Kaouthar; Guivarc'h, Anne; Lebreton, Sandrine; Planchais, Séverine; Klodmann, Jennifer; Eubel, Holger; Crilat, Emilie; Lefebvre-De Vos, Delphine; Ghelis, Thanos; Richard, Luc; Abdelly, Chedly; Carol, Pierre; Braun, Hans-Peter; Savouré, Arnould

    2016-09-01

    Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain. PMID:27303048

  15. Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

    PubMed Central

    Bröer, Stefan; Bailey, Charles G.; Kowalczuk, Sonja; Ng, Cynthia; Vanslambrouck, Jessica M.; Rodgers, Helen; Auray-Blais, Christiane; Cavanaugh, Juleen A.; Bröer, Angelika; Rasko, John E.J.

    2008-01-01

    Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes. PMID:19033659

  16. Oxidation of proline decreases immunoreactivity and alters structure of barley prolamin.

    PubMed

    Huang, Xin; Sontag-Strohm, Tuula; Stoddard, Frederick L; Kato, Yoji

    2017-01-01

    Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC-MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins. PMID:27507515

  17. Proline-rich peptides: multifunctional bioactive molecules as new potential therapeutic drugs.

    PubMed

    Vitali, Alberto

    2015-01-01

    Proline-rich peptides (PRPs) include a large and heterogeneous group of small-medium sized peptides characterized by the presence of proline residues often constituting peculiar sequences. This feature confers them a typical structure that determines the various biological functions endowed by these molecules. In particular the left-handed-polyproline-II helix is essential for the expression of the antimicrobial, immunomodulatory, antioxidant properties and to finely modulate protein-protein interactions, thus playing crucial roles in many cell signal transduction pathways. These peptides are widely diffuse in the animal kingdom and in humans, where they are present in many tissues and biological fluids. This review highlights the most relevant biological properties of these peptides, focusing on the potential therapeutic role that the PRPs may play as a promising source of new peptidebased novel drugs. PMID:25692951

  18. DEVELOPMENTAL TOXICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS OF ALIPHATIC ACIDS, INCLUDING DOSE-RESPONSE ASSESSMENT OF VALPROIC ACID IN MICE AND RATS

    EPA Science Inventory

    The anticonvulsant valproic acid (VPA), or 2-propylpentanoic acid, is a short-chain aliphatic acid that is teratogenic in humans and rodents. PA and 14 related using the Chernoff/Kavlock assay Sprague-Dawley rats were gavaged with the test agent in corn oil once daily organogenes...

  19. Susceptibility to Dental Caries and the Salivary Proline-Rich Proteins

    PubMed Central

    Levine, Martin

    2011-01-01

    Early childhood caries affects 28% of children aged 2–6 in the US and is not decreasing. There is a well-recognized need to identify susceptible children at birth. Caries-free adults neutralize bacterial acids in dental biofilms better than adults with severe caries. Saliva contains acidic and basic proline-rich proteins (PRPs) which attach to oral streptococci. The PRPs are encoded within a small region of chromosome 12. An acidic PRP allele (Db) protects Caucasian children from caries but is more common in African Americans. Some basic PRP allelic phenotypes have a three-fold greater frequency in caries-free adults than in those with severe caries. Early childhood caries may associate with an absence of certain basic PRP alleles which bind oral streptococci, neutralize biofilm acids, and are in linkage disequilibrium with Db in Caucasians. The encoding of basic PRP alleles is updated and a new technology for genotyping them is described. PMID:22190937

  20. Susceptibility to dental caries and the salivary proline-rich proteins.

    PubMed

    Levine, Martin

    2011-01-01

    Early childhood caries affects 28% of children aged 2-6 in the US and is not decreasing. There is a well-recognized need to identify susceptible children at birth. Caries-free adults neutralize bacterial acids in dental biofilms better than adults with severe caries. Saliva contains acidic and basic proline-rich proteins (PRPs) which attach to oral streptococci. The PRPs are encoded within a small region of chromosome 12. An acidic PRP allele (Db) protects Caucasian children from caries but is more common in African Americans. Some basic PRP allelic phenotypes have a three-fold greater frequency in caries-free adults than in those with severe caries. Early childhood caries may associate with an absence of certain basic PRP alleles which bind oral streptococci, neutralize biofilm acids, and are in linkage disequilibrium with Db in Caucasians. The encoding of basic PRP alleles is updated and a new technology for genotyping them is described. PMID:22190937

  1. Requirement of proline synthesis during Arabidopsis reproductive development

    PubMed Central

    2012-01-01

    Background Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development. Results In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR. Conclusions Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for

  2. Fatty acid composition, including CLA's isomers and cholesterol content of m. longissimus lumborum and m. semimebranosus of Katahdin, Suffolk, Katahdin x Suffolk, and Suffolk x Katahdin lambs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipids in meat products have important human health implications. Muscle tissues from Katahdin (KK), Suffolk (SS), Katahdin x Suffolk (KS), and Suffolk x Katahdin (SS) lambs were analyzed to determine the effect of breed-type on muscle fatty acid composition, including conjugated linoleic acid (CLA)...

  3. Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle, Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Lü, Dong; Guo, Wen-Chao; Ahmat, Tursun; Yang, Lu; Mu, Li-Li; Li, Guo-Qing

    2014-04-01

    Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5'UTR and coding region, but differed in the lengths of 3'UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3'UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3'UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults. PMID:23956209

  4. Identification and Characterization of Bifunctional Proline Racemase/Hydroxyproline Epimerase from Archaea: Discrimination of Substrates and Molecular Evolution

    PubMed Central

    Watanabe, Seiya; Tanimoto, Yoshiaki; Nishiwaki, Hisashi; Watanabe, Yasuo

    2015-01-01

    Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily. PMID:25786142

  5. Rapid identification of triterpenoid sulfates and hydroxy fatty acids including two new constituents from Tydemania expeditionis by LC-MS

    PubMed Central

    Zhang, Jian-Long; Kubanek, Julia; Hay, Mark E.; Aalbersberg, William; Ye, Wen-Cai; Jiang, Ren-Wang

    2011-01-01

    Tydemania expeditionis Weber-van Bosse (Udoteaceae) is a weakly calcified green alga. In the present paper, liquid chromatography coupled with photodiode array detection and electrospray mass spectrometry was developed to identify the fingerprint components. A total of four triterpenoid sulfates and three hydroxy fatty acids in the ethyl acetate fraction of the crude extract were structurally characterized on the basis of retention time, online UV spectrum and mass fragmentation pattern. Furthermore, detailed LC-MS analysis revealed two new hydroxy fatty acids, which were then prepared and characterized by extensive NMR analyses. The proposed method provides a scientific and technical platform for the rapid identification of triterpenoid sulfates and hydroxy fatty acids in similar marine algae and terrestrial plants. PMID:21915955

  6. Analysis of salt-induced physiological and proline changes in 46 switchgrass (Panicum virgatum) lines indicates multiple response modes.

    PubMed

    Kim, Jeongwoon; Liu, Yiming; Zhang, Xunzhong; Zhao, Bingyu; Childs, Kevin L

    2016-08-01

    Switchgrass (Panicum virgatum) is targeted as a biofuel feedstock species that may be grown on marginal lands including those with saline soils. Our study investigated salt stress responses in 46 switchgrass lines from the lowland and upland ecotypes by assessing physiological phenotypes and proline concentrations. Lowland switchgrass lines demonstrated less severe responses to salt stress than most upland switchgrass lines, but a number of upland lines performed as well as lowland individuals. Photosynthetic rate (Pn), the most important physiological trait measured, was reduced by salt treatment in all lines. Tolerant lines showed ∼50% reduction in Pn under salt stress, and sensitive lines exhibited ∼90% reduction in Pn after salt stress. Proline analysis showed the largest amount of variation under salt stress with some lines exhibiting minor increases in proline, but some salt-sensitive lines demonstrated more than 5000-fold increase in proline concentration in response to salt treatment. Clustering of salt-stress phenotypic responses revealed five groups of switchgrass. Lowland lines were present in two of the phenotypic clusters, but upland lines were found in all five of the phenotypic clusters. These results suggest that there are multiple modes of salt response in switchgrass including two distinct modes of salt tolerance. PMID:27111258

  7. Exclusive Use of trans-Editing Domains Prevents Proline Mistranslation*

    PubMed Central

    Vargas-Rodriguez, Oscar; Musier-Forsyth, Karin

    2013-01-01

    Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to cognate tRNAs. Although the accuracy of this process is critical for overall translational fidelity, similar sizes of many amino acids provide a challenge to ARSs. For example, prolyl-tRNA synthetases (ProRSs) mischarge alanine and cysteine onto tRNAPro. Many bacterial ProRSs possess an alanine-specific proofreading domain (INS) but lack the capability to edit Cys-tRNAPro. Instead, Cys-tRNAPro is cleared by a single-domain homolog of INS, the trans-editing YbaK protein. A global bioinformatics analysis revealed that there are six types of “INS-like” proteins. In addition to INS and YbaK, four additional single-domain homologs are widely distributed throughout bacteria: ProXp-ala (formerly named PrdX), ProXp-x (annotated as ProX), ProXp-y (annotated as YeaK), and ProXp-z (annotated as PA2301). The last three are domains of unknown function. Whereas many bacteria encode a ProRS containing an INS domain in addition to YbaK, many other combinations of INS-like proteins exist throughout the bacterial kingdom. Here, we focus on Caulobacter crescentus, which encodes a ProRS with a truncated INS domain that lacks catalytic activity, as well as YbaK and ProXp-ala. We show that C. crescentus ProRS can readily form Cys- and Ala-tRNAPro, and deacylation studies confirmed that these species are cleared by C. crescentus YbaK and ProXp-ala, respectively. Substrate specificity of C. crescentus ProXp-ala is determined, in part, by elements in the acceptor stem of tRNAPro and further ensured through collaboration with elongation factor Tu. These results highlight the diversity of approaches used to prevent proline mistranslation and reveal a novel triple-sieve mechanism of editing that relies exclusively on trans-editing factors. PMID:23564458

  8. Proline induces heat tolerance in chickpea (Cicer arietinum L.) plants by protecting vital enzymes of carbon and antioxidative metabolism.

    PubMed

    Kaushal, Neeru; Gupta, Kriti; Bhandhari, Kalpna; Kumar, Sanjeev; Thakur, Prince; Nayyar, Harsh

    2011-07-01

    Chickpea is a heat sensitive crop hence its potential yield is considerably reduced under high temperatures exceeding 35 °C. In the present study, we evaluated the efficacy of proline in countering the damage caused by heat stress to growth and to enzymes of carbon and antioxidative metabolism in chickpea. The chickpea seeds were raised without (control) and with proline (10 μM) at temperatures of 30/25 °C, 35/30 °C, 40/35 °C and 45/40 °C as day/ night (12 h/12 h) in a growth chamber. The shoot and root length at 40/35 °C decreased by 46 and 37 %, respectively over control while at 45/40 °C, a decrease of 63 and 47 %, respectively over control was observed. In the plants growing in the presence of 10 μM proline at 40/35 °C and 45/40 °C, the shoot length showed improvement of 32 and 53 %, respectively over untreated plants, while the root growth was improved by 22 and 26 %, respectively. The stress injury (as membrane damage) increased with elevation of temperatures while cellular respiration, chlorophyll content and relative leaf water content reduced as the temperature increased to 45/40 °C. The endogenous proline was elevated to 46 μmol g(-1) dw at 40/35 °C but declined to 19 μmol g(-1) dw in plants growing at 45/40 °C that was associated with considerable inhibition of growth at this temperature. The oxidative damage measured as malondialdehyde and hydrogen peroxide content increased manifolds in heat stressed plants coupled with inhibition in the activities of enzymatic (superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase) and levels of non-enzymatic (ascorbic acid, glutathione, proline) antioxidants. The enzymes associated with carbon fixation (RUBISCO), sucrose synthesis (sucrose phosphate synthase) and sucrose hydrolysis (invertase) were strongly inhibited at 45/40 °C. The plants growing in the presence of proline accumulated proline up to 63 μmol g(-1) dw and showed less injury to

  9. Experimental sink removal induces stress responses, including shifts in amino acid and phenylpropanoid metabolism, in soybean leaves

    PubMed Central

    Turner, Glenn W.; Cuthbertson, Daniel J.; Voo, Siau Sie; Settles, Matthew L.; Grimes, Howard D.

    2012-01-01

    The repeated removal of flower, fruit, or vegetative buds is a common treatment to simulate sink limitation. These experiments usually lead to the accumulation of specific proteins, which are degraded during later stages of seed development, and have thus been designated as vegetative storage proteins. We used oligonucleotide microarrays to assess global effects of sink removal on gene expression patterns in soybean leaves and found an induction of the transcript levels of hundreds of genes with putative roles in the responses to biotic and abiotic stresses. In addition, these data sets indicated potential changes in amino acid and phenylpropanoid metabolism. As a response to sink removal we detected an induced accumulation of γ-aminobutyric acid, while proteinogenic amino acid levels decreased. We also observed a shift in phenylpropanoid metabolism with an increase in isoflavone levels, concomitant with a decrease in flavones and flavonols. Taken together, we provide evidence that sink removal leads to an up-regulation of stress responses in distant leaves, which needs to be considered as an unintended consequence of this experimental treatment. PMID:22109846

  10. Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

    PubMed Central

    Alexandropoulos, K; Cheng, G; Baltimore, D

    1995-01-01

    To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another. Images Fig. 1 Fig. 2 Fig. 3 PMID:7536925

  11. Exogenous proline mediates alleviation of cadmium stress by promoting photosynthetic activity, water status and antioxidative enzymes activities of young date palm (Phoenix dactylifera L.).

    PubMed

    Zouari, M; Ben Ahmed, Ch; Zorrig, W; Elloumi, N; Rabhi, M; Delmail, D; Ben Rouina, B; Labrousse, P; Ben Abdallah, F

    2016-06-01

    The ability of exogenous compatible solutes, such as proline, to counteract cadmium (Cd) inhibitory effects in young date palm plants (Phoenix dactylifera L. cv Deglet Nour) was investigated. Two-year-old date palm plants were subjected for five months at different Cd stress levels (0, 10 and 30 mg CdCl2 kg(-1) soil) whether supplied or not with exogenous proline (20mM) added through the irrigation water. Different levels of Cd stress altered plant growth, gas exchanges and chlorophyll content as well as water status, but at different extent among them. In contrast, an increase of antioxidant enzymes activities of Cd-treated plants in association with high amounts of proline content, hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) and electrolyte leakage (EL) were observed. Interestingly, exogenous proline mitigated the adverse effects of Cd on young date palm. Indeed, it alleviated the oxidative damage induced by Cd accumulation and established better levels of plant growth, water status and photosynthetic activity. Moreover, proline-treated plants showed high antioxidant enzymes activities (superoxide dismutase, catalase and glutathione peroxydase) in roots and leaves as compared to Cd-treated plants. PMID:26901506

  12. Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity.

    PubMed

    Astafieva, Alexandra A; Enyenihi, Atim A; Rogozhin, Eugene A; Kozlov, Sergey A; Grishin, Eugene V; Odintsova, Tatyana I; Zubarev, Roman A; Egorov, Tsezi A

    2015-09-01

    Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications. PMID:26259198

  13. Fatty acid composition of ruminal digesta and longissimus muscle from lambs fed silage mixtures including red clover, sainfoin, and timothy.

    PubMed

    Campidonico, L; Toral, P G; Priolo, A; Luciano, G; Valenti, B; Hervás, G; Frutos, P; Copani, G; Ginane, C; Niderkorn, V

    2016-04-01

    This work investigated the effects of feeding silage mixtures of a plant containing polyphenol oxidase (PPO; red clover [; RC]), a plant containing tannins (sainfoin [; SF]), and a grass species not containing these compounds (timothy [; T]) on ruminal and intramuscular (i.m.) fatty acids of lambs. Forty 4-mo-old castrated male Romane lambs, divided into 5 groups, received 1 of the following silages: 1) T (100%), 2) a binary mixture of timothy and tannin-containing sainfoin ( cv. Perly; 50:50 [T-SF]), 3) a binary mixture of timothy and PPO-containing red clover ( cv. Mervius; 50:50 [T-RC]), 4) a ternary mixture of timothy, sainfoin, and red clover containing both tannins and PPO (50:25:25, respectively [T-SF-RC]), and 5) a binary mixture of tannin-containing sainfoin and PPO-containing red clover (50:50 [SF-RC]). In the rumen digesta, the partial or total replacement of T with forage legumes was associated with greater concentrations of PUFA ( < 0.001) and 1esser concentrations of MUFA ( < 0.001). The inclusion of forage legumes in the silage favored the accumulation of 18:3 -3 ( < 0.001), with the greatest concentrations being observed in SF-RC. This latter diet also led to the greatest percentage of 18:2 -6 ( < 0.001). Forage legumes decreased the -11 18:1 to 30% of T in rumen digesta ( < 0.001). Forage legumes decreased the total concentration of branched-chain fatty acids in the rumen digesta (on average, -28%; < 0.001), this effect being less marked (-17%; = 0.014) in T-RC in comparison with T. The dietary treatment tended to affect the proportion of MUFA ( = 0.081) and of PUFA ( = 0.079) in the i.m. fat of the LM, respectively, at the highest and lowest numerical value in the T group. The sum of -3 fatty acids was less in the T and T-SF groups compared with the mixture of legumes without T (SF-RC; < 0.001 and < 0.008, respectively). The latter group had also a lesser -6-to--3 ratio than the T-SF group ( = 0.01). -11 18:1 was greater ( < 0.03) in lambs given T

  14. Liquid chromatographic resolution of amino acid esters of acyclovir including racemic valacyclovir on crown ether-based chiral stationary phases.

    PubMed

    Ahn, Seong Ae; Hyun, Myung Ho

    2015-03-01

    Valacyclovir, a potential prodrug for the treatment of patients with herpes simplex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs) based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 covalently bonded to silica gel. In order to find out an appropriate mobile phase condition, various mobile phases consisting of various organic modifiers in water containing various acidic modifiers were applied to the resolution of valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved quite well on the two CSPs with the separation factors (α) in the range of 2.49 ~ 6.35 and resolutions (RS ) in the range of 2.95 ~ 12.21. Between the two CSPs, the CSP containing residual silanol protecting n-octyl groups on the silica surface was found to be better than the CSP containing residual silanol groups. PMID:25626672

  15. Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus.

    PubMed

    Cao, Huihui; Ke, Tao; Liu, Renhu; Yu, Jingyin; Dong, Caihua; Cheng, Mingxing; Huang, Junyan; Liu, Shengyi

    2015-01-01

    Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application

  16. Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus

    PubMed Central

    Liu, Renhu; Yu, Jingyin; Dong, Caihua; Cheng, Mingxing; Huang, Junyan; Liu, Shengyi

    2015-01-01

    Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs’ mechanism and application

  17. The Binding of Syndapin SH3 Domain to Dynamin Proline-rich Domain Involves Short and Long Distance Elements.

    PubMed

    Luo, Lin; Xue, Jing; Kwan, Ann; Gamsjaeger, Roland; Wielens, Jerome; von Kleist, Lisa; Cubeddu, Liza; Guo, Zhong; Stow, Jennifer L; Parker, Michael W; Mackay, Joel P; Robinson, Phillip J

    2016-04-29

    Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction. PMID:26893375

  18. Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level

    PubMed Central

    Majumdar, Rajtilak; Barchi, Boubker; Turlapati, Swathi A.; Gagne, Maegan; Minocha, Rakesh; Long, Stephanie; Minocha, Subhash C.

    2016-01-01

    The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells. PMID:26909083

  19. Proline derivatives in fruits of bergamot (Citrus bergamia Risso et Poit): presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine.

    PubMed

    Servillo, Luigi; Giovane, Alfonso; Balestrieri, Maria Luisa; Cautela, Domenico; Castaldo, Domenico

    2011-01-12

    The content of proline and various compounds deriving from its metabolism (4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine) was determined in fruits and seeds of Bergamot (Citrus bergamia Risso et Poit), growing in the Calabria region (South Italy). A HPLC-ESI-tandem mass spectrometry method, which allowed rapid determination of L-proline, 4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine in juice and extracts of bergamot fruit with minimum sample preparation and short analysis time (about 10 min), is presented. Proline and 4-hydroxy-L-proline levels in the samples were also determined by HPLC analysis with fluorescence detection and the results compared to those obtained with HPLC-ESI-tandem mass spectrometry. For the first time, the presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine in the fruits of a plant of the Citrus genus is reported. PMID:21128667

  20. Skin aging and photoaging alter fatty acids composition, including 11,14,17-eicosatrienoic acid, in the epidermis of human skin.

    PubMed

    Kim, Eun Ju; Kim, Min-Kyoung; Jin, Xing-Ji; Oh, Jang-Hee; Kim, Ji Eun; Chung, Jin Ho

    2010-06-01

    We investigated the alterations of major fatty acid components in epidermis by natural aging and photoaging processes, and by acute ultraviolet (UV) irradiation in human skin. Interestingly, we found that 11,14,17-eicosatrienoic acid (ETA), which is one of the omega-3 polyunsaturated acids, was significantly increased in photoaged human epidermis in vivo and also in the acutely UV-irradiated human skin in vivo, while it was significantly decreased in intrinsically aged human epidermis. The increased ETA content in the epidermis of photoaged human skin and acute UV-irradiated human skin is associated with enhanced expression of human elongase 1 and calcium-independent phosphodiesterase A(2). We demonstrated that ETA inhibited matrix metalloproteinase (MMP)-1 expression after UV-irradiation, and that inhibition of ETA synthesis using EPTC and NA-TCA, which are elongase inhibitors, increased MMP-1 expression. Therefore, our results suggest that the UV increases the ETA levels, which may have a photoprotective effect in the human skin. PMID:20514327

  1. Skin Aging and Photoaging Alter Fatty Acids Composition, Including 11,14,17-eicosatrienoic Acid, in the Epidermis of Human Skin

    PubMed Central

    Kim, Eun Ju; Kim, Min-Kyoung; Jin, Xing-Ji; Oh, Jang-Hee; Kim, Ji Eun

    2010-01-01

    We investigated the alterations of major fatty acid components in epidermis by natural aging and photoaging processes, and by acute ultraviolet (UV) irradiation in human skin. Interestingly, we found that 11,14,17-eicosatrienoic acid (ETA), which is one of the omega-3 polyunsaturated acids, was significantly increased in photoaged human epidermis in vivo and also in the acutely UV-irradiated human skin in vivo, while it was significantly decreased in intrinsically aged human epidermis. The increased ETA content in the epidermis of photoaged human skin and acute UV-irradiated human skin is associated with enhanced expression of human elongase 1 and calcium-independent phophodiesterase A2. We demonstrated that ETA inhibited matrix metalloproteinase (MMP)-1 expression after UV-irradiation, and that inhibition of ETA synthesis using EPTC and NA-TCA, which are elongase inhibitors, increased MMP-1 expression. Therefore, our results suggest that the UV increases the ETA levels, which may have a photoprotective effect in the human skin. PMID:20514327

  2. Interaction of serum amyloid P component with hexanoyl bis(d-proline) (CPHPC)

    SciTech Connect

    Kolstoe, Simon E.; Jenvey, Michelle C.; Purvis, Alan; Light, Mark E.; Thompson, Darren; Hughes, Peter; Pepys, Mark B.; Wood, Stephen P.

    2014-08-01

    Serum amyloid P component is a pentameric plasma glycoprotein that recognizes and binds to amyloid fibres in a calcium-dependent fashion and is likely to contribute to their deposition and persistence in vivo. Five molecules of the drug CPHPC avidly cross-link pairs of protein pentamers and the decameric complex is rapidly cleared in vivo. Crystal structures of the protein in complex with a bivalent drug and cadmium ions, which improve crystal quality, allow the definition of the preferred bound drug isomers. Under physiological conditions, the pentameric human plasma protein serum amyloid P component (SAP) binds hexanoyl bis(d-proline) (R-1-(6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl) pyrrolidine-2-carboxylic acid; CPHPC) through its d-proline head groups in a calcium-dependent interaction. Cooperative effects in binding lead to a substantial enhancement of affinity. Five molecules of the bivalent ligand cross-link and stabilize pairs of SAP molecules, forming a decameric complex that is rapidly cleared from the circulation by the liver. Here, it is reported that X-ray analysis of the SAP complex with CPHPC and cadmium ions provides higher resolution detail of the interaction than is observed with calcium ions. Conformational isomers of CPHPC observed in solution by HPLC and by X-ray analysis are compared with the protein-bound form. These are discussed in relation to the development of CPHPC to provide SAP depletion for the treatment of amyloidosis and other indications.

  3. Ameliorating Effects of Exogenously Applied Proline on Seed Composition, Seed Oil Quality and Oil Antioxidant Activity of Maize (Zea mays L.) under Drought Stress

    PubMed Central

    Ali, Qasim; Anwar, Farooq; Ashraf, Muhammad; Saari, Nazamid; Perveen, Rashida

    2013-01-01

    This study was carried out to appraise whether or not the exogenous application of a potential osmoprotectant, proline, could ameliorate the adverse effects of drought stress on maize seed and seed oil composition, as well as oil antioxidant activity. Water stress reduced the kernel sugar, oil, protein and moisture contents and most of the seed macro- and micro-elements analyzed in both maize cultivars but it increased the contents of seed fiber and ash. Water stress increased the oil oleic acid content with a subsequent decrease in the amount of linoleic acid, resulting in an increased oil oleic/linoleic ratio for both maize cultivars. However, no variation was observed in oil stearic and palmitic acids content due to water stress. A considerable drought induced an increase in seed oil α-, γ-, δ- and total tocopherols and flavonoids were observed in both maize cultivars. However, oil phenolic and carotenoid content as well as 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity decreased. Foliar-applied proline significantly increased the content of seed sugar, oil, protein, moisture, fiber and ash in both maize cultivars under well irrigated and water deficit conditions. Furthermore, exogenous application of proline increased the oil oleic and linoleic acid contents. The concentrations of antioxidant compounds namely phenolics, carotenoids, flavonoids and tocopherols estimated in the seed oil increased due to foliar-applied proline under water deficit conditions that was positively correlated with the enhanced oil DPPH free radical scavenging activity. Moreover, the increase in the contents of these antioxidant compounds and oil antioxidant activity due to the foliar application of proline was noted to be more pronounced under water deficit conditions. PMID:23344043

  4. Pharmacokinetic and Pharmacodynamic Interaction of Boswellic Acids and Andrographolide with Glyburide in Diabetic Rats: Including Its PK/PD Modeling.

    PubMed

    Samala, Sujatha; Veeresham, Ciddi

    2016-03-01

    The effect of boswellic acids (BA) and andrographolide (AD) on the pharmacokinetics and pharmacodynamics of glyburide in normal as well as in streptozotocin-induced diabetic rats was studied. In normal and diabetic rats, the combination of glyburide with BA or AD increased significantly (p < 0.01) all the pharmacokinetic parameters, such as Cmax, AUC0-n, AUCtotal, t1/2, and mean residence time, and decreased the clearance, Vd, markedly as compared with the control group. In rat liver, microsomes BA and AD have shown CYP3A4 inhibitory activity significantly (p < 0.01), compared with the vehicle group. The increase in hypoglycemic action by concomitant administration of glyburide with BA or AD was more in diabetic rats than when the drugs were used singly and with the control group, which suggests the enhancement of glucose reduction capacity of glyburide in diabetic rats along with BA or AD. In PK/PD modeling of BA and AD with glyburide, the predicted PK and PD parameters are in line with the observed PK and PD parameters. The results revealed that BA and AD led to the PK/PD changes because of glyburide-increased bioavailability and because of the inhibition of CYP3A4 enzyme. In conclusion, add-on preparations containing BA or AD may increase the bioavailability of glyburide, and hence the dose should be monitored. PMID:26762235

  5. Retinoic Acid Induced 1, RAI1: A Dosage Sensitive Gene Related to Neurobehavioral Alterations Including Autistic Behavior

    PubMed Central

    Carmona-Mora, Paulina; Walz, Katherina

    2010-01-01

    Genomic structural changes, such as gene Copy Number Variations (CNVs) are extremely abundant in the human genome. An enormous effort is currently ongoing to recognize and catalogue human CNVs and their associations with abnormal phenotypic outcomes. Recently, several reports related neuropsychiatric diseases (i.e. autism spectrum disorders, schizophrenia, mental retardation, behavioral problems, epilepsy) with specific CNV. Moreover, for some conditions, both the deletion and duplication of the same genomic segment are related to the phenotype. Syndromes associated with CNVs (microdeletion and microduplication) have long been known to display specific neurobehavioral traits. It is important to note that not every gene is susceptible to gene dosage changes and there are only a few dosage sensitive genes. Smith-Magenis (SMS) and Potocki-Lupski (PTLS) syndromes are associated with a reciprocal microdeletion and microduplication within chromosome 17p11.2. in humans. The dosage sensitive gene responsible for most phenotypes in SMS has been identified: the Retinoic Acid Induced 1 (RAI1). Studies on mouse models and humans suggest that RAI1 is likely the dosage sensitive gene responsible for clinical features in PTLS. In addition, the human RAI1 gene has been implicated in several neurobehavioral traits as spinocerebellar ataxia (SCA2), schizophrenia and non syndromic autism. In this review we discuss the evidence of RAI1 as a dosage sensitive gene, its relationship with different neurobehavioral traits, gene structure and mutations, and what is known about its molecular and cellular function, as a first step in the elucidation of the mechanisms that relate dosage sensitive genes with abnormal neurobehavioral outcomes. PMID:21629438

  6. Proline rich motifs as drug targets in immune mediated disorders.

    PubMed

    Srinivasan, Mythily; Dunker, A Keith

    2012-01-01

    The current version of the human immunome network consists of nearly 1400 interactions involving approximately 600 proteins. Intermolecular interactions mediated by proline-rich motifs (PRMs) are observed in many facets of the immune response. The proline-rich regions are known to preferentially adopt a polyproline type II helical conformation, an extended structure that facilitates transient intermolecular interactions such as signal transduction, antigen recognition, cell-cell communication and cytoskeletal organization. The propensity of both the side chain and the backbone carbonyls of the polyproline type II helix to participate in the interface interaction makes it an excellent recognition motif. An advantage of such distinct chemical features is that the interactions can be discriminatory even in the absence of high affinities. Indeed, the immune response is mediated by well-orchestrated low-affinity short-duration intermolecular interactions. The proline-rich regions are predominantly localized in the solvent-exposed regions such as the loops, intrinsically disordered regions, or between domains that constitute the intermolecular interface. Peptide mimics of the PRM have been suggested as potential antagonists of intermolecular interactions. In this paper, we discuss novel PRM-mediated interactions in the human immunome that potentially serve as attractive targets for immunomodulation and drug development for inflammatory and autoimmune pathologies. PMID:22666276

  7. Proline Rich Motifs as Drug Targets in Immune Mediated Disorders

    PubMed Central

    Srinivasan, Mythily; Dunker, A. Keith

    2012-01-01

    The current version of the human immunome network consists of nearly 1400 interactions involving approximately 600 proteins. Intermolecular interactions mediated by proline-rich motifs (PRMs) are observed in many facets of the immune response. The proline-rich regions are known to preferentially adopt a polyproline type II helical conformation, an extended structure that facilitates transient intermolecular interactions such as signal transduction, antigen recognition, cell-cell communication and cytoskeletal organization. The propensity of both the side chain and the backbone carbonyls of the polyproline type II helix to participate in the interface interaction makes it an excellent recognition motif. An advantage of such distinct chemical features is that the interactions can be discriminatory even in the absence of high affinities. Indeed, the immune response is mediated by well-orchestrated low-affinity short-duration intermolecular interactions. The proline-rich regions are predominantly localized in the solvent-exposed regions such as the loops, intrinsically disordered regions, or between domains that constitute the intermolecular interface. Peptide mimics of the PRM have been suggested as potential antagonists of intermolecular interactions. In this paper, we discuss novel PRM-mediated interactions in the human immunome that potentially serve as attractive targets for immunomodulation and drug development for inflammatory and autoimmune pathologies. PMID:22666276

  8. Multiple gas-phase conformations of proline-containing peptides: is it always cis/trans isomerization?

    PubMed

    Lietz, Christopher B; Chen, Zhengwei; Yun Son, Chang; Pang, Xueqin; Cui, Qiang; Li, Lingjun

    2016-08-01

    Ion mobility-mass spectrometry (IM-MS) is often employed to look at the secondary, tertiary, and quaternary structures of naked peptides and proteins in the gas-phase. Recently, it has offered a unique glimpse into proline-containing peptides and their cis/trans Xxx-Pro isomers. An experimental "signature" has been identified wherein a proline-containing peptide has its Pro residues substituted with another amino acid and the presence or absence of conformations in the IM-MS spectra is observed. Despite the high probability that one could attribute these conformations to cis/trans isomers, it is also possible that cis/trans isomers are not the cause of the additional conformations in proline-containing peptides. However, the experimental evidence of such a system has not been demonstrated or reported. Herein, we present the IM-MS analysis of Neuropeptide Y's wild-type (WT) signal sequence and Leu7Pro (L7P) mutant. Although comparison of arrival times and collision cross-sections of [M + 4H](4+) ions yields the cis/trans "signature", molecular dynamics indicates that a cis-Pro7 is not very stable and that trans-Pro7 conformations of the same cross-section arise with equal frequency. We believe that this work further underscores the importance of theoretical calculations in IM-MS structural assignments. PMID:27434776

  9. Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.

    PubMed

    Jensen, Jaide V K; Eberhardt, Dorit; Wendisch, Volker F

    2015-11-20

    The glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. Corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. Here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and arginine, and the diamine putrescine was demonstrated. Feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of glutamate dehydrogenase gene gdh, lowering expression of phosphoglucoisomerase gene pgi, along with the introduction of a second copy of the arginine biosynthesis operon argCJB(A49V,M54V)D into the chromosome resulted in a C. glutamicum strain producing ornithine with a yield of 0.52 g ornithine per g glucose, an increase of 71% as compared to the parental ΔargFRG strain. Strains capable of producing 0.41 g citrulline per g glucose, 0.29 g proline per g glucose, 0.30 g arginine per g glucose, and 0.17 g putrescine per g glucose were derived from the ornithine-producing platform strain by plasmid-based overexpression of appropriate pathway modules with one to three genes. PMID:26393954

  10. Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism.

    PubMed

    Nasuno, Ryo; Hirano, Yoshinori; Itoh, Takafumi; Hakoshima, Toshio; Hibi, Takao; Takagi, Hiroshi

    2013-07-16

    Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ(1)-pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ(1)-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs. PMID:23818613

  11. Proline is required for male gametophyte development in Arabidopsis

    PubMed Central

    2012-01-01

    Background In crosses between the proline-deficient mutant homozygous for p5cs1 and heterozygous for p5cs2 (p5cs1 p5cs2/P5CS2), used as male, and different Arabidopsis mutants, used as females, the p5cs2 mutant allele was rarely transmitted to the outcrossed progeny, suggesting that the fertility of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised. Results To confirm the fertility defects of pollen from p5cs1 p5cs2/P5CS2 mutants, transmission of mutant alleles through pollen was tested in two ways. First, the number of progeny inheriting a dominant sulfadiazine resistance marker linked to p5cs2 was determined. Second, the number of p5cs2/p5cs2 embryos was determined. A ratio of resistant to susceptible plantlets close to 50%, and the absence of aborted embryos were consistent with the hypothesis that the male gametophyte carrying both p5cs1 and p5cs2 alleles is rarely transmitted to the offspring. In addition, in reciprocal crosses with wild type, about 50% of the p5cs2 mutant alleles were transmitted to the sporophytic generation when p5cs1 p5cs2/P5CS2 was used as a female, while less than 1% of the p5cs2 alleles could be transmitted to the outcrossed progeny when p5cs1 p5cs2/P5CS2 was used as a male. Morphological and functional analysis of mutant pollen revealed a population of small, degenerated, and unviable pollen grains, indicating that the mutant homozygous for p5cs1 and heterozygous for p5cs2 is impaired in pollen development, and suggesting a role for proline in male gametophyte development. Consistent with these findings, we found that pollen from p5cs1 homozygous mutants, display defects similar to, but less pronounced than pollen from p5cs1 p5cs2/P5CS2 mutants. Finally, we show that pollen from p5cs1 p5cs2/P5CS2 plants contains less proline than wild type and that exogenous proline supplied from the beginning of another development can partially complement both morphological and functional pollen defects

  12. A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

    PubMed Central

    Opitz, Robert; Müller, Matthias; Reuter, Cédric; Barone, Matthias; Soicke, Arne; Roske, Yvette; Piotukh, Kirill; Huy, Peter; Beerbaum, Monika; Wiesner, Burkhard; Beyermann, Michael; Schmieder, Peter; Freund, Christian; Volkmer, Rudolf; Oschkinat, Hartmut; Schmalz, Hans-Günther; Kühne, Ronald

    2015-01-01

    Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity. PMID:25848013

  13. Omega-3 fatty acid concentrate from Dunaliella salina possesses anti-inflammatory properties including blockade of NF-κB nuclear translocation.

    PubMed

    Chitranjali, T; Anoop Chandran, P; Muraleedhara Kurup, G

    2015-02-01

    The health benefits of omega-3 polyunsaturated fatty acids (ω-3 PUFA), mainly eicosapentaenoic acid (EPA 20:5) and docosahexaenoic acid (DHA, 22:6), have been long known. Although various studies have demonstrated the health benefits of ω-3 PUFA, the mechanisms of action of ω-3 PUFAs are still not completely understood. While the major commercial source is marine fish oil, in this study we suggest the marine micro algae, Dunaliella salina as an alternate source of omega-3 fatty acids. Treatment with this algal omega-3 fatty acid concentrate (Ds-ω-3 FA) resulted in significant down-regulation of LPS-induced production of TNF-α and IL-6 by peripheral blood mononuclear cells (PBMCs). The concentrate was also found to be a potent blocker of cyclooxygenase (COX-2) and matrix metalloproteinase (MMP-2 and MMP-9) expression. The present study reveals the anti-inflammatory properties of Ds-ω-3 FA concentrate including the inhibition of NF-κB translocation. PMID:25391558

  14. New CE-ESI-MS analytical method for the separation, identification and quantification of seven phenolic acids including three isomer compounds in virgin olive oil.

    PubMed

    Nevado, Juan José Berzas; Peñalvo, Gregorio Castañeda; Robledo, Virginia Rodríguez; Martínez, Gabriela Vargas

    2009-10-15

    A sensitive and expeditious CE-ESI-MS analytical method for the separation, identification and determination of seven selected antioxidants (cinnamic and benzoic acids), including three isomers of coumaric acid (ortho-, meta- and para-) has been developed. In order to obtain the analytical separation, capillary electrophoresis and CE-MS interface parameters (e.g., buffer pH and composition, sheath liquid and gas flow rates, sheath liquid composition, electrospray voltage, etc.) were carefully optimized. The polar fraction containing the selected phenolic acids was obtained using a previously optimized SPE pretreatment. An MS detector in order to extract structural information about the target compounds and facilitate their qualitative analysis was used in the negative ion mode. The proposed off-line SPE CE-ESI-MS method was validated by assessing its precision, LODs and LOQs, linearity range and accuracy. The optimized and validated method was used in order to quantify the selected antioxidants in various samples of virgin olive oil and extra-virgin olive oil obtained from the main olive varieties cropped in Castilla-La Mancha, Spain. Salicylic acid was used as internal standard throughout in order to ensure reproducibility in the quantitative analysis of the oil samples. The results confirmed the presence of hydroxyphenyl acetic, p-coumaric, ferulic and vanillic acids in substantial amounts (microg g(-1) level) in all samples. PMID:19635353

  15. Purification, Characterization, and Application of a Novel Dye-Linked l-Proline Dehydrogenase from a Hyperthermophilic Archaeon, Thermococcus profundus

    PubMed Central

    Sakuraba, Haruhiko; Takamatsu, Yoshinori; Satomura, Takenori; Kawakami, Ryushi; Ohshima, Toshihisa

    2001-01-01

    The distribution of dye-linked l-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked l-proline dehydrogenase (dye-l-proDH, l-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (α2 β2) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the α-subunit (50-kDa subunit) and the β-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-l-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70°C for 10 min, and ca. 40% of the activity still remained after heating at 80°C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50°C for 10 min. The enzyme exclusively catalyzed l-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for l-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Δ1-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of l-proline at concentrations from 0.10 to 2.5 mM using the stable dye-l-proDH was achieved. PMID:11282592

  16. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

  17. Carbon Monoxide Modulates α–Smooth Muscle Actin and Small Proline Rich-1a Expression in Fibrosis

    PubMed Central

    Zheng, Liang; Zhou, Zhihong; Lin, Ling; Alber, Sean; Watkins, Simon; Kaminski, Naftali; Choi, Augustine M. K.; Morse, Danielle

    2009-01-01

    Carbon monoxide (CO) is a biologically active molecule produced in the body by the stress-inducible enzyme, heme oxygenase. We have previously shown that CO suppresses fibrosis in a murine bleomycin model. To investigate the mechanisms by which CO opposes fibrogenesis, we performed gene expression profiling of fibroblasts treated with transforming growth factor-β1 and CO. The most highly differentially expressed categories of genes included those related to muscular system development and the small proline-rich family of proteins. We confirmed in vitro, and in an in vivo bleomycin model of lung fibrosis, that CO suppresses α–smooth muscle actin expression and enhances small proline-rich protein-1a expression. We further show that these effects of CO depend upon signaling via the extracellular signal–regulated kinase pathway. Our results demonstrate novel transcriptional targets for CO and further elucidate the mechanism by which CO suppresses fibrosis. PMID:19097987

  18. High-speed civil transport impact: Role of sulfate, nitric acid trihydrate, and ice aerosols studied with a two-dimensional model including aerosol physics

    SciTech Connect

    Pitari, G.; Ricciardulli, L.; Visconti, G.; Rizi, V.

    1993-12-20

    The authors discuss a two-dimensional model used to study the atmospheric interactions of ozone with exhaust gases from high speed civil transport (HSCT) fleets. Their model encompases the stratosphere and troposphere, includes photochemical reactions as part of the sulfur cycle, and models sulfuric acid aerosols. The inclusion of heterogeneous chemistry effects tempers the impact of nitrogen oxide emissions from HSCT on ozone depletion, in support of previous work from other studies.

  19. The prebiotic synthesis of amino acids - interstellar vs. atmospheric mechanisms

    NASA Astrophysics Data System (ADS)

    Meierhenrich, U. J.; Muñoz Caro, G. M.; Schutte, W. A.; Barbier, B.; Arcones Segovia, A.; Rosenbauer, H.; Thiemann, W. H.-P.; Brack, A.

    2002-11-01

    Until very recently, prebiotic amino acids were believed to have been generated in the atmosphere of the early Earth, as successfully simulated by the Urey-Miller experiments. Two independent studies now identified ice photochemistry in the interstellar medium as a possible source of prebiotic amino acids. Ultraviolet irradiation of ice mixtures containing identified interstellar molecules (such as H2O, CO2, CO, CH3OH, and NH3) in the conditions of vacuum and low temperature found in the interstellar medium generated amino acid structures including glycine, alanine, serine, valine, proline, and aspartic acid. After warmup, hydrolysis and derivatization, our team was able to identify 16 amino acids as well as furans and pyrroles. Enantioselective analyses of the amino acids showed racemic mixtures. A prebiotic interstellar origin of amino acid structures is now discussed to be a plausible alternative to the Urey-Miller mechanism.

  20. Role of Conserved Proline Residues in Human Apolipoprotein A-IV Structure and Function*

    PubMed Central

    Deng, Xiaodi; Walker, Ryan G.; Morris, Jamie; Davidson, W. Sean; Thompson, Thomas B.

    2015-01-01

    Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members. PMID:25733664

  1. Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans

    SciTech Connect

    Corley, R.A.; Saghir, S.A.; Bartels, M.J.; Hansen, S.C.; Creim, J.; McMartin, K.E.; Snellings, W.M.

    2011-02-01

    A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed male Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200 mg/m{sup 3}. While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.

  2. Phytoremediation of cadmium improved with the high production of endogenous phenolics and free proline contents in Parthenium hysterophorus plant treated exogenously with plant growth regulator and chelating agent.

    PubMed

    Ali, Nasir; Hadi, Fazal

    2015-09-01

    Pot experiments were conducted to evaluate the effects of gibberellic acid (GA3) and ethylenediaminetetraacetic acid (EDTA) on growth parameters, cadmium (Cd) phytoextraction, total phenolics, free proline and chlorophyll content of Parthenium hysterophorus plant grown in Cd-contaminated (100 mg/kg) soil. GA3 was applied as foliar spray (10(-2), 10(-4) and 10(-6) M) while EDTA (40 mg/kg soil) was added to soil as single and in split doses. Results showed decrease in growth parameters due to Cd stress but P. hysterophorus plant demonstrated Cd hyperaccumulator potential based on bioconcentration factor (BCF). Lower concentration of GA3 (10(-6) M) showed highest significant increase in the growth parameters while Cd concentration, accumulation (1.97 ± 0.11 mg/DBM) and bioconcentration (9.75 ± 0.34) was significantly higher in the treatment T11 (GA3 10(-2) + split doses of EDTA). Cadmium significantly increased the root free proline while total phenolic concentration was significantly high in all parts of the plant. Chlorophyll contents were significantly reduced by Cd. GA3 showed significant increase in phenolic and chlorophyll contents in plant. Cadmium accumulation in plant tissues showed positive correlation with free proline (R (2) = 0.527, R (2) = 0.630) and total phenolics (R (2) = 0.554, R (2) = 0.723) in roots and leaves, respectively. Cd contents negatively correlated with biomass, chlorophyll and total water contents. Proline and phenolic contents showed positive correlation with dry biomass of plant. These findings suggest further investigation to study the role of endogenous phenolics and proline in heavy metal phytoremediation. PMID:25940488

  3. Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population.

    PubMed

    Lang, Julien; Faure, Denis

    2016-05-01

    Accumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations. PMID:27110651

  4. In Vitro Antibacterial and Antibiofilm Activities of Chlorogenic Acid against Clinical Isolates of Stenotrophomonas maltophilia including the Trimethoprim/Sulfamethoxazole Resistant Strain

    PubMed Central

    Karunanidhi, Arunkumar; Thomas, Renjan; van Belkum, Alex; Neela, Vasanthakumari

    2013-01-01

    The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim/sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 μg mL−1 and 16 to 32 μg mL−1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia. PMID:23509719

  5. First Evidence for Substrate Channeling between Proline Catabolic Enzymes

    PubMed Central

    Sanyal, Nikhilesh; Arentson, Benjamin W.; Luo, Min; Tanner, John J.; Becker, Donald F.

    2015-01-01

    Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyze the four-electron oxidation of proline to glutamate via the intermediates P5C and l-glutamate-γ-semialdehyde (GSA). In Gram-negative bacteria, PRODH and P5CDH are fused together in the bifunctional enzyme proline utilization A (PutA) whereas in other organisms PRODH and P5CDH are expressed as separate monofunctional enzymes. Substrate channeling has previously been shown for bifunctional PutAs, but whether the monofunctional enzymes utilize an analogous channeling mechanism has not been examined. Here, we report the first evidence of substrate channeling in a PRODH-P5CDH two-enzyme pair. Kinetic data for the coupled reaction of PRODH and P5CDH from Thermus thermophilus are consistent with a substrate channeling mechanism, as the approach to steady-state formation of NADH does not fit a non-channeling two-enzyme model. Furthermore, inactive P5CDH and PRODH mutants inhibit NADH production and increase trapping of the P5C intermediate in coupled assays of wild-type PRODH-P5CDH enzyme pairs, indicating that the mutants disrupt PRODH-P5CDH channeling interactions. A dissociation constant of 3 μm was estimated for a putative PRODH-P5CDH complex by surface plasmon resonance (SPR). Interestingly, P5CDH binding to PRODH was only observed when PRODH was immobilized with the top face of its (βα)8 barrel exposed. Using the known x-ray crystal structures of PRODH and P5CDH from T. thermophilus, a model was built for a proposed PRODH-P5CDH enzyme channeling complex. The structural model predicts that the core channeling pathway of bifunctional PutA enzymes is conserved in monofunctional PRODH-P5CDH enzyme pairs. PMID:25492892

  6. Steady-State Kinetic Mechanism of the Proline:Ubiquinone Oxidoreductase Activity of Proline Utilization A (PutA) from Escherichia coli

    PubMed Central

    Moxley, Michael A.; Tanner, John J.; Becker, Donald F.

    2011-01-01

    The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains. In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a β8α8-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest kcat/Km was obtained with CoQ2. The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ1, combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites. PMID:22040654

  7. RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata

    PubMed Central

    Peng, Ting; Jia, Mao-Mao; Liu, Ji-Hong

    2015-01-01

    Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis. PMID:26483822

  8. RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

    PubMed

    Peng, Ting; Jia, Mao-Mao; Liu, Ji-Hong

    2015-01-01

    Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis. PMID:26483822

  9. Targeted mutation of Δ12 and Δ15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil.

    PubMed

    Bielecka, Monika; Kaminski, Filip; Adams, Ian; Poulson, Helen; Sloan, Raymond; Li, Yi; Larson, Tony R; Winzer, Thilo; Graham, Ian A

    2014-06-01

    We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven Δ12 desaturases and one of three Δ15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have Δx+3 activity, while CSFAD3A activity was exclusively at the Δ15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major Δ12 and Δ15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1(Δ9) ) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2(Δ6,9) and 18:2(Δ9,15), were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2(Δ9,15) from endogenous 18:1(Δ9) when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop. PMID:24506492

  10. Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens.

    PubMed

    Goytia, Maira; Chamond, Nathalie; Cosson, Alain; Coatnoan, Nicolas; Hermant, Daniel; Berneman, Armand; Minoprio, Paola

    2007-01-01

    The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host. PMID:17849014

  11. Molecular and Structural Discrimination of Proline Racemase and Hydroxyproline-2-Epimerase from Nosocomial and Bacterial Pathogens

    PubMed Central

    Goytia, Maira; Chamond, Nathalie; Cosson, Alain; Coatnoan, Nicolas; Hermant, Daniel; Berneman, Armand; Minoprio, Paola

    2007-01-01

    The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for ‘proline racemase’ virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not ‘one way’ directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on contraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host. PMID:17849014

  12. Structure analysis of an amyloid-forming model peptide by a systematic glycine and proline scan.

    PubMed

    Gerling, Ulla I M; Brandenburg, Enrico; von Berlepsch, Hans; Pagel, Kevin; Koksch, Beate

    2011-08-01

    The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high β-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the β-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement. PMID:21726080

  13. Thermoresponsive and Mechanical Properties of Poly(L-proline) Gels.

    PubMed

    Gkikas, Manos; Avery, Reginald K; Olsen, Bradley D

    2016-02-01

    Gelation of the left helical N-substituted homopolypeptide poly(L-proline) (PLP) in water was explored, employing rheological and small-angle scattering studies at different temperatures and concentrations in order to investigate the network structure and its mechanical properties. Stiff gels were obtained at 10 wt % or higher at 5 °C, the first time gelation has been observed for homopolypeptides. The secondary structure and helical rigidity of PLP has large structural similarities to gelatin but as gels the two materials show contrasting trends with temperature. With increasing temperature in D2O, the network stiffens, with broad scattering features of similar correlation length for all concentrations and molar masses of PLP. A thermoresponsive transition was also achieved between 5 and 35 °C, with moduli at 35 °C higher than gelatin at 5 °C. The brittle gels could tolerate strains of 1% before yielding with a frequency-independent modulus over the observed range, similar to natural proline-rich proteins, suggesting the potential for thermoresponsive or biomaterial-based applications. PMID:26736072

  14. Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

    PubMed Central

    Zhang, Lu; Alfano, James R.

    2014-01-01

    The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity. PMID:25384482

  15. Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

    PubMed Central

    Molenaar, D; Hagting, A; Alkema, H; Driessen, A J; Konings, W N

    1993-01-01

    Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed. PMID:8366030

  16. T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis.

    PubMed

    Brill, Jeanette; Hoffmann, Tamara; Putzer, Harald; Bremer, Erhard

    2011-04-01

    Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ-ProA-ProH route is responsible for the production of proline as an osmoprotectant, and the ProB-ProA-ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine. PMID:21233158

  17. Asymmetric Synthesis, Structure, and Reactivity of Unexpectedly Stable Spiroepoxy-β-Lactones Including Facile Conversion to Tetronic Acids: Application to (+)-Maculalactone A

    PubMed Central

    Duffy, Richard J.; Morris, Kay A.; Vallakati, Ravikrishna; Zhang, Wei; Romo, Daniel

    2009-01-01

    A novel class of small spirocyclic heterocycles, spiroepoxy-β-lactones (1,4-dioxaspiro[2.3]-hexan-5-ones), is described that exhibit a number of interesting reactivity patterns. These spiroheterocycles, including an optically active series, are readily synthesized by epoxidation of ketene dimers (4-alkylidene-2-oxetanones) available from homo- or heteroketene dimerization. An analysis of bond lengths in these systems by X-ray crystallography and comparison to data for known spirocycles and those determined computationally, suggest that anomeric effects in these systems may be more pronounced due to their rigidity and may contribute to their surprising stability. The synthetic utility of spiroepoxy-β-lactones was explored and one facile rearrangement identified under several conditions provides a 3-step route from acid chlorides to optically active tetronic acids, ubiquitous heterocycles in bioactive natural products. The addition of various nucleophiles to these spirocycles leads primarily to addition at C5 and C2. The utility of an optically active spiroepoxy-β-lactone was demonstrated in the concise, enantioselective synthesis of the anti-fouling agent, (+)-maculalactone A, which proceeds in 5 steps from hydrocinnamoyl chloride by way of a tetronic acid intermediate. PMID:19453152

  18. Asymmetric synthesis, structure, and reactivity of unexpectedly stable spiroepoxy-beta-lactones including facile conversion to tetronic acids: application to (+)-maculalactone A.

    PubMed

    Duffy, Richard J; Morris, Kay A; Vallakati, Ravikrishna; Zhang, Wei; Romo, Daniel

    2009-07-01

    A novel class of small spirocyclic heterocycles, spiroepoxy-beta-lactones (1,4-dioxaspiro[2.3]-hexan-5-ones), is described that exhibit a number of interesting reactivity patterns. These spiroheterocycles, including an optically active series, are readily synthesized by epoxidation of ketene dimers (4-alkylidene-2-oxetanones) available from homo- or heteroketene dimerization. An analysis of bond lengths in these systems by X-ray crystallography and comparison to data for known spirocycles and those determined computationally suggest that anomeric effects in these systems may be more pronounced due to their rigidity and may contribute to their surprising stability. The synthetic utility of spiroepoxy-beta-lactones was explored, and one facile rearrangement identified under several conditions provides a three-step route from acid chlorides to optically active tetronic acids, ubiquitous heterocycles in bioactive natural products. The addition of various nucleophiles to these spirocycles leads primarily to addition at C5 and C2. The utility of an optically active spiroepoxy-beta-lactone was demonstrated in the concise, enantioselective synthesis of the antifouling agent, (+)-maculalactone A, which proceeds in five steps from hydrocinnamoyl chloride by way of a tetronic acid intermediate. PMID:19453152

  19. Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake

    PubMed Central

    Cheng, Zhihui; Lin, Mingqun

    2014-01-01

    ABSTRACT How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth. PMID:25425236

  20. Survey analysis and chemical characterization of solid inhomogeneous samples using a general homogenization procedure including acid digestion, drying, grinding and briquetting together with X-ray fluorescence.

    PubMed

    Sahlin, Eskil; Magnusson, Bertil

    2012-08-15

    A survey analysis and chemical characterization methodology for inhomogeneous solid waste samples of relatively large samples (typically up to 100g) using X-ray fluorescence following a general homogenization procedure is presented. By using a combination of acid digestion and grinding various materials can be homogenized e.g. pure metals, alloys, salts, ores, plastics, organics. In the homogenization step, solid material is fully or partly digested in a mixture of nitric acid and hydrochloric acid in an open vessel. The resulting mixture is then dried, grinded, and finally pressed to a wax briquette. The briquette is analyzed using wave-length dispersive X-ray fluorescence with fundamental parameters evaluation. The recovery of 55 elements were tested by preparing samples with known compositions using different alloys, pure metals or elements, oxides, salts and solutions of dissolved compounds. It was found that the methodology was applicable to 49 elements including Na, Mg, Al, Si, P, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, Cs, Ba, La, Ce, Ta, W, Re, Ir, Pt, Au, Tl, Pb, Bi, and Th, that all had recoveries >0.8. 6 elements were lost by volatilization, including Br, I, Os, and Hg that were completely lost, and S and Ge that were partly lost. Since all lanthanides are chemically similar to La and Ce, all actinides are chemically similar to Th, and Hf is chemically similar to Zr, it is likely that the method is applicable to 77 elements. By using an internal standard such as strontium, added as strontium nitrate, samples containing relatively high concentrations of elements not measured by XRF (hydrogen to fluorine), e.g. samples containing plastics, can be analyzed. PMID:22841048

  1. Trypanosoma cruzi Proline Transport Presents a Cell Density-dependent Regulation.

    PubMed

    Sayé, Melisa; Miranda, Mariana R; Reigada, Chantal; Pereira, Claudio A

    2016-07-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is mainly obtained from extracellular medium by transport proteins, in this work we studied the regulation of the T. cruzi proline transporter TcAAAP069. Proline uptake and intracellular concentration presented oscillations during epimastigote growth phases, increasing during the early exponential phase (322 pmol/min) and decreasing to undetectable levels during the late exponential phase. Transporter expression rate correlated with proline uptake, and its subcellular localization alternated from both, the plasma membrane and close to the flagellar pocket, when the transport is higher, to only the flagellar pocket region, when the transport decreased until proline uptake and TcAAAP069 protein became undetectable at the end of the growth curve. Interestingly, when parasites were treated with conditioned medium or were concentrated to artificially increase the culture density, the proline transport was completely abolished resembling the effects observed in late exponential phase. These data highlight for the first time the existence of a density-associated regulation of relevant physiological processes such as proline metabolism. PMID:26750517

  2. Cotton Effect in Copper-Proline Complexes in the Visible Region

    ERIC Educational Resources Information Center

    Volkov, Victor; Pfister, Rolf

    2005-01-01

    The electronic properties of Cu(II) complex with proline are considered to demonstrate the Cotton effect in the visible region. A series of experiments in optical rotatory dispersion spectroscopy with free D- and L-proline and their complexes with the Cu(II) ion in aqueous solution is suggested.

  3. A proline-derived transannular N-cap for nucleation of short α-helical peptides.

    PubMed

    Tian, Yuan; Wang, Dongyuan; Li, Jingxu; Shi, Chuan; Zhao, Hui; Niu, Xiaogang; Li, Zigang

    2016-07-28

    We report herein a proline-derived transannular N-cap as a helix nucleating template in diverse bio-related peptide sequences via macrolactamization on resin. This approach takes advantage of synergistic stabilization effects of both N-capping properties of proline and substitution of a main chain hydrogen bond with a covalent bond. PMID:27357119

  4. Influence of proline position upon the ion channel activity of alamethicin.

    PubMed Central

    Kaduk, C; Duclohier, H; Dathe, M; Wenschuh, H; Beyermann, M; Molle, G; Bienert, M

    1997-01-01

    Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable. PMID:9129817

  5. Protective effects of proline against cadmium toxicity in micropropagated hyperaccumulator, Solanum nigrum L.

    PubMed

    Xu, Jin; Yin, HengXia; Li, Xia

    2009-02-01

    Solanum nigrum is a newly discovered Cd-hyperaccumulator. In the present study, the protective effects of proline against cadmium toxicity of callus and regenerated shoots of S. nigrum are investigated based on a high frequency in vitro shoot regeneration system. Proline pretreatment reduces the reactive oxygen species levels and protects the plasma membrane integrity of callus under cadmium stress, and therefore improves the cadmium tolerance in S. nigrum. Inductively coupled plasma mass spectroscopy analysis shows that exogenous proline increases the cadmium accumulation in callus and regenerated shoots of S. nigrum. Further analysis indicates that the improvement of cadmium tolerance caused by proline pretreatment is correlated with an increase of superoxide dismutase and catalase activity and intracellular total glutathione content. The interaction between proline and enzymic or non-enzymic antioxidants is discussed. PMID:19043719

  6. Prediction and Biochemical Demonstration of a Catabolic Pathway for the Osmoprotectant Proline Betaine

    PubMed Central

    Kumar, Ritesh; Zhao, Suwen; Vetting, Matthew W.; Wood, B. McKay; Sakai, Ayano; Cho, Kyuil; Solbiati, José; Almo, Steven C.; Sweedler, Jonathan V.; Jacobson, Matthew P.; Gerlt, John A.; Cronan, John E.

    2014-01-01

    ABSTRACT Through the use of genetic, enzymatic, metabolomic, and structural analyses, we have discovered the catabolic pathway for proline betaine, an osmoprotectant, in Paracoccus denitrificans and Rhodobacter sphaeroides. Genetic and enzymatic analyses showed that several of the key enzymes of the hydroxyproline betaine degradation pathway also function in proline betaine degradation. Metabolomic analyses detected each of the metabolic intermediates of the pathway. The proline betaine catabolic pathway was repressed by osmotic stress and cold stress, and a regulatory transcription factor was identified. We also report crystal structure complexes of the P. denitrificans HpbD hydroxyproline betaine epimerase/proline betaine racemase with l-proline betaine and cis-hydroxyproline betaine. PMID:24520058

  7. Multi-Biomarkers for Early Detection of Type 2 Diabetes, Including 10- and 12-(Z,E)-Hydroxyoctadecadienoic Acids, Insulin, Leptin, and Adiponectin

    PubMed Central

    Umeno, Aya; Yoshino, Kohzoh; Hashimoto, Yoshiko; Shichiri, Mototada; Kataoka, Masatoshi; Yoshida, Yasukazu

    2015-01-01

    We have previously found that fasting plasma levels of totally assessed 10- and 12-(Z,E)-hydroxyoctadecadienoic acid (HODE) correlated well with levels of glycated hemoglobin (HbA1c) and glucose during oral glucose tolerance tests (OGTT); these levels were determined via liquid chromatography—mass spectrometry after reduction and saponification. However, 10- and 12-(Z,E)-HODE alone cannot perfectly detect early impaired glucose tolerance (IGT) and/or insulin resistance, which ultimately lead to diabetes. In this study, we randomly recruited healthy volunteers (n = 57) who had no known history of any diseases, and who were evaluated using the OGTT, the HODE biomarkers, and several additional proposed biomarkers, including retinol binding protein 4 (RBP4), adiponectin, leptin, insulin, glycoalbumin, and high sensitivity-C-reactive protein. The OGTT revealed that our volunteers included normal individuals (n = 44; Group N), “high-normal” individuals (fasting plasma glucose 100–109 mg/dL) with IGT (n = 11; Group HN+IGT), and diabetic individuals (n = 2; Group D). We then used these groups to evaluate the potential biomarkers for the early detection of type 2 diabetes. Plasma levels of RBP4 and glycoalbumin were higher in Group HN+IGT, compared to those in Group N, and fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids were significantly correlated with levels of RBP4 (p = 0.003, r = 0.380) and glycoalbumin (p = 0.006, r = 0.316). Furthermore, we developed a stepwise multiple linear regression models to predict the individuals’ insulin resistance index (the Matsuda Index 3). Fasting plasma levels of 10- and 12-(Z,E)-HODE/linoleic acids, glucose, insulin, and leptin/adiponectin were selected as the explanatory variables for the models. The risks of type 2 diabetes, early IGT, and insulin resistance were perfectly predicted by comparing fasting glucose levels to the estimated Matsuda Index 3 (fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids, insulin

  8. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

    PubMed Central

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-01-01

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  9. Effect of l-Proline on Sake Brewing and Ethanol Stress in Saccharomyces cerevisiae

    PubMed Central

    Takagi, Hiroshi; Takaoka, Miki; Kawaguchi, Akari; Kubo, Yoshito

    2005-01-01

    During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular l-proline in cells of S. cerevisiae grown under ethanol stress. An l-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1D154N, which encodes the Asp154Asn mutant γ-glutamyl kinase. This mutation increases the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase, which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the l-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of l-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for l-proline utilization, and replacing the wild-type PRO1 allele with the pro1D154N allele. The resultant strain accumulated l-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate l-proline to brew sake containing five times more l-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles. PMID:16332860

  10. Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

    PubMed Central

    Hoffmann, Tamara; von Blohn, Carsten; Stanek, Agnieszka; Moses, Susanne; Barzantny, Helena

    2012-01-01

    Bacillus subtilis synthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, an opuE mutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of the opuE mutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of the opuE mutant was considerably lower than that of its opuE+ parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels of B. subtilis participated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered. PMID:22685134

  11. Enteric Bacterial Metabolites Propionic and Butyric Acid Modulate Gene Expression, Including CREB-Dependent Catecholaminergic Neurotransmission, in PC12 Cells - Possible Relevance to Autism Spectrum Disorders

    PubMed Central

    Nankova, Bistra B.; Agarwal, Raj; MacFabe, Derrick F.; La Gamma, Edmund F.

    2014-01-01

    Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as

  12. Evaluation of the role of proline residues flanking the RGD motif of dendroaspin, an inhibitior of platelet aggregation and cell adhesion.

    PubMed Central

    Lu, X; Sun, Y; Shang, D; Wattam, B; Egglezou, S; Hughes, T; Hyde, E; Scully, M; Kakkar, V

    2001-01-01

    The effect of a panel of proline mutants of dendroaspin, an inhibitor of platelet aggregation and cell adhesion, including A(42)-dendroaspin, A(47)-dendroaspin, A(49)-dendroaspin, A(42,47)-dendroaspin and A(47,49)-dendroaspin, was investigated using platelet-aggregation and cell-adhesion assays. Here we show that a single alanine-for-proline substitution did not affect potency when measured as the ability either to inhibit platelet aggregation induced by ADP (IC(50) approximately 170 nM) or to block transfected A375-SM cell adhesion to fibrinogen in the presence of Mn(2+) as compared with wild-type dendroaspin. By comparison, double proline substitution with alanines significantly reduced the potency in both assays by approx. 5-8-fold. These observations, therefore, suggest that proline residues flanking the RGD motif in dendroaspin and other RGD-containing venom proteins, e.g. disintegrins, may contribute to maintaining a favourable conformation for the solvent-exposed RGD site for its recognition by integrin receptors. PMID:11311124

  13. Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

    PubMed

    Tada, Y; Yano, N; Takahashi, H; Yuzawa, K; Ando, H; Kubo, Y; Nagasawa, A; Ohashi, N; Ogata, A; Nakae, D

    2010-10-01

    L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions. PMID:20447433

  14. Overexpression of Ste20-related proline/alanine-rich kinase exacerbates experimental colitis in mice.

    PubMed

    Yan, Yutao; Laroui, Hamed; Ingersoll, Sarah A; Ayyadurai, Saravanan; Charania, Moiz; Yang, Stephen; Dalmasso, Guillaume; Obertone, Tracy S; Nguyen, Hang; Sitaraman, Shanthi V; Merlin, Didier

    2011-08-01

    Inflammatory bowel disease, mainly Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease. PMID:21705622

  15. Sensitive determination of D-amino acids in mammals and the effect of D-amino-acid oxidase activity on their amounts.

    PubMed

    Hamase, Kenji; Konno, Ryuichi; Morikawa, Akiko; Zaitsu, Kiyoshi

    2005-09-01

    The determination of small amounts of D-amino acids in mammalian tissues is still a challenging theme in the separation sciences. In this review, various gas-chromatographic and high-performance liquid chromatographic methods are discussed including highly selective and sensitive column-switching procedures. Based on these methods, the distributions of D-aspartic acid, D-serine, D-alanine, D-leucine and D-proline have been clarified in the mouse brain. As the regulation mechanisms of D-amino acid amounts in mammals, we focused on the D-amino-acid oxidase, which catalyzes the degradation of D-amino acids. Using the mutant mouse strain lacking D-amino-acid oxidase activity, the effects of the enzymatic activity on the amounts and distributions of various D-amino acids have been investigated. PMID:16141519

  16. A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V105DLP108 Immunoactivity.

    PubMed

    Wang, Heng; Ji, Fangxiao; Liang, Huanbin; Gu, Honglang; Ning, Zhangyong; Liu, Rongchang; Zhang, Guihong

    2015-01-01

    The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region. PMID:26177202

  17. Potentiometric determination of the dissociation constants of an asymmetric sorbent containing l-proline, and the stability constants of its Cu(II) complexes.

    PubMed

    Zolotarev, Y A; Kurganov, A A; Davankov, V A

    1978-09-01

    The dissociation constants of the carboxyl groups (pK(a1) = 2.2, n = 1.8) and amino groups (pK(a2) = 9.5, n(2) = 1.6) of a sorbent prepared by reacting l-proline with a cross-linked chloromethylated styrene polymer have been determined by potentiometric titration. The potentiometrically measured stability constants of the Cu(II) complexes of the resin (logbeta(1) = 6.9 and log beta(2) = 12.4) were found to be close to the values for the Cu(II) complexes of N-benzyl-l-proline. For complexed resins of alpha-amino-acid type the pH-values of decomplexation do not appear to be directly correlated with the stability constants. PMID:18962307

  18. Formation of intermediate cementum. III: /sup 3/H-tryptophan and /sup 3/H-proline uptake into the epithelial root sheath of Hertwig in vitro

    SciTech Connect

    Lindskog, S.; Hammarstroem, L.

    1982-01-01

    The intermediate cementum is a narrow, mineralized tissue between the cementum and dentin. Recent studies have shown that this tissue is mineralized by the epithelial root sheath in a way similar to the mineralization of the innermost layer of aprismatic enamel. In the present investigation uptake of proline and tryptophan into the epithelial root sheath was studied with autoradiography. Tryptophan is an amino acid that is incorporated into enamel matrix but not into collagen. Tryptophan uptake was significant in the whole epithelial root sheath, but not into the odontoblasts or predentin. Proline was incorporated into the predentin while the root sheath was unlabeled. This indicated that the matrix of the intermediate cementum was formed by the epithelial root sheath of Hertwig, and that this matrix was a noncollagenous matrix possibly of the same nature as enamel matrix.

  19. Comparative study of the use of sarcosine, proline and glycine as acrylamide inhibitors in ripe olive processing.

    PubMed

    Sánchez, Antonio Higinio; Beato, Víctor Manuel; López-López, Antonio; Montaño, Alfredo

    2014-01-01

    The main purpose of this study was to evaluate the inhibitory effect on acrylamide (AA) formation and the impact on sensory characteristics in ripe olives of three selected amino acids (sarcosine, proline and glycine), which previously showed high AA inhibition rates in an olive model system. Each amino acid was separately added to packing solutions to give 100 or 200 mM at equilibrium, prior to a sterilisation treatment at 121°C. The results showed that sarcosine at 100 mM may be a good candidate for reducing the AA content in ripe olives with a limited effect on sensory characteristics. Studies with a model solution of AA and sarcosine heated at 121°C for 30 min suggested that the main mechanism for the inhibitory effect of sarcosine on AA formation was the Michael reaction. PMID:24294998

  20. Functional reconstitution of a purified proline permease from Candida albicans: interaction with the antifungal cispentacin.

    PubMed

    Jethwaney, D; Höfer, M; Khaware, R K; Prasad, R

    1997-02-01

    We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent Km for L-proline binding to the purified protein was 153 microM. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaleimide, were very similar to those of intact cells, The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-proline binding and translocation in reconstituted proteoliposomes. However, the uptake of L-proline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent Km of 40 microM. PMID:9043117

  1. Plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to Bacillus subtilis.

    PubMed

    Bashir, Abdallah; Hoffmann, Tamara; Kempf, Bettina; Xie, Xiulan; Smits, Sander H J; Bremer, Erhard

    2014-10-01

    L-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the stress-protective potential of the plant-derived L-proline derivatives N-methyl-L-proline, L-proline betaine (stachydrine), trans-4-L-hydroxproline and trans-4-hydroxy-L-proline betaine (betonicine) for cells challenged by high salinity or extremes in growth temperature. l-Proline betaine and betonicine conferred salt stress protection, but trans-4-L-hydroxyproline and N-methyl-L-proline was unable to do so. Except for L-proline, none of these compounds served as a nutrient for B. subtilis. L-Proline betaine was a considerably better osmostress protectant than betonicine, and its import strongly reduced the l-proline pool produced by B. subtilis under osmotic stress conditions, whereas a supply of betonicine affected the L-proline pool only modestly. Both compounds downregulated the transcription of the osmotically inducible opuA operon, albeit to different extents. Mutant studies revealed that L-proline betaine was taken up via the ATP-binding cassette transporters OpuA and OpuC, and the betaine-choline-carnitine-transporter-type carrier OpuD; betonicine was imported only through OpuA and OpuC. L-Proline betaine and betonicine also served as temperature stress protectants. A striking difference between these chemically closely related compounds was observed: L-proline betaine was an excellent cold stress protectant, but did not provide heat stress protection, whereas the reverse was true for betonicine. Both compounds were primarily imported in temperature-challenged cells via the high-capacity OpuA transporter. We developed an in silico model for the OpuAC-betonicine complex based on the crystal structure of the OpuAC solute receptor complexed with L-proline betaine. PMID:25012968

  2. Functional characterization and expression analysis of rice δ1-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism

    PubMed Central

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; Funck, Dietmar

    2015-01-01

    While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ1-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD+ as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism. PMID:26300893

  3. Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

    PubMed

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; Funck, Dietmar

    2015-01-01

    While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism. PMID:26300893

  4. Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

    PubMed Central

    Rinehart, C A; Hubbard, J S

    1976-01-01

    When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate. PMID:956126

  5. [Amino acids in saliva].

    PubMed

    Klinger, G; Gruhn, K

    1984-01-01

    Total amino acids in saliva and free and peptide-bound amino acids from 21 saliva samples were determined. The contents of amino acids was 25 mmol/1; total nitrogen content was 78-80 mmol/1. Amino acids consist of Prolin in 25%. Some patients were examined before and after application of the depot estrogen ethinyl estradiosulfonat, which stimulates the assimilation of protein. After application, amino acids increased and the authors found a shift between the single amino acids. Estrogen medication induced an increase in proteins with the character of collagens. Clinical effects are discussed. (author's modified) PMID:6240853

  6. Coordinated activation of the Rac-GAP β2-chimaerin by an atypical proline-rich domain and diacylglycerol.

    PubMed

    Gutierrez-Uzquiza, Alvaro; Colon-Gonzalez, Francheska; Leonard, Thomas A; Canagarajah, Bertram J; Wang, HongBin; Mayer, Bruce J; Hurley, James H; Kazanietz, Marcelo G

    2013-01-01

    Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region. PMID:23673634

  7. Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

    PubMed Central

    Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

    2014-01-01

    Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to “trapping and clamping” by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same “clamping” phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

  8. Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis.

    PubMed

    Méjean, Annick; Dalle, Klervi; Paci, Guillaume; Bouchonnet, Stéphane; Mann, Stéphane; Pichon, Valérie; Ploux, Olivier

    2016-07-22

    LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the ana cluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment. PMID:27340731

  9. Bio-mimicking of proline-rich motif applied to carbon nanotube reveals unexpected subtleties underlying nanoparticle functionalization.

    PubMed

    Zhang, Yuanzhao; Jimenez-Cruz, Camilo A; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

    2014-01-01

    Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to "trapping and clamping" by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same "clamping" phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

  10. A highly acid-resistant novel strain of Lactobacillus johnsonii No. 1088 has antibacterial activity, including that against Helicobacter pylori, and inhibits gastrin-mediated acid production in mice

    PubMed Central

    Aiba, Yuji; Nakano, Yasuhiro; Koga, Yasuhiro; Takahashi, Kenji; Komatsu, Yasuhiko

    2015-01-01

    A novel strain of Lactobacillus johnsonii No. 1088 was isolated from the gastric juice of a healthy Japanese male volunteer, and characterized for its effectiveness in the stomach environment. Lactobacillus johnsonii No. 1088 was found to have the strongest acid resistance among several lactobacilli examined (>10% of cells survived at pH 1.0 after 2 h), and such a high acid resistance property was a specific characteristic of this strain of L. johnsonii. When cultured with various virulent bacteria, L. johnsonii No. 1088 inhibited the growth of Helicobacter pylori,Escherichia coli O-157, Salmonella Typhimurium, and Clostridium difficile, in which case its effectiveness was more potent than that of a type strain of L. johnsonii,JCM2012. In addition to its effect in vitro, L. johnsonii No. 1088 inhibited the growth of H. pylori in human intestinal microbiota-associated mice in both its live and lyophilized forms. Moreover, L. johnsonii No. 1088 suppressed gastric acid secretion in mice via decreasing the number of gastrin-positive cells in the stomach. These results taken together suggest that L. johnsonii No. 1088 is a unique lactobacillus having properties beneficial for supporting H. pylori eradication by triple therapy including the use of a proton pump inhibitor (PPI) and also for prophylaxis of gastroesophageal reflux disease possibly caused after H. pylori eradication as a side effect of PPI. PMID:25771812

  11. Nicotianamine and histidine/proline are, respectively, the most important copper chelators in xylem sap of Brassica carinata under conditions of copper deficiency and excess.

    PubMed

    Irtelli, B; Petrucci, W A; Navari-Izzo, F

    2009-01-01

    The effect of two different copper conditions (deficiency and excess) on the amino acid composition in B. carinata xylem sap was analysed. When the Cu in the nutrient solution was increased from 0.12 to 2.5 or 5 microM, the concentrations of histidine, threonine, glutamine, proline, methionine, and glycine were much increased in the xylem sap. When Cu was made deficient in the nutrient solution by decreasing its concentration from 0.12 microM to 0 microM, nicotianamine, glutamine, and threonine were significantly increased in the xylem sap. Aqueous solutions containing different Cu-amino acid complexes (simulated saps) responded in a specific way to the changes in pH, providing a signature that was used to evaluate, by comparison with the real xylem sap, the importance of each amino acid in the xylem transport of Cu. For a single amino acid, the free solution Cu(2+) concentration versus pH titration curves for histidine and proline were the most similar to that for xylem under Cu excess. Under Cu deficiency, this Cu concentration versus pH titration curve appeared to be very similar to that for nicotianamine. It is concluded that increased Cu concentrations induced the selective synthesis of certain amino acids in the sap, of which histidine and proline are the most important. Under Cu deficiency, the concentration of nicotianamine was induced the most. The fact that nicotianamine is induced under Cu starvation and not under Cu excess, is in contrast to similar studies indicating species-specific reactions. However, the induction of nicotianamine under Cu starvation is in line with recent molecular data of the role of nicotianamine in intracellular Cu delivery. PMID:19033552

  12. Ornithine-δ-aminotransferase is essential for Arginine Catabolism but not for Proline Biosynthesis

    PubMed Central

    Funck, Dietmar; Stadelhofer, Bettina; Koch, Wolfgang

    2008-01-01

    Background Like many other plant species, Arabidopsis uses arginine (Arg) as a storage and transport form of nitrogen, and proline (Pro) as a compatible solute in the defence against abiotic stresses causing water deprivation. Arg catabolism produces ornithine (Orn) inside mitochondria, which was discussed controversially as a precursor for Pro biosynthesis, alternative to glutamate (Glu). Results We show here that ornithine-δ-aminotransferase (δOAT, At5g46180), the enzyme converting Orn to pyrroline-5-carboxylate (P5C), is localised in mitochondria and is essential for Arg catabolism. Wildtype plants could readily catabolise supplied Arg and Orn and were able to use these amino acids as the only nitrogen source. Deletion mutants of δOAT, however, accumulated urea cycle intermediates when fed with Arg or Orn and were not able to utilize nitrogen provided as Arg or Orn. Utilisation of urea and stress induced Pro accumulation were not affected in T-DNA insertion mutants with a complete loss of δOAT expression. Conclusion Our findings indicate that δOAT feeds P5C exclusively into the catabolic branch of Pro metabolism, which yields Glu as an end product. Conversion of Orn to Glu is an essential route for recovery of nitrogen stored or transported as Arg. Pro biosynthesis occurs predominantly or exclusively via the Glu pathway in Arabidopsis and does not depend on Glu produced by Arg and Orn catabolism. PMID:18419821

  13. Effect of exogenous proline, betaine, and carnitine on growth of Listeria monocytogenes in a minimal medium.

    PubMed

    Beumer, R R; Te Giffel, M C; Cox, L J; Rombouts, F M; Abee, T

    1994-04-01

    Three Listeria monocytogenes strains isolated from food or food-processing environments were used to assess the response of this species to salinity in a chemically defined minimal medium. Growth in a minimal medium containing five essential amino acids and glucose as a carbon and energy source was comparable to growth in a rich medium (brain heart infusion broth). In the absence and presence of 3% NaCl the final cell numbers reached in minimal medium were 10(9) and 10(7) CFU/ml, respectively. Growth under the latter conditions could not be detected by spectrophotometry by measuring A660. Apparently, this technique was not suitable for these experiments since the detection level was > 10(7) CFU/ml. Exogenously added proline (10 mM), trimethylglycine (betaine) (1 mM), and beta-hydroxy-gamma-N-trimethyl aminobutyrate (carnitine) (1 mM) significantly stimulated growth under osmotic stress conditions in minimal medium at both 37 and 10 degrees C. Betaine and carnitine are present in foods derived from plants and animals, respectively. Therefore, these compounds can contribute significantly to growth of L. monocytogenes in various foods at high osmolarities. PMID:8017923

  14. N-all-trans-retinoyl-L-proline inhibits metastatic potential of hepatocellular carcinoma cells.

    PubMed

    Wu, Xing Zhong; Shi, Peng-Chen; Hu, Ping; Chen, Yi; Ding, Sheng-Song

    2006-08-01

    Tumor metastasis is usually a serious problem in tumor patients because of the lack of therapeutic approaches. A new compound, N-all-trans-retinoyl-L-proline (ATRP), has been developed and its metastasis inhibition activity has been studied. Low concentrations of ATRP have already been found to inhibit hepatocellular carcinoma cells (HCC) in a dose- and time-dependent manner by inducing the expression of p27(kip). We found that ATRP inhibited metastasis-associated behaviors in Hep3B cells, such as cell migration, invasion, collagen adhesion and gelatinase expression, more significantly than retinoic acid. Further, such inhibitory activities were observed in the regulation of cellular surface fucosylated epitope functions, such as binding of ulex europaeus lectin, expression of Lewis x, y and b, and activity of alpha1,3 fucosyltransferase. Hep3B cells pretreated with ATRP showed a significantly reduced incidence of experimental intrahepatic metastasis in nude mice. We conclude that ATRP is an alternative inhibitor and potential therapeutic agent for HCC metastasis with a different mechanism of action from ATRP. PMID:16806999

  15. Knockout of Ste20-like proline/alanine-rich kinase (SPAK) attenuates intestinal inflammation in mice.

    PubMed

    Zhang, Yuchen; Viennois, Emilie; Xiao, Bo; Baker, Mark T; Yang, Stephen; Okoro, Ijeoma; Yan, Yutao

    2013-05-01

    Inflammatory bowel diseases are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Herein, SPAK knockout (KO) C57BL/6 mice exhibited significant increases in intestinal transepithelial resistance, a marked decrease in paracellular permeability to fluorescence isothiocyanate-dextran, and altered apical side tight junction sodium ion selectivity, compared with wild-type mice. Furthermore, the expression of junction protein, claudin-2, decreased. In contrast, expressions of occludin, E-cadherin, β-catenin, and claudin-5 increased significantly, whereas no obvious change of claudin-1, claudin-4, zonula occludens protein 1, and zonula occludens protein 2 expressions was observed. In murine models of colitis induced by dextran sulfate sodium and trinitrobenzene sulfuric acid, KO mice were more tolerant than wild-type mice, as demonstrated by colonoscopy features, histological characteristics, and myeloperoxidase activities. Consistent with these findings, KO mice showed increased IL-10 levels and decreased proinflammatory cytokine secretion, ameliorated bacterial translocation on treatment with dextran sulfate sodium, and regulation of with no lysine (WNK) kinase activity. Together, these features may reduce epithelial permeability. In conclusion, SPAK deficiency increases intestinal innate immune homeostasis, which is important for control or attenuation of pathological responses in inflammatory bowel diseases. PMID:23499375

  16. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

    PubMed Central

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S.; Harlow, Mark L.

    2015-01-01

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission. PMID:26446566

  17. Association of purified skeletal-muscle AMP deaminase with a histidine-proline-rich-glycoprotein-like molecule.

    PubMed Central

    Ranieri-Raggi, M; Montali, U; Ronca, F; Sabbatini, A; Brown, P E; Moir, A J; Raggi, A

    1997-01-01

    Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea atpH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75-80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine-proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472-477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed. PMID:9307011

  18. Metabolic evidence of vitamin B-12 deficiency, including high homocysteine and methylmalonic acid and low holotranscobalamin, is more pronounced in older adults with elevated plasma folate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: An analysis of data from the National Health and Nutrition Examination Survey indicated that in older adults exposed to folic acid fortification, the combination of low serum vitamin B-12 and elevated folate is associated with higher concentrations of homocysteine and methylmalonic acid ...

  19. Chemiluminescence Detection of Serine, Proline, Glycine, Asparagine, Leucine, and Histidine by Using Corresponding Aminoacyl-tRNA Synthetases as Recognition Elements.

    PubMed

    Kugimiya, Akimitsu; Fukada, Rie

    2015-06-01

    Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. However, currently available methods are time consuming, potentially delaying diagnosis. Therefore, the development of more rapid analytical tools is needed. In this study, a chemiluminescence detection method for amino acids was developed, and the conditions for the enzyme reaction and assay were examined. For the recognition of each amino acid (here, serine, proline, glycine, asparagine, leucine, and histidine), the corresponding aminoacyl-tRNA synthetase (aaRS) was employed, and multiple enzymatic reactions were combined with a luminol chemiluminescence reaction. This method provided selective quantification from 1 to 20 μM for serine, proline, glycine, and leucine; 1 to 60 μM for asparagine; and 1 to 150 μM for histidine. This assay, which utilized aaRSs for the detection of amino acids, could be useful for simple and rapid analysis of amino acids in clinical diagnostics. PMID:25935222

  20. Regulation of amino acid transporters in pluripotent cell populations in the embryo and in culture; novel roles for sodium-coupled neutral amino acid transporters.

    PubMed

    Tan, Boon Siang Nicholas; Rathjen, Peter D; Harvey, Alexandra J; Gardner, David K; Rathjen, Joy

    2016-08-01

    The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment, and inappropriate concentrations of amino acids, or the loss of amino acid-sensing mechanisms, can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells, a cell population derived from the blastocyst, has been shown in culture. l-proline acts as a signalling molecule, exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2, Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo, reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore, SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation, an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. PMID:27373508

  1. Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors*

    PubMed Central

    Rienzo, Matthew; Rocchi, Angela R.; Threatt, Stephanie D.; Dougherty, Dennis A.; Lummis, Sarah C. R.

    2016-01-01

    Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC. PMID:26668320

  2. Influences of Proline and Cysteine Residues on Fragment Yield in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Smargiasso, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2014-06-01

    Matrix-assisted laser desorption/ionization in-source decay produces highly informative fragments for the sequencing of peptides/proteins. Among amino acids, cysteine and proline residues were found to specifically influence the fragment yield. As they are both frequently found in small peptide structures for which de novo sequencing is mandatory, the understanding of their specific behaviors would allow useful fragmentation rules to be established. In the case of cysteine, a c•/ w fragment pair originating from Xxx-Cys is formed by side-chain loss from the cysteine residue. The presence of a proline residue contributes to an increased yield of ISD fragments originating from N-Cα bond cleavage at Xxx1-Xxx2Pro, which is attributable to the cyclic structure of the proline residue. Our results suggest that the aminoketyl radical formed by MALDI-ISD generally induces the homolytic N-Cα bond cleavage located on the C-terminal side of the radical site. In contrast, N-Cα bond cleavage at Xxx-Pro produces no fragments and the N-Cα bond at the Xxx1-Xxx2Pro bond is alternatively cleaved via a heterolytic cleavage pathway.

  3. Amino Acid and Protein Metabolism in Bermuda Grass During Water Stress 12

    PubMed Central

    Barnett, N. M.; Naylor, A. W.

    1966-01-01

    The ability of Arizona Common and Coastal Bermuda grass [Cynodon dactylon (L.) Pers.] to synthesize amino acids and proteins during water stress was investigated. Amino acids were continually synthesized during the water stress treatments, but protein synthesis was inhibited and protein levels decreased. Water stress induced a 10- to 100-fold accumulation of free proline in shoots and a 2- to 6-fold accumulation of free asparagine, both of which are characteristic responses of water-stressed plants. Valine levels increased, and glutamic acid and alanine levels decreased. 14C labeling experiments showed that free proline turns over more slowly than any other free amino acid during water stress. This proline is readily synthesized and accumulated from glutamic acid. It is suggested that during water stress free proline functions as a storage compound. No significant differences were found in the amino acid and protein metabolism of the 2 varieties of Bermuda grass. PMID:16656387

  4. cis–trans-Amide isomerism of the 3,4-dehydroproline residue, the ‘unpuckered’ proline

    PubMed Central

    2016-01-01

    Summary Proline (Pro) is an outstanding amino acid in various biochemical and physicochemical perspectives, especially when considering the cis–trans isomerism of the peptidyl-Pro amide bond. Elucidation of the roles of Pro in chemical or biological systems and engineering of its features can be addressed with various Pro analogues. Here we report an experimental work investigating the basic physicochemical properties of two Pro analogues which possess a 3,4-double bond: 3,4-dehydroproline and 4-trifluoromethyl-3,4-dehydroproline. Both indicate a flat pyrroline ring in their crystal structures, in agreement with previous theoretical calculations. In solution, the peptide mimics exhibit an almost unchanged equilibrium of the trans/cis ratios compared to that of Pro and 4-trifluoromethylproline derivatives. Finally we demonstrate that the 3,4-double bond in the investigated structures leads to an increase of the amide rotational barriers, presumably due to an interplay with the transition state. PMID:27340450

  5. N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

    PubMed

    Manconi, Barbara; Cabras, Tiziana; Sanna, Monica; Piras, Valentina; Liori, Barbara; Pisano, Elisabetta; Iavarone, Federica; Vincenzoni, Federica; Cordaro, Massimo; Faa, Gavino; Castagnola, Massimo; Messana, Irene

    2016-05-01

    In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213. PMID:26991339

  6. Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2.

    PubMed

    Perlikowska, Renata; Piekielna, Justyna; Mazur, Marzena; Koralewski, Robert; Olczak, Jacek; do Rego, Jean-Claude; Fichna, Jakub; Modranka, Jakub; Janecki, Tomasz; Janecka, Anna

    2014-09-01

    In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the μ-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-β-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies. PMID:25047937

  7. Proline rich-oligopeptides: diverse mechanisms for antihypertensive action.

    PubMed

    Morais, Katia L P; Ianzer, Danielle; Miranda, José Rodolfo R; Melo, Robson L; Guerreiro, Juliano R; Santos, Robson A S; Ulrich, Henning; Lameu, Claudiana

    2013-10-01

    Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b and -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches. PMID:23933300

  8. Analysis of the free amino acid content in pollen of nine Asteraceae species of known allergenic activity.

    PubMed

    Mondal, A K; Parui, S; Mandal, S

    1998-01-01

    The study reports the free amino acid composition of the pollen of nine members of the family Asteraceae, i.e. Ageratum conyzoides L., Blumea oxyodonta DC., Eupatorium odoratum L., Gnaphalium indicum L., Mikania scandens Willd., Parthenium hysterophorus L., Spilanthes acmella Murr., Vernonia cinerea (L.) Lees. and Xanthium strumarium L. by thin layer chromatography. The amino acid content was found to vary from 0.5-4.0% of the total dry weight. Fourteen amino acids were identified, among which amino-n-butyric acid, aspartic acid and proline were present in almost all pollen samples. The other major amino acids present in free form included arginine, cystine, glutamic acid, glycine, isoleucine, leucine, methionine, ornithine, tryptophan and tyrosine. PMID:9852488

  9. Human milk nonprotein nitrogen components: changing patterns of free amino acids and urea in the course of early lactation.

    PubMed

    Harzer, G; Franzke, V; Bindels, J G

    1984-08-01

    Free amino acids and urea were analyzed in 78 human milk samples obtained during the first 5 wk of lactation from 10 mothers delivering at term. Significant differences (p less than 0.05) in the concentrations between colostral and mature milk were found for glutamic acid, glutamine, alanine, glycine, cystine, and phosphoethanolamine which increased, and with serine, phosphoserine, aspartic acid + asparagine, arginine, lysine, isoleucine, phenylalanine, proline, methionine, tryptophan, and beta-alanine which decreased. Some of these changes occurred within the first 5 days of lactation, so that differences between transitional and mature milk became negligible (glutamic acid, alanine, and serine, aspartic acid + asparagine, lysine, isoleucine, methionine, tryptophan, respectively). No significant differences between any of the three stages of lactation were found regarding the concentrations of total free amino acids, urea, taurine, threonine, valine, leucine, histidine, and tyrosine. Possible relevances for free amino acids, including nonprotein ones, in human milk are discussed. PMID:6147084

  10. Contrasting changes in transport of glycine vs proline at fertilization and during preimplantation development of mouse embryos

    SciTech Connect

    Haghighat, N.; Van Winkle, L.J.

    1987-05-01

    Na/sup +/ dependent glycine transport decreased steadily during cleavage of mouse embryos and then increased dramatically upon formation of early blastocysts (approx. 80 h post coitus), while proline uptake increased several-fold upon fertilization of eggs and then decreased through the blastocyst stage. V/sub max/ and K/sub m/ values for Gly transport in unfertilized eggs, 8-cell embryos and blastocysts were 9.5, 4.0 and 20 fmol. (egg or embryo)/sup -1/ min/sup -1/ and 93, 94 and 30 ..mu..M, respectively. Gly transport in 2-cell embryos was Cl-dependent and sigmoidally related to the (Na/sup +/), whereas Cl/sup -/-dependent Gly uptake was linearly related to (Na/sup +/) in blastocysts. Uptake of 1.0 ..mu..M (/sup 3/H)Gly in cleavage stages was inhibited by 10 mM sarcosine but not by Glu, Ser, or Lys and only weakly by MeAIB, BCO and pipecolate, whereas BCO, Ser, Lys, Pipecolate, Ala and Leu strongly inhibited transport in blastocysts; and Lys inhibition was unequivocally competitive (K/sub i/ approx. 70 ..mu..M). Na/sup +/-dependent uptake of 0.9 ..mu..M L-(/sup 3/H)Pro was inhibited strongly by only pipecolate in unfertilized eggs, but MeAIB and BCO were also strong inhibitors in zygotes. Fertilization was also accompanied by an increase in the V/sub max/ (0.9 vs 6.7 fmol. cell/sup -1/ min/sup -1/) and K/sub m/ (66 vs 230 ..mu..m) values for proline transport. This appears to be the first report of a change in amino acid transport upon fertilization of mammalian eggs, although transport of several amino acids increases dramatically in sea urchin zygotes.

  11. Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)*

    PubMed Central

    Moxley, Michael A.; Sanyal, Nikhilesh; Krishnan, Navasona; Tanner, John J.; Becker, Donald F.

    2014-01-01

    PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to Δ1-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ1-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD+-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed. PMID:24352662

  12. Sinorhizobium meliloti Chemoreceptor McpU Mediates Chemotaxis toward Host Plant Exudates through Direct Proline Sensing

    PubMed Central

    Webb, Benjamin A.; Hildreth, Sherry; Helm, Richard F.

    2014-01-01

    Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcpU deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 μM to McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing. PMID:24657863

  13. Evidence that COMT genotype and proline interact on negative-symptom outcomes in schizophrenia and bipolar disorder.

    PubMed

    Clelland, C L; Drouet, V; Rilett, K C; Smeed, J A; Nadrich, R H; Rajparia, A; Read, L L; Clelland, J D

    2016-01-01

    Elevated peripheral proline is associated with psychiatric disorders, and there is evidence that proline is a neuromodulator. The proline dehydrogenase (PRODH) gene, which encodes the enzyme that catalyzes proline catabolism, maps to human chromosome 22q11.2, a region conferring risk of schizophrenia. In the Prodh-null mouse, an interaction between elevated peripheral proline and another 22q11.2 gene, catechol-O-methyltransferase (COMT), on neurotransmission and behavior has been reported. We explored the relationship between fasting plasma proline levels and COMT Val(158)Met genotype on symptoms (positive, negative and total) in schizophrenia patients. In an exploratory study we also examined symptom change in patients with bipolar disorder. There was a significant interaction between peripheral proline and COMT on negative symptoms in schizophrenia (P<0.0001, n=95). In COMT Val/Val patients, high proline was associated with low Scale for the Assessment of Negative Symptom (SANS) scores. In contrast, high proline was associated with high SANS scores in patients carrying a Met allele. The relationship between proline and COMT also appears to modify negative symptoms across psychiatric illness. In bipolar disorder, a significant interaction was also observed on negative-symptom change (P=0.007, n=43). Negative symptoms are intractable and largely unaddressed by current medications. These data indicate a significant interaction between peripheral proline and COMT genotype, influencing negative symptoms in schizophrenia and bipolar disorder. That high proline has converse effects on symptoms by COMT genotype, may have implications for therapeutic decisions. PMID:27622935

  14. Arsenic Metabolites, Including N-Acetyl-4-hydroxy-m-arsanilic Acid, in Chicken Litter from a Roxarsone-Feeding Study Involving 1600 Chickens.

    PubMed

    Yang, Zonglin; Peng, Hanyong; Lu, Xiufen; Liu, Qingqing; Huang, Rongfu; Hu, Bin; Kachanoski, Gary; Zuidhof, Martin J; Le, X Chris

    2016-07-01

    The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROX-fed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of As(III) and As(V)) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizer. PMID:26876684

  15. An Investigation of the Influence of Chain Length on the Interfacial Ordering of L-Lysine and L-Proline and Their Homopeptides at Hydrophobic and Hydrophilic Interfaces Studied by Sum Frequency Generation and Quartz Crystal Microbalance

    SciTech Connect

    York, R.L.; Holinga, G.J.; Somorjai, G.A.

    2009-02-23

    Sum frequency generation vibrational spectroscopy (SFG) and quartz crystal microbalance with dissipation monitoring (QCM-D) are employed to study the interfacial structure and adsorbed amount of the amino acids l-lysine and l-proline and their corresponding homopeptides, poly-l-lysine and poly-l-proline, at two liquid-solid interfaces. SFG and QCM-D experiments of these molecules are carried out at the interface between phosphate buffered saline at pH 7.4 (PBS) and the hydrophobic deuterated polystyrene (d{sub 8}-PS) surface as well as the interface between PBS and hydrophilic fused silica (SiO{sub 2}). The SFG spectra of the amino acids studied here are qualitatively similar to their corresponding homopeptides; however, the SFG signal from amino acids at the solid/PBS interface is smaller in magnitude relative to their more massive homopeptides at the concentrations studied here. Substantial differences are observed in SFG spectra for each species between the hydrophobic d{sub 8}-PS and the hydrophilic SiO{sub 2} liquid-solid interfaces, suggesting surface-dependent interfacial ordering of the biomolecules. Over the range of concentrations used in this study, QCM-D measurements also indicate that on both surfaces poly-l-lysine adsorbs to a greater extent than its constituent amino acid l-lysine. The opposite trend is demonstrated by poly-l-proline which sticks to both surfaces less extensively than its corresponding amino acid, l-proline. Lastly, we find that the adsorption of the molecules studied here can have a strong influence on interfacial water structure as detected in the SFG spectra.

  16. Effect of hypothalamic proline-rich peptide (PRP-1) on neuronal and bone marrow cell apoptosis.

    PubMed

    Galoyan, Armen A; Krieglstein, Josef; Klumpp, Susanne; Danielian, Kristina E; Galoian, Karine A; Kremers, Wolfram; Bezirganyan, Kristina B; Davtyan, Tigran K

    2007-11-01

    The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects including immunoregulatory, hematopoietic, antimicrobial and anti-neurodegenerative properties. Here we investigated the influence of PRP-1 on staurosporine-induced apoptosis of postnatal hippocampal cells and on doxorubicin-induced bone marrow granulocyte- and monocyte apoptosis. The intention was to further characterize the effect of PRP-1 on the survival rate of neurons and in context with myelopoiesis. We demonstrate that PRP-1 significantly reduced apoptosis of postnatal hippocampal cells induced by staurosporine. The protective effect of PRP-1 against apoptotic cell death was shown to be both time- and dose-dependent. Neuroprotection was more pronounced after prolonged pretreatment of the cells with PRP-1 before the induction of apoptosis with staurosporine. The related peptide [arg(8)]vasopressin did not reveal neuroprotection. PRP-1 also significantly reduced apoptosis of bone marrow monocytes and granulocytes induced by doxorubicin. This protective effect lasted for 2-4 h and was not detectable anymore after 24 h when PRP-1 and doxorubicin were added simultaneously. Previously obtained data and results of the current studies suggested that the hypothalamic PRP-1 possibly represents an endogenous peptide whose primary functions are to regulate myelopoiesis and neuron survival as we provide evidence that PRP can differentially reduce both staurosporine- and doxorubicin-induced hippocampal and bone marrow cell apoptosis. PMID:17549627

  17. Clinical Manifestations in Paroxysmal Kinesigenic Dyskinesia Patients with Proline-Rich Transmembrane Protein 2 Gene Mutation

    PubMed Central

    Youn, Jinyoung; Kim, Ji Sun; Lee, Munhyang; Lee, Jeehun; Roh, Hakjae

    2014-01-01

    Background and Purpose Given the diverse phenotypes including combined non-dyskinetic symptoms in patients harboring mutations of the gene encoding proline-rich transmembrane protein 2 (PRRT2), the clinical significance of these mutations in paroxysmal kinesigenic dyskinesia (PKD) is questionable. In this study, we investigated the clinical characteristics of PKD patients with PRRT2 mutations. Methods Familial and sporadic PKD patients were enrolled and PRRT2 gene sequencing was performed. Demographic and clinical data were compared between PKD patients with and without a PRRT2 mutation. Results Among the enrolled PKD patients (8 patients from 5 PKD families and 19 sporadic patients), PRRT2 mutations were detected in 3 PKD families (60%) and 2 sporadic cases (10.5%). All familial patients with a PRRT2 gene mutation had the c.649dupC mutation, which is the most commonly reported mutation. Two uncommon mutations (c.649delC and c.629dupC) were detected only in the sporadic cases. PKD patients with PRRT2 mutation were younger at symptom onset and had more non-dyskinetic symptoms than those without PRRT2 mutation. However, the characteristics of dyskinetic movement did not differ between the two groups. Conclusions This is the first study of PRRT2 mutations in Korea. The presence of a PRRT2 mutation was more strongly related to familial PKD, and was clinically related with earlier age of onset and common non-dyskinetic symptoms in PKD patients. PMID:24465263

  18. Membrane interactions of proline-rich antimicrobial peptide, Chex1-Arg20, multimers.

    PubMed

    Li, Wenyi; Sani, Marc-Antoine; Jamasbi, Elaheh; Otvos, Laszlo; Hossain, Mohammed Akhter; Wade, John D; Separovic, Frances

    2016-06-01

    The increasing prevalence of antibiotic-resistant pathogens requires the development of new antibiotics. Proline-rich antimicrobial peptides (PrAMPs), including native apidaecins, Bac7, and oncocins or designed A3APO, show multi-modal actions against pathogens together with immunostimulatory activities. The interactions of the designed PrAMP, Chex1-Arg20, and its dimeric and tetrameric oligomers with different model membranes were investigated by circular dichroism spectroscopy, dynamic light scattering, zeta potential, differential scanning calorimetry, and dye leakage. Chex1-Arg20 oligomers showed stronger affinity and preferential binding to negatively charged phospholipid bilayers and led to lipid aggregation and neutralization. Fluorescence microscopy of negatively charged giant unilamellar vesicles with AlexFluor-647-labeled Chex1-Arg20 dimers and tetramers displayed aggregation at a peptide/lipid low ratio of 1:200 and at higher peptide concentrations (1:100/1:50) for Chex1-Arg20 monomer. Such interactions, aggregation, and neutralization of PrAMP oligomers additionally showed the importance of interactions of PrAMPs with negatively charged membranes. PMID:26926423

  19. Is foliar spray of proline sufficient for mitigation of salt stress in Brassica juncea cultivars?

    PubMed

    Wani, A S; Ahmad, A; Hayat, S; Tahir, I

    2016-07-01

    The effects of foliar application of proline (20 mM) on growth, physio-biochemical, and yield parameters were assessed in two Brassica juncea (L.) Czern & Coss cultivars, namely, Varuna and RH-30, at different levels (2.8, 4.2, or 5.6 dsm(-1)) of NaCl in soil. At 29 days after sowing (DAS), plants were sprayed with either 20 mM proline or water in the presence or absence of NaCl stress. The NaCl negatively affected parameters related to growth, photosynthesis, and yield in both varieties but more in RH-30 than in Varuna. Exogenous application of proline counteracted the effects of salt stress in Varuna only, by increasing the antioxidative capacity of the plants. Moreover, proline was not effective in alleviating the detrimental effects of higher salt concentrations on the studied parameters. Proline application to unstressed plants increased growth, photosynthesis, and yield parameters in both varieties; however, the effects were more prominent in Varuna than in RH-30. PMID:27026543

  20. Structural and thermodynamic basis of proline-induced transmembrane complex stabilization

    PubMed Central

    Schmidt, Thomas; Situ, Alan J.; Ulmer, Tobias S.

    2016-01-01

    In membrane proteins, proline-mediated helix kinks are indispensable for the tight packing of transmembrane (TM) helices. However, kinks invariably affect numerous interhelical interactions, questioning the acceptance of proline substitutions and evolutionary origin of kinks. Here, we present the structural and thermodynamic basis of proline-induced integrin αIIbβ3 TM complex stabilization to understand the introduction of proline kinks in membrane proteins. In phospholipid bicelles, the A711P substitution in the center of the β3 TM helix changes the direction of adjacent helix segments to form a 35 ± 2° angle and predominantly repacks the segment in the inner membrane leaflet due to a swivel movement. This swivel repacks hydrophobic and electrostatic interhelical contacts within intracellular lipids, resulting in an overall TM complex stabilization of −0.82 ± 0.01 kcal/mol. Thus, proline substitutions can directly stabilize membrane proteins and such substitutions are proposed to follow the structural template of integrin αIIbβ3(A711P). PMID:27436065

  1. A unique cis-3-hydroxy-l-proline dehydratase in the enolase superfamily.

    PubMed

    Zhang, Xinshuai; Kumar, Ritesh; Vetting, Matthew W; Zhao, Suwen; Jacobson, Matthew P; Almo, Steven C; Gerlt, John A

    2015-02-01

    The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8 ). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7 ; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9 ; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ(1)-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources. PMID:25608448

  2. Direct linking of metabolism and gene expression in the proline utilization A protein from Escherichia coli

    PubMed Central

    Zhou, Yuzhen; Zhu, Weidong; Bellur, Padmanetra S.; Rewinkel, Dustin; Becker, Donald F.

    2009-01-01

    Summary The control of gene expression by enzymes provides a direct pathway for cells to respond to fluctuations in metabolites and nutrients. One example is the proline utilization A (PutA) protein from Escherichia coli. PutA is a membrane-associated enzyme that catalyzes the oxidation of L-proline to glutamate using a flavin containing proline dehydrogenase domain and a NAD+ dependent Δ1-pyrroline-5-carboxylate dehydrogenase domain. In some Gram-negative bacteria such as E. coli, PutA is also endowed with a ribbon-helix-helix DNA-binding domain and acts as a transcriptional repressor of the proline utilization genes. PutA switches between transcriptional repressor and enzymatic functions in response to proline availability. Molecular insights into the redox based mechanism of PutA functional switching from recent studies are reviewed. In addition, new results from cell-based transcription assays are presented which correlate PutA membrane localization with put gene expression levels. General membrane localization of PutA, however, is not sufficient to activate the put genes. PMID:18324349

  3. Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N-Propargylglycine†‡

    PubMed Central

    White, Tommi A.; Johnson, William H.; Whitman, Christian P.; Tanner, John J.

    2009-01-01

    The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 Å resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the ε-amino group of Lys99 via a 3-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25°, which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this α,β-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a 3-carbon chain. The presumed non-enzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely. PMID:18426222

  4. The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.

    PubMed Central

    Reinhard, M; Giehl, K; Abel, K; Haffner, C; Jarchau, T; Hoppe, V; Jockusch, B M; Walter, U

    1995-01-01

    Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases, revealed the presence of a proline-rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline-rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation. Images PMID:7737110

  5. UV Photodissociation of Proline-containing Peptide Ions: Insights from Molecular Dynamics

    NASA Astrophysics Data System (ADS)

    Girod, Marion; Sanader, Zeljka; Vojkovic, Marin; Antoine, Rodolphe; MacAleese, Luke; Lemoine, Jérôme; Bonacic-Koutecky, Vlasta; Dugourd, Philippe

    2015-03-01

    UV photodissociation of proline-containing peptide ions leads to unusual product ions. In this paper, we report laser-induced dissociation of a series of proline-containing peptides at 213 nm. We observe specific fragmentation pathways corresponding to the formation of (y-2), (a + 2) and (b + 2) fragment ions. This was not observed at 266 nm or for peptides which do not contain proline residues. In order to obtain insights into the fragmentation dynamics at 213 nm, a small peptide (RPK for arginine-proline-lysine) was studied both theoretically and experimentally. Calculations of absorption spectra and non-adiabatic molecular dynamics (MD) were made. Second and third excited singlet states, S2 and S3, lie close to 213 nm. Non-adiabatic MD simulation starting from S2 and S3 shows that these transitions are followed by C-C and C-N bond activation close to the proline residue. After this first relaxation step, consecutive rearrangements and proton transfers are required to produce unusual (y-2), (a + 2) and (b + 2) fragment ions. These fragmentation mechanisms were confirmed by H/D exchange experiments.

  6. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

    PubMed

    Salwiczek, Mario; Nyakatura, Elisabeth K; Gerling, Ulla I M; Ye, Shijie; Koksch, Beate

    2012-03-21

    Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references). PMID:22130572

  7. Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region.

    PubMed

    Desrochers, Guillaume; Lussier-Price, Mathieu; Omichinski, James G; Angers, Annie

    2015-12-22

    Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both in vivo and in vitro, with a calculated KD placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested. PMID:26613292

  8. Motional timescale predictions by molecular dynamics simulations: Case study using proline and hydroxyproline sidechain dynamics

    PubMed Central

    Aliev, Abil E; Kulke, Martin; Khaneja, Harmeet S; Chudasama, Vijay; Sheppard, Tom D; Lanigan, Rachel M

    2014-01-01

    We propose a new approach for force field optimizations which aims at reproducing dynamics characteristics using biomolecular MD simulations, in addition to improved prediction of motionally averaged structural properties available from experiment. As the source of experimental data for dynamics fittings, we use 13C NMR spin-lattice relaxation times T1 of backbone and sidechain carbons, which allow to determine correlation times of both overall molecular and intramolecular motions. For structural fittings, we use motionally averaged experimental values of NMR J couplings. The proline residue and its derivative 4-hydroxyproline with relatively simple cyclic structure and sidechain dynamics were chosen for the assessment of the new approach in this work. Initially, grid search and simplexed MD simulations identified large number of parameter sets which fit equally well experimental J couplings. Using the Arrhenius-type relationship between the force constant and the correlation time, the available MD data for a series of parameter sets were analyzed to predict the value of the force constant that best reproduces experimental timescale of the sidechain dynamics. Verification of the new force-field (termed as AMBER99SB-ILDNP) against NMR J couplings and correlation times showed consistent and significant improvements compared to the original force field in reproducing both structural and dynamics properties. The results suggest that matching experimental timescales of motions together with motionally averaged characteristics is the valid approach for force field parameter optimization. Such a comprehensive approach is not restricted to cyclic residues and can be extended to other amino acid residues, as well as to the backbone. Proteins 2014; 82:195–215. © 2013 Wiley Periodicals, Inc. PMID:23818175

  9. Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth.

    PubMed

    Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

    2016-03-01

    Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

  10. Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth

    PubMed Central

    Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

    2016-01-01

    Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

  11. Effect of including carob pulp in the diet of fattening pigs on the fatty acid composition and oxidative stability of pork.

    PubMed

    Inserra, L; Luciano, G; Bella, M; Scerra, M; Cilione, C; Basile, P; Lanza, M; Priolo, A

    2015-02-01

    The effect of feeding pigs with carob pulp on meat quality was investigated. Nine pigs were finished on a conventional concentrate-based diet (control), while two groups received a diet comprising of the same ingredients with the inclusion of 8% or 15% carob pulp (Carob 8% and Carob 15%, respectively). Feeding carob-containing diets reduced the concentration of saturated fatty acids in the muscle, increased the concentration of monounsaturated fatty acids in meat (P < 0.01) and of n-3 polyunsaturated fatty acids (PUFAs) and reduced the n-6/n-3 PUFA ratio (P < 0.001). The meat underwent slow oxidative deterioration over 9 days of storage. However, the Carob 15% treatment increased meat susceptibility to lipid oxidation across storage (P = 0.03), while the dietary treatment did not affect meat colour stability. In conclusion, feeding pigs with carob pulp could represent a strategy,in the Mediterranean areas, to naturally improve meat nutritional value and to promote the exploitation of this local feed resource. PMID:25460134

  12. Modulation by Amino Acids: Toward Superior Control in the Synthesis of Zirconium Metal-Organic Frameworks.

    PubMed

    Gutov, Oleksii V; Molina, Sonia; Escudero-Adán, Eduardo C; Shafir, Alexandr

    2016-09-12

    The synthesis of zirconium metal-organic frameworks (Zr MOFs) modulated by various amino acids, including l-proline, glycine, and l-phenylalanine, is shown to be a straightforward approach toward functional-group incorporation and particle-size control. High yields in Zr-MOF synthesis are achieved by employing 5 equivalents of the modulator at 120 °C. At lower temperatures, the method provides a series of Zr MOFs with increased particle size, including many suitable for single-crystal X-ray diffraction studies. Furthermore, amino acid modulators can be incorporated at defect sites in Zr MOFs with an amino acid/ligand ratio of up to 1:1, depending on the ligand structure and reaction conditions. The MOFs obtained through amino acid modulation exhibit an improved CO2 -capture capacity relative to nonfunctionalized materials. PMID:27482849

  13. Flanking proline residues identify the L-type Ca2+ channel binding site of calciseptine and FS2.

    PubMed

    Kini, R M; Caldwell, R A; Wu, Q Y; Baumgarten, C M; Feher, J J; Evans, H J

    1998-06-23

    Calciseptine and FS2 are 60-amino acid polypeptides, isolated from venom of the black mamba (Dendroaspis polylepis polylepis), that block voltage-dependent L-type Ca2+ channels. We predicted that these polypeptides contain an identical functional site between residues 43 and 46 by searching for proline residues that mark the flanks of protein-protein interaction sites [Kini, R. M., and Evans, H. J. (1966) FEBS Lett. 385, 81-86]. The predicted Ca2+ channel binding site also occurs in closely related toxins, C10S2C2 and S4C8. Therefore, it is likely that these toxins also will block L-type Ca2+ channels. To test the proposed binding site on calciseptine and FS2, an eight-residue peptide, named L-calchin (L-type calcium channel inhibitor), was synthesized and examined for biological activity. As expected for an L-type Ca2+ channel blocker, L-calchin reduced peak systolic and developed pressure in isolated rat heart Langendorff preparations without affecting diastolic pressure or heart rate. Furthermore, L-calchin caused a voltage-independent block of L-type Ca2+ channel currents in whole-cell patch-clamped rabbit ventricular myocytes. Thus the synthetic peptide exhibits the L-type Ca2+ channel blocking properties of the parent molecules, calciseptine and FS2, but with a lower potency. These results strongly support the identification of a site in calciseptine and FS2 that is important for binding to L-type Ca2+ channels and reinforce the importance of proline brackets flanking protein-protein interaction sites. PMID:9636051

  14. Active sites of salivary proline-rich protein for binding to Porphyromonas gingivalis fimbriae.

    PubMed Central

    Kataoka, K; Amano, A; Kuboniwa, M; Horie, H; Nagata, H; Shizukuishi, S

    1997-01-01

    Porphyromonas gingivalis fimbriae specifically bind salivary acidic proline-rich protein 1 (PRP1) through protein-protein interactions. The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were analyzed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 64:1631-1637, 1996). In this study, we investigated the sites of binding of the PRP1 molecules to the fimbriae. PRP1 (amino acid residues 1 to 150) was proteolysed to three fragments (residues 1 to 74 [fragment 1-74], 75 to 129, and 130 to 150). 125I-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments also inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) beads by 50 and 83%, respectively. However, the N-terminal fragment failed to show any effect. Analogous peptides corresponding to residues 75 to 89, 90 to 106, 107 to 120, 121 to 129, and 130 to 150 of PRP1 were synthesized. The fimbriae significantly bound peptide 130-150, immobilized on 96-well plates, and the peptide also inhibited binding of 125I-labeled fimbriae to PRP1-coated HAP beads by almost 100%. Peptides 75-89, 90-106, and 121-129, immobilized on plates, showed considerable ability to bind fimbriae. For further analysis of active sites in residues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGPPQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%. This amino acid sequence was shared in the alignment of residues 75 to 89, 90 to 106, and 107 to 120. Six synthetic peptides were prepared by serial deletions of individual residues from the N and C termini of peptide GRPQGPPQ. Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ. Further deletions of the dipeptide Pro-Gln from the N and C termini of peptide PQGPPQ resulted in significant loss of the inhibitory effect. These results strongly suggest that PQGPPQ

  15. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.

    PubMed

    Doerfel, Lili K; Wohlgemuth, Ingo; Kothe, Christina; Peske, Frank; Urlaub, Henning; Rodnina, Marina V

    2013-01-01

    Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells. PMID:23239624

  16. A Thioether-Stabilized d-Proline-l-Proline-Induced β-Hairpin Peptide of Defensin Segment Increases Its Anti-Candida albicans Ability.

    PubMed

    Zhao, Bingchuan; Yang, Dan; Wong, Jack Ho; Wang, Jianpeng; Yin, Cuiming; Zhu, Yuxia; Fan, Shangrong; Ng, Tzi Bun; Xia, Jiang; Li, Zigang

    2016-08-01

    We report a β-hairpin dual stabilizing strategy: a d-proline-l-proline (d-Pro-l-Pro) dipeptide as the nucleating turn, and a thioether tether as a side-chain linkage at a precisely designed position to stabilize the β-hairpin. This method was used to modify the C-terminal β-hairpin moiety of the plant defensin, pv-defensin, in order to obtain a stabilized peptide with enhanced anti-Candida albicans activity (MIC 84-3.0 μm), high serum stability (50 % remaining after 48 h) and low hemolysis (<10 % at 152 μm). This modified peptide penetrated the C. albicans cell membrane within 5 min and showed high activity against clinically isolated antibiotic-resistant C. albicans and Candida glabrata strains. PMID:27194395

  17. Incomplete tricarboxylic acid cycle in a type I methylotroph, Methylococcus capsulatus.

    PubMed Central

    Patel, R; Hoare, L; Hoare, D S; Taylor, B F

    1975-01-01

    Alpha-Ketoglutaratedehydrogenase was undetectable in extracts of Methylococcus capsulatus. Cells incorporated [1-14-C] acetate into only four protein amino acids (glutamate, proline, arginine, and leucine) and the C5, but not C1, of glutamate. PMID:806581

  18. Unconventional N-H…N Hydrogen Bonds Involving Proline Backbone Nitrogen in Protein Structures.

    PubMed

    Deepak, R N V Krishna; Sankararamakrishnan, Ramasubbu

    2016-05-10

    Contrary to DNA double-helical structures, hydrogen bonds (H-bonds) involving nitrogen as the acceptor are not common in protein structures. We systematically searched N-H…N H-bonds in two different sets of protein structures. Data set I consists of neutron diffraction and ultrahigh-resolution x-ray structures (0.9 Å resolution or better) and the hydrogen atom positions in these structures were determined experimentally. Data set II contains structures determined using x-ray diffraction (resolution ≤ 1.8 Å) and the positions of hydrogen atoms were generated using a computational method. We identified 114 and 14,347 potential N-H…N H-bonds from these two data sets, respectively, and 56-66% of these were of the Ni+1-Hi+1…Ni type, with Ni being the proline backbone nitrogen. To further understand the nature of such unusual contacts, we performed quantum chemical calculations on the model compound N-acetyl-L-proline-N-methylamide (Ace-Pro-NMe) with coordinates taken from the experimentally determined structures. A potential energy profile generated by varying the ψ dihedral angle in Ace-Pro-NMe indicates that the conformation with the N-H…N H-bond is the most stable. An analysis of H-bond-forming proline residues reveals that more than 30% of the proline carbonyl groups are also involved in n → π(∗) interactions with the carbonyl carbon of the preceding residue. Natural bond orbital analyses demonstrate that the strength of N-H…N H-bonds is less than half of that observed for a conventional H-bond. This study clearly establishes the H-bonding capability of proline nitrogen and its prevalence in protein structures. We found many proteins with multiple instances of H-bond-forming prolines. With more than 15% of all proline residues participating in N-H…N H-bonds, we suggest a new, to our knowledge, structural role for proline in providing stability to loops and capping regions of secondary structures in proteins. PMID:27166805

  19. Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium.

    PubMed

    Zhang, Mi; Huang, He; Dai, Silan

    2014-03-10

    Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ(1)-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum×morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136-KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development. PMID:24434369

  20. Molecular mechanisms for the reaction between (˙)OH radicals and proline: insights on the role as reactive oxygen species scavenger in plant stress.

    PubMed

    Signorelli, Santiago; Coitiño, E Laura; Borsani, Omar; Monza, Jorge

    2014-01-01

    The accumulation of proline (Pro) and overproduction of reactive oxygen species (ROS) by plants exposed to stress is well-documented. In vitro assays show that enzyme inactivation by hydroxyl radicals ((•)OH) can be avoided in the presence of Pro, suggesting this amino acid might act as a (•)OH scavenger. Although production of hydroxyproline (Hyp) has been hypothesized in connection with such antioxidant activity, no evidence on the detailed mechanism of scavenging has been reported. To elucidate whether and how Hyp might be produced, we used density functional theory calculations coupled to a polarizable continuum model to explore 27 reaction channels including H-abstraction by (•)OH and (•)OH/H2O addition. The structure and energetics of stable species and transition states for each reaction channel were characterized at the PCM-(U)M06/6-31G(d,p) level in aqueous solution. Evidence is found for a main pathway in which Pro scavenges (•)OH by successive H-abstractions (ΔG(‡,298) = 4.1 and 7.5 kcal mol(-1)) to yield 3,4-Δ-Pro. A companion pathway with low barriers yielding Δ(1)-pyrroline-5-carboxylate (P5C) is also supported, linking with 5-Hyp through hydration. However, this connection remains unlikely in stressed plants because P5C would be efficiently recycled to Pro (contributing to its accumulation) by P5C reductase, hypothesis coined here as the "Pro-Pro cycle". PMID:24328335

  1. Exogenous γ-aminobutyric acid treatment affects citrate and amino acid accumulation to improve fruit quality and storage performance of postharvest citrus fruit.

    PubMed

    Sheng, Ling; Shen, Dandan; Luo, Yi; Sun, Xiaohua; Wang, Jinqiu; Luo, Tao; Zeng, Yunliu; Xu, Juan; Deng, Xiuxin; Cheng, Yunjiang

    2017-02-01

    The loss of organic acids during postharvest storage is one of the major factors that reduces the fruit quality and economic value of citrus. Citrate is the most important organic acid in citrus fruits. Molecular evidence has proved that γ-aminobutyric acid (GABA) shunt plays a key role in citrate metabolism. Here, we investigated the effects of exogenous GABA treatment on citrate metabolism and storage quality of postharvest citrus fruit. The content of citrate was significantly increased, which was primarily attributed to the inhibition of the expression of glutamate decarboxylase (GAD). Amino acids, including glutamate, alanine, serine, aspartate and proline, were also increased. Moreover, GABA treatment decreased the fruit rot rate. The activities of antioxidant enzymes and the content of energy source ATP were affected by the treatment. Our results indicate that GABA treatment is a very effective approach for postharvest quality maintenance and improvement of storage performance in citrus production. PMID:27596402

  2. The role of calcium in improving photosynthesis and related physiological and biochemical attributes of spring wheat subjected to simulated acid rain.

    PubMed

    Dolatabadian, Aria; Sanavy, Seyed Ali Mohammad Modarres; Gholamhoseini, Majid; Joghan, Aydin Khodaei; Majdi, Mohammad; Kashkooli, Arman Beyraghdar

    2013-04-01

    The response of photosynthesis parameters, catalase, superoxide dismutase and peroxidase activity, malondialdehyde, proline, chlorophyll, yield and yield components to foliar application of calcium and simulated acid rain in wheat were investigated. Foliar treatment of calcium led to significant increases in the photosynthesis rate, transpiration rate, stomatal conductance, proline, chlorophyll, yield and yield components in plants subjected to acid rain. Antioxidant enzyme activity and lipid peroxidation in the wheat leaves decreased because of calcium foliar application. Calcium hindered degradation of the rubisco subunits under acid rain treatment compared with water-treated plants. Results suggest that acid rain induces the production of free radicals resulting in lipid peroxidation of the cell membrane so that significant increase in antioxidant enzyme activity was observed. In addition, photosynthetic parameters i.e. photosynthesis rate, transpiration rate and stomatal conductance were drastically suppressed by acid rain. The cellular damage caused by free radicals might be reduced or prevented by a protective metabolism including antioxidative enzymes and calcium. We report that foliar application of calcium before acid rain may ameliorate the adverse effects of acid rain in wheat plants. PMID:24431486

  3. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

    PubMed

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-03-18

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  4. New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket.

    PubMed

    Perera, Erick; Pons, Tirso; Hernandez, Damir; Moyano, Francisco J; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2010-09-01

    Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed. PMID:20649906

  5. Osteogenesis from Dental Pulp Derived Stem Cells: A Novel Conditioned Medium Including Melatonin within a Mixture of Hyaluronic, Butyric, and Retinoic Acids

    PubMed Central

    Maioli, Margherita; Basoli, Valentina; Santaniello, Sara; Cruciani, Sara; Delitala, Alessandro Palmerio; Pinna, Roberto; Milia, Egle; Grillari-Voglauer, Regina; Fontani, Vania; Rinaldi, Salvatore; Muggironi, Roberta; Pigliaru, Gianfranco; Ventura, Carlo

    2016-01-01

    Human dental pulp stem cells (hDPSCs) have shown relevant potential for cell therapy in the orthopedic and odontoiatric fields. The optimization of their osteogenic potential is currently a major challenge. Vascular endothelial growth factor A (VEGF A) has been recently reported to act as a major conductor of osteogenesis in vitro and in vivo. Here, we attempted to prime endogenous VEGF A expression without the need for viral vector mediated gene transfer technologies. We show that hDPSCs exposure to a mixture of hyaluronic, butyric, and retinoic acids (HA + BU + RA) induced the transcription of a gene program of osteogenesis and the acquirement of an osteogenic lineage. Such response was also elicited by cell exposure to melatonin, a pleiotropic agent that recently emerged as a remarkable osteogenic inducer. Interestingly, the commitment to the osteogenic fate was synergistically enhanced by the combinatorial exposure to a conditioned medium containing both melatonin and HA + BU + RA. These in vitro results suggest that in vivo osteogenesis might be improved and further studies are needed. PMID:26880937

  6. Proteolytic activity of Escherichia coli oligopeptidase B against proline-rich antimicrobial peptides.

    PubMed

    Mattiuzzo, Maura; De Gobba, Cristian; Runti, Giulia; Mardirossian, Mario; Bandiera, Antonella; Gennaro, Renato; Scocchi, Marco

    2014-02-28

    Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes the bacterial cells specifically less susceptible to several proline-rich antimicrobial peptides known to penetrate into the bacterial cytosol, and that its level of activity directly correlates with the degree of resistance. We established that E. coli OpdB can efficiently hydrolyze in vitro cationic antimicrobial peptides up to 30 residues in length, even though they contained several prolines, shortening them to inactive fragments. Two consecutive basic residues are a preferred cleavage site for the peptidase. In the case of a single basic residue, there is no cleavage if proline residues are present in the P1 and P2 positions. These results also indicate that cytosolic peptidases may cause resistance to antimicrobial peptides that have an intracellular mechanism of action, such as the proline-rich peptides, and may contribute to define the substrate specificity of the E. coli OpdB. PMID:24225368

  7. Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

    PubMed Central

    Bae, J H; Miller, K J

    1992-01-01

    The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity. PMID:1610171

  8. A new charge-tagged proline-based organocatalyst for mechanistic studies using electrospray mass spectrometry

    PubMed Central

    Willms, J Alexander; Beel, Rita; Schmidt, Martin L; Mundt, Christian

    2014-01-01

    Summary A new 4-hydroxy-L-proline derivative with a charged 1-ethylpyridinium-4-phenoxy substituent has been synthesized with the aim of facilitating mechanistic studies of proline-catalyzed reactions by ESI mass spectrometry. The charged residue ensures a strongly enhanced ESI response compared to neutral unmodified proline. The connection by a rigid linker fixes the position of the charge tag far away from the catalytic center in order to avoid unwanted interactions. The use of a charged catalyst leads to significantly enhanced ESI signal abundances for every catalyst-derived species which are the ones of highest interest present in a reacting solution. The new charged proline catalyst has been tested in the direct asymmetric inverse aldol reaction between aldehydes and diethyl ketomalonate. Two intermediates in accordance with the List–Houk mechanism for enamine catalysis have been detected and characterized by gas-phase fragmentation. In addition, their temporal evolution has been followed using a microreactor continuous-flow technique. PMID:25246962

  9. Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

    PubMed Central

    Srivastava, Dhiraj; Schuermann, Jonathan P.; White, Tommi A.; Krishnan, Navasona; Sanyal, Nikhilesh; Hura, Greg L.; Tan, Anmin; Henzl, Michael T.; Becker, Donald F.; Tanner, John J.

    2010-01-01

    The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site. PMID:20133651

  10. Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

    SciTech Connect

    Srivastava, Dhiraj; Schuermann, Jonathan P.; White, Tommi A.; Krishnan, Navasona; Sanyal, Nikhilesh; Hura, Greg L.; Tan, Anmin; Henzl, Michael T.; Becker, Donald F.; Tanner, John J.

    2010-04-26

    The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD{sup +}-dependent {Delta}{sup 1}-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 {angstrom} resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 {angstrom}. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 {angstrom} and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, {Delta}{sup 1}-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of {Delta}{sup 1}-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.

  11. Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma.

    PubMed

    Sherrier, D J; Taylor, G S; Silverstein, K A T; Gonzales, M B; VandenBosch, K A

    2005-04-01

    Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis. PMID:15868212

  12. Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin.

    PubMed

    Eugster, Philippe J; Salamin, Karine; Grouzmann, Eric; Monod, Michel

    2015-12-01

    Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus. PMID:26464108

  13. Binding of the proline-rich region of the epithelial Na+ channel to SH3 domains and its association with specific cellular proteins.

    PubMed Central

    McDonald, F J; Welsh, M J

    1995-01-01

    The amiloride-sensitive epithelial Na+ channel (ENaC) is an important component of the Na(+)-reabsorption pathway in many epithelia. The identification of three subunits of ENaC (alpha, beta and gamma), as well as results from a number of functional and biochemical studies, suggests that functional Na+ channels are composed of a complex of proteins. To learn about possible interactions of the channel with other proteins, we studied the alpha-subunit of rat and human ENaC. We found that the proline-rich C-terminal domains of both rat and human alpha-ENaC, expressed as glutathione S-transferase fusion proteins, bound to SH3 domains in vitro. A 116 kDa protein from a human lung adenocarcinoma cell line (H441) was specifically bound by the human alpha-ENaC C-terminal fusion protein and by a shorter 18-amino acid proline-rich peptide derived from the larger fusion protein. The 116 kDa protein was not glycosylated and was not phosphorylated on tyrosine or by cyclic AMP-dependent protein kinase (PKA). A 134 kDa protein which was also bound by the human alpha-ENaC C-terminal fusion protein was a substrate for phosphorylation by PKA. These data suggest that the proline-rich C-terminal tail of alpha-ENaC may interact with other proteins that control its function, regulation or localization. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8526861

  14. Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from Escherichia coli*

    PubMed Central

    Singh, Ranjan K.; Larson, John D.; Zhu, Weidong; Rambo, Robert P.; Hura, Greg L.; Becker, Donald F.; Tanner, John J.

    2011-01-01

    The trifunctional flavoprotein proline utilization A (PutA) links metabolism and gene regulation in Gram-negative bacteria by catalyzing the two-step oxidation of proline to glutamate and repressing transcription of the proline utilization regulon. Small-angle x-ray scattering (SAXS) and domain deletion analysis were used to obtain solution structural information for the 1320-residue PutA from Escherichia coli. Shape reconstructions show that PutA is a symmetric V-shaped dimer having dimensions of 205 × 85 × 55 Å. The particle consists of two large lobes connected by a 30-Å diameter cylinder. Domain deletion analysis shows that the N-terminal DNA-binding domain mediates dimerization. Rigid body modeling was performed using the crystal structure of the DNA-binding domain and a hybrid x-ray/homology model of residues 87–1113. The calculations suggest that the DNA-binding domain is located in the connecting cylinder, whereas residues 87–1113, which contain the two catalytic active sites, reside in the large lobes. The SAXS data and amino acid sequence analysis suggest that the Δ1-pyrroline-5-carboxylate dehydrogenase domains lack the conventional oligomerization flap, which is unprecedented for the aldehyde dehydrogenase superfamily. The data also provide insight into the function of the 200-residue C-terminal domain. It is proposed that this domain serves as a lid that covers the internal substrate channeling cavity, thus preventing escape of the catalytic intermediate into the bulk medium. Finally, the SAXS model is consistent with a cloaking mechanism of gene regulation whereby interaction of PutA with the membrane hides the DNA-binding surface from the put regulon thereby activating transcription. PMID:22013066

  15. A fluorescence-based demonstration of intestinal villi and epithelial cell in chickens fed dietary silicic acid powder including bamboo vinegar compound liquid.

    PubMed

    Ruttanavut, J; Matsumoto, Y; Yamauchi, K

    2012-10-01

    This study investigates the combined effect of silicic acid and bamboo vinegar compound liquid (SPV) on the growth and intestinal histological alterations in poultry. Forty-eight 7-day-old male Sanuki Cochin chickens were fed a commercial mash diet supplemented with SPV at 0, 0.1, 0.2, and 0.3% level ad libitum for 112 days. Body weight gain tended to improve with increased concentrations of dietary SPV, although these results were not statistically significant (P<0.1). Tissue observation by light microscopy revealed that the jejunal villus height (P<0.01) and duodenal and jejunal villus area (P<0.05) increased in the 0.2 and 0.3% SPV groups, respectively, compared with the control. Cell mitosis within the duodenum and jejunum also increased in the 0.2 and 0.3% SPV groups. Scanning electron microscopy revealed a prominent increase in the number of protuberant cells on the villus apical surface of the duodenum and jejunum for the 0.2 and 0.3% SPV groups compared with the control. Poultry in the 0.3% SPV group had the highest body weight gain and hypertrophied histological alterations of intestinal villi. Fluorescent microscopic images of cell mitosis and protuberant cells in the duodenal crypt clearly confirmed positive reactions for the activator protein 2α (AP-2α) and proliferating cell nuclear antigen (PCNA), compared with the control. The present results indicate that dietary SPV stimulates adsorption by the epithelial cells, which activate cell proliferation and self-renewal and regulate the expression of cell cycle regulators AP-2α and PCNA, resulting in higher body weight gain. Thus, we can conclude that a concentration of 0.3% dietary SPV is ideal for promoting growth in poultry. PMID:22936452

  16. Dihydropyrimidinone positive modulation of delta-subunit-containing gamma-aminobutyric acid type A receptors, including an epilepsy-linked mutant variant.

    PubMed

    Lewis, Ryan W; Mabry, John; Polisar, Jason G; Eagen, Kyle P; Ganem, Bruce; Hess, George P

    2010-06-15

    Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells. Using the three-component Biginelli reaction, 13 DHPMs with structural features similar to those of the barbiturate phenobarbital were synthesized; one DHPM used (monastrol) is commercially available. An up to approximately 3-fold increase in the current from recombinant alpha1beta2delta receptors was observed with the DHPM compound JM-II-43A or monastrol when co-applied with saturating GABA concentrations, similar to the current potentiation observed with the nonselective potentiating compounds phenobarbital and tracazolate. No agonist activity was observed for the DHPMs at the concentrations tested. A kinetic model was used in conjunction with dose-dependent measurements to calculate apparent dissociation constant values for JM-II-43A (400 muM) and monastrol (200 microM) at saturating GABA concentrations. We examined recombinant receptors composed of combinations of subunits alpha1, alpha4, alpha5, alpha6, beta2, beta3, gamma2L, and delta with JM-II-43A to demonstrate the preference for potentiation of delta-subunit-containing receptors. Lastly, reduced currents from receptors containing the mutated delta(E177A) subunit, described by Dibbens et al. [(2004) Hum. Mol. Genet. 13, 1315-1319] as a heritable susceptibility allele for generalized epilepsy with febrile seizures plus, are also potentiated by these DHPMs. PMID:20450160

  17. Tyrosine Nitration within the Proline-Rich Region of Tau in Alzheimer's Disease

    PubMed Central

    Reyes, Juan F.; Fu, Yifan; Vana, Laurel; Kanaan, Nicholas M.; Binder, Lester I.

    2011-01-01

    A substantial body of evidence suggests that nitrative injury contributes to neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative disorders. Previously, we showed in vitro that within the tau protein the N-terminal tyrosine residues (Y18 and Y29) are more susceptible to nitrative modifications than other tyrosine sites (Y197 and Y394). Using site-specific antibodies to nitrated tau at Y18 and Y29, we identified tau nitrated in both glial (Y18) and neuronal (Y29) tau pathologies. In this study, we report the characterization of two novel monoclonal antibodies, Tau-nY197 and Tau-nY394, recognizing tau nitrated at Y197 and Y394, respectively. By Western blot analysis, Tau-nY197 labeled soluble tau and insoluble paired helical filament proteins (PHF-tau) nitrated at Y197 from control and AD brain samples. Tau-nY394 failed to label soluble tau isolated from control or severe AD samples, but labeled insoluble PHF-tau to a limited extent. Immunohistochemical analysis using Tau-nY197 revealed the hallmark tau pathology associated with AD; Tau-nY394 did not detect any pathological lesions characteristic of the disorder. These data suggest that a subset of the hallmark pathological inclusions of AD contain tau nitrated at Y197. However, nitration at Y197 was also identified in soluble tau from all control samples, including those at Braak stage 0, suggesting that nitration at this site in the proline-rich region of tau may have normal biological functions in the human brain. PMID:21514440

  18. Interaction of proline, sugars, and anthocyanins during photosynthetic acclimation of Arabidopsis thaliana to drought stress.

    PubMed

    Sperdouli, Ilektra; Moustakas, Michael

    2012-04-15

    The relationships among photosynthetic acclimation, proline (Pro), soluble sugar (SS), and anthocyanin (An) accumulation in Arabidopsis thaliana leaves to the onset of drought stress (OnDS), mild (MiDS) and moderate drought stress (MoDS), were evaluated. As leaf water content (LWC) decreased, metabolic concentrations (Pro, SS, and An) increased and were negatively and significantly correlated with LWC. Thus, these metabolites may have an important role in the acclimation process to drought stress (DS). No correlations among Pro, SS and An accumulation with the quantum efficiency of PSII photochemistry (Φ(PSII)) and the excitation pressure (1-q(P)) were observed under DS. This implies that, while metabolites increased in a drought-dependent way, PSII activity did not decrease in the same pattern. Our results indicated that, under MoDS, A. thaliana leaves were able to maintain oxidative compounds such as malondialdeyde, an end product of lipid peroxidation, within the range of control leaves, and to cope with oxidative damage, as was evident by the decreased excitation pressure (1-q(P)) and similar (ns difference) Φ(PSII) to that of control leaves. In addition, a statistically significant increased accumulation of Pro, SS and An was recorded only under MoDS compared to controls. The better PSII functioning of MoDS Arabidopsis leaves may reflect the greater capacity of these leaves to undertake key metabolic adjustments, including increased Pro, SS and An accumulation, to maintain a higher antioxidant protection and a better balance between light capture and energy use. PMID:22305050

  19. [Evaluation of ten fish species to be included as part of renal diet, due to their protein, phosphorus and fatty acids content].

    PubMed

    Castro-González, Maria Isabel; Maafs-Rodríguez, Ana Gabriela; Pérez-Gil Romo, Fernando

    2012-06-01

    Because renal disease is highly complex, its nutritional treatment is complicated and many foods are restricted, including fish because its phosphorus content. The aim of the present study was to analyze ten fillet fish species, commonly consumed in Mexico (Cyprinus carpio carpio, Ophichthus rex, Symphurus elongatus, Eucinostomus entomelas, Chirostoma patzcuaro, Bairdiella chrysoura, Salmo salar Oreochromis urolepis hornorum, Sphyraena guachancho, Istiophorus albicans), to determine their phosphorus (P), protein (Pr), cholesterol, sodium, potassium, vitamins D3 and E, and n-3 PUFA (EPA+DHA) according to the AOAC techniques, in order to identify which species could be included in renal diet; particularly because of their risk:benefit relations (calculated with those results). Protein values ranged from 16.5 to 33.5g/100 g of fillet; the specie with the highest phosphorus contest was Salmo salar, and with the lowest, Symphurus elongatus. EPA+DHA quantity ranged from 79.64 mg/100 g to 1,381.53 mg/100 g. Considering de P/Pr relation recommended to renal patients, all analyzed species (except Salmo salar, Ophichthus rex and Istiophorus albicans) could be included in their diet. As for the P/EPA+DHA relation, the species most recommended to renal patients are Symphurus elongatus, Bairdiella chrysoura and Sphyraena guachancho. PMID:23610899

  20. Arginine and proline applied as food additives stimulate high freeze tolerance in larvae of Drosophila melanogaster.

    PubMed

    Koštál, Vladimír; Korbelová, Jaroslava; Poupardin, Rodolphe; Moos, Martin; Šimek, Petr

    2016-08-01

    The fruit fly Drosophila melanogaster is an insect of tropical origin. Its larval stage is evolutionarily adapted for rapid growth and development under warm conditions and shows high sensitivity to cold. In this study, we further developed an optimal acclimation and freezing protocol that significantly improves larval freeze tolerance (an ability to survive at -5°C when most of the freezable fraction of water is converted to ice). Using the optimal protocol, freeze survival to adult stage increased from 0.7% to 12.6% in the larvae fed standard diet (agar, sugar, yeast, cornmeal). Next, we fed the larvae diets augmented with 31 different amino compounds, administered in different concentrations, and observed their effects on larval metabolomic composition, viability, rate of development and freeze tolerance. While some diet additives were toxic, others showed positive effects on freeze tolerance. Statistical correlation revealed tight association between high freeze tolerance and high levels of amino compounds involved in arginine and proline metabolism. Proline- and arginine-augmented diets showed the highest potential, improving freeze survival to 42.1% and 50.6%, respectively. Two plausible mechanisms by which high concentrations of proline and arginine might stimulate high freeze tolerance are discussed: (i) proline, probably in combination with trehalose, could reduce partial unfolding of proteins and prevent membrane fusions in the larvae exposed to thermal stress (prior to freezing) or during freeze dehydration; (ii) both arginine and proline are exceptional among amino compounds in their ability to form supramolecular aggregates which probably bind partially unfolded proteins and inhibit their aggregation under increasing freeze dehydration. PMID:27489218

  1. Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

    PubMed

    Coutts, S J; Kelly, T A; Snow, R J; Kennedy, C A; Barton, R W; Adams, J; Krolikowski, D A; Freeman, D M; Campbell, S J; Ksiazek, J F; Bachovchin, W W

    1996-05-10

    A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms. PMID:8642568

  2. Two novel PRPF31 premessenger ribonucleic acid processing factor 31 homolog mutations including a complex insertion-deletion identified in Chinese families with retinitis pigmentosa

    PubMed Central

    Dong, Bing; Chen, Jieqiong; Zhang, Xiaohui; Pan, Zhe; Bai, Fengge

    2013-01-01

    Objective To identify the causative mutations in two Chinese families with retinitis pigmentosa (RP), and to describe the associated phenotype. Methods Individuals from two unrelated families underwent full ophthalmic examinations. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Linkage analysis was performed on the known genetic loci for autosomal dominant retinitis pigmentosa with a panel of polymorphic markers in the two families, and then all coding exons of the PRP31 premessenger ribonucleic acid processing factor 31 homolog (PRPF31) gene were screened for mutations with direct sequencing of PCR-amplified DNA fragments. Allele-specific PCR was used to validate a substitution in all available family members and 100 normal controls. A large deletion was detected with real-time quantitative PCR (RQ-PCR) using a panel of primers from regions around the PRPF31 gene. Long-range PCR, followed by DNA sequencing, was used to define the breakpoints. Results Clinical examination and pedigree analysis revealed two four-generation families (RP24 and RP106) with autosomal dominant retinitis pigmentosa. A significant two-point linkage odd disequilibrium score was generated at marker D19S926 (Zmax=3.55, θ=0) for family RP24 and D19S571 (Zmax=3.21, θ=0) for family RP106, and further linkage and haplotype studies confined the disease locus to chromosome 19q13.42 where the PRPF31 gene is located. Mutation screening of the PRPF31 gene revealed a novel deletion c.1215delG (p.G405fs+7X) in family RP106. The deletion cosegregated with the family’s disease phenotype, but was not found in 100 normal controls. No disease-causing mutation was detected in family RP24 with PCR-based sequencing analysis. RQ-PCR and long-range PCR analysis revealed a complex insertion-deletion (indel) in the patients of family RP24. The deletion is more than 19 kb and encompasses part of the PRPF31 gene (exons 1–3), together with three adjacent

  3. Use of Cis-[18F]Fluoro-Proline for Assessment of Exercise-Related Collagen Synthesis in Musculoskeletal Connective Tissue

    PubMed Central

    Skovgaard, Dorthe; Kjaer, Andreas; Heinemeier, Katja Maria; Brandt-Larsen, Malene; Madsen, Jacob; Kjaer, Michael

    2011-01-01

    Protein turnover in collagen rich tissue is influenced by exercise, but can only with difficulty be studied in vivo due to use of invasive procedure. The present study was done to investigate the possibility of applying the PET-tracer, cis-[18F]fluoro-proline (cis-Fpro), for non-invasive assessment of collagen synthesis in rat musculoskeletal tissues at rest and following short-term (3 days) treadmill running. Musculoskeletal collagen synthesis was studied in rats at rest and 24 h post-exercise. At each session, rats were PET scanned at two time points following injection of cis-FPro: (60 and 240 min p.i). SUV were calculated for Achilles tendon, calf muscle and tibial bone. The PET-derived results were compared to mRNA expression of collagen type I and III. Tibial bone had the highest SUV that increased significantly (p<0.001) from the early (60 min) to the late (240 min) PET scan, while SUV in tendon and muscle decreased (p<0.001). Exercise had no influence on SUV, which was contradicted by an increased gene expression of collagen type I and III in muscle and tendon. The clearly, visible uptake of cis-Fpro in the collagen-rich musculoskeletal tissues is promising for multi-tissue studies in vivo. The tissue-specific differences with the highest basal uptake in bone are in accordance with earlier studies relying on tissue incorporation of isotopic-labelled proline. A possible explanation of the failure to demonstrate enhanced collagen synthesis following exercise, despite augmented collagen type I and III transcription, is that SUV calculations are not sensitive enough to detect minor changes in collagen synthesis. Further studies including kinetic compartment modeling must be performed to establish whether cis-Fpro can be used for non-invasive in-vivo assessment of exercise-induced changes in musculoskeletal collagen synthesis. PMID:21347251

  4. Effects of Heat Shock on Amino Acid Metabolism of Cowpea Cells 1

    PubMed Central

    Mayer, Randall R.; Cherry, Joe H.; Rhodes, David

    1990-01-01

    When cowpea (Vigna unguiculata) cells maintained at 26°C are transferred to 42°C, rapid accumulation of γ-aminobutyrate (>10-fold) is induced. Several other amino acids (including β-alanine, alanine, and proline) are also accumulated, but less extensively than γ-aminobutyrate. Total free amino acid levels are increased approximately 1.5-fold after 24 hours at 42°C. Heat shock also leads to release of amino acids into the medium, indicating heat shock damage to the integrity of the plasmalemma. Some of the changes in metabolic rates associated with heat shock were estimated by monitoring the 15N labeling kinetics of free intracellular, extracellular and protein-bound amino acids of cultures supplied with 15NH4+, and analyzing the labeling data by computer simulation. Preliminary computer simulation models of nitrogen flux suggest that heat shock induces an increase in the γ-aminobutyrate synthesis rate from 12.5 nanomoles per hour per gram fresh weight in control cells maintained at 26°C, to as high as 800 nanomoles per hour per gram fresh weight within the first 2 hours of heat shock. This 64-fold increase in the γ-aminobutyrate synthesis rate greatly exceeds the expected (Q10) change of metabolic rate of 2.5- to 3-fold due to a 16°C increase in temperature. We suggest that this metabolic response may in part involve an activation of glutamate decarboxylase in vivo, perhaps mediated by a transient cytoplasmic acidification. Proline appears to be synthesized from glutamate and not from ornithine in cowpea cells. Proline became severalfold more heavily labeled than ornithine, citrulline and arginine in both control and heat-shocked cultures. Proline synthesis rate was increased 2.7-fold by heat shock. Alanine, β-alanine, valine, leucine, and isoleucine synthesis rates were increased 1.6-, 3.5-, 2.0-, 5.0-, and 6.0-fold, respectively, by heat shock. In contrast, the phenylalanine synthesis rate was decreased by 50% in response to heat shock. The

  5. Prediction of individual milk proteins including free amino acids in bovine milk using mid-infrared spectroscopy and their correlations with milk processing characteristics.

    PubMed

    McDermott, A; Visentin, G; De Marchi, M; Berry, D P; Fenelon, M A; O'Connor, P M; Kenny, O A; McParland, S

    2016-04-01

    The aim of this study was to evaluate the effectiveness of mid-infrared spectroscopy in predicting milk protein and free amino acid (FAA) composition in bovine milk. Milk samples were collected from 7 Irish research herds and represented cows from a range of breeds, parities, and stages of lactation. Mid-infrared spectral data in the range of 900 to 5,000 cm(-1) were available for 730 milk samples; gold standard methods were used to quantify individual protein fractions and FAA of these samples with a view to predicting these gold standard protein fractions and FAA levels with available mid-infrared spectroscopy data. Separate prediction equations were developed for each trait using partial least squares regression; accuracy of prediction was assessed using both cross validation on a calibration data set (n=400 to 591 samples) and external validation on an independent data set (n=143 to 294 samples). The accuracy of prediction in external validation was the same irrespective of whether undertaken on the entire external validation data set or just within the Holstein-Friesian breed. The strongest coefficient of correlation obtained for protein fractions in external validation was 0.74, 0.69, and 0.67 for total casein, total β-lactoglobulin, and β-casein, respectively. Total proteins (i.e., total casein, total whey, and total lactoglobulin) were predicted with greater accuracy then their respective component traits; prediction accuracy using the infrared spectrum was superior to prediction using just milk protein concentration. Weak to moderate prediction accuracies were observed for FAA. The greatest coefficient of correlation in both cross validation and external validation was for Gly (0.75), indicating a moderate accuracy of prediction. Overall, the FAA prediction models overpredicted the gold standard values. Near-unity correlations existed between total casein and β-casein irrespective of whether the traits were based on the gold standard (0.92) or mid

  6. Discovery of spirocyclic proline tryptophan hydroxylase-1 inhibitors.

    PubMed

    Goldberg, Daniel R; De Lombaert, Stéphane; Aiello, Robert; Bourassa, Patricia; Barucci, Nicole; Zhang, Qing; Paralkar, Vishwas; Valentine, James; Zavadoski, William

    2016-02-15

    The central role of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) as a neurotransmitter with important cognitive and behavioral functions is well known. However, 5-HT produced in the brain only accounts for approximately 5% of the total amount of 5-HT generated in the body. At the onset of our work, it appeared that substituted phenylalanine derivatives or related aryl amino acids were required to produce potent inhibitors of TPH1, as significant losses of inhibitory activity were noted in the absence of this structural element. We disclose herein the discovery of a new class of TPH1 inhibitors that significantly lower peripherally 5-HT. PMID:26821821

  7. Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

    PubMed Central

    Thouta, Samrat; Sokolov, Stanislav; Abe, Yuki; Clark, Sheldon J.; Cheng, Yen M.; Claydon, Tom W.

    2014-01-01

    In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile655 to Tyr667 revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln664 marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement. PMID:24606930

  8. The mechanism of proline/glutamate antiport in rat kidney mitochondria. Energy dependence and glutamate-carrier involvement.

    PubMed

    Atlante, A; Passarella, S; Pierro, P; Di Martino, C; Quagliariello, E

    1996-10-01

    Proline/glutamate antiport in rat kidney mitochondria has been studied in terms of two different features: energy dependence and glutamate-carrier contribution to accomplish proline movement across the mitochondrial membrane. Energy dependence of the proline/glutamate antiporter in rat kidney mitochondria has been investigated by means of both spectroscopic measurements and isotopic techniques, using either normal or [14C]glutamate-loaded mitochondria. The sensitivity of the proline/glutamate antiport to the ionophores valinomycin and nigericin, under conditions in which delta psi and delta pH are selectively affected, shows that the exchange is energy dependent. Measurements of both membrane potential and proton movement across the mitochondrial membrane suggest that proline/glutamate antiport is driven by the electrochemical proton gradient via the delta psi dependent proline/glutamate translocator and delta pH-dependent glutamate/OH- carrier. Such a carrier provides for re-uptake of glutamate that has already passed out of the mitochondria in exchange with incoming proline, made possible by the existence of a separate pool of glutamate in the intermembrane space, directly shown by means of HPLC measurements. PMID:8898903

  9. Context of action of Proline Dehydrogenase (ProDH) in the Hypersensitive Response of Arabidopsis

    PubMed Central

    2014-01-01

    Background Proline (Pro) dehydrogenase (ProDH) potentiates the oxidative burst and cell death of the plant Hypersensitive Response (HR) by mechanisms not yet elucidated. ProDH converts Pro into ∆1 pyrroline-5-carboxylate (P5C) and can act together with P5C dehydrogenase (P5CDH) to produce Glu, or with P5C reductase (P5CR) to regenerate Pro and thus stimulate the Pro/P5C cycle. To better understand the effects of ProDH in HR, we studied the enzyme at three stages of the defense response differing in their ROS and cell death levels. In addition, we tested if ProDH requires P5CDH to potentiate HR. Results Control and infected leaves of wild type and p5cdh plants were used to monitor ProDH activity, in vivo Pro catabolism, amino acid content, and gene expression. Wild type plants activated ProDH at all HR stages. They did not consume Pro during maximal ROS accumulation, and maintained almost basal P5C levels at all conditions. p5cdh mutants activated ProDH as wild type plants. They achieved maximum oxidative burst and cell death levels producing normal HR lesions, but evidenced premature defense activation. Conclusion ProDH activation has different effects on HR. Before the oxidative burst it leads to Pro consumption involving the action of P5CDH. During the oxidative burst, ProDH becomes functionally uncoupled to P5CDH and apparently works with P5CR. The absence of P5CDH does not reduce ROS, cell death, or pathogen resistance, indicating this enzyme is not accompanying ProDH in the potentiation of these defense responses. In contrast, p5cdh infected plants displayed increased ROS burst and earlier initiation of HR cell death. In turn, our results suggest that ProDH may sustain HR by participating in the Pro/P5C cycle, whose action on HR must be formally evaluated in a future. PMID:24410747

  10. 3-(1H-Indol-3-yl)-2-(2-nitro­benzene­sulfonamido)­propanoic acid including an unknown solvate

    PubMed Central

    Khan, Islam Ullah; Mubashar-ur-Rehman, Hafiz; Aziz, Salman; Harrison, William T. A.

    2012-01-01

    In the title compound, C17H15N3O6S, which crystallized with highly disordered methanol and/or water solvent mol­ecules, the dihedral angle between the the indole and benzene ring systems is 5.3 (2)°, which allows for the formation of intra­molecular π–π stacking inter­actions [centroid–centroid separations = 3.641 (3) and 3.694 (3) Å] and an approximate overall U-shape for the mol­ecule. In the crystal, dimers linked by pairs of Ns—H⋯Oc (s = sulfonamide and c = carboxyl­ate) hydrogen bonds generate R 2 2(10) loops, whereas Ni—H⋯π (i = indole) inter­actions lead to chains propagating in [100] or [010]. Together, these lead to a three-dimensional network in which the solvent voids are present as inter­secting (two-dimensional) systems of [100] and [010] channels. The title compound was found to contain a heavily disordered solvent mol­ecule, which could be methanol or water or a mixture of the two. Due to its uncertain nature and the unresolvable disorder, the data were processed with the SQUEEZE option in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148–155], which revealed 877.8 Å3 of solvent-accessible volume per unit cell and 126 electron-units of scattering density or 109.7 Å3 (16 electron units) per organic mol­ecule.. This was not included in the calculations of overall formula weight, density and absorption coefficient. PMID:22807845

  11. Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores

    PubMed Central

    Zutz, Christoph; Bacher, Markus; Parich, Alexandra; Kluger, Bernhard; Gacek-Matthews, Agnieszka; Schuhmacher, Rainer; Wagner, Martin; Rychli, Kathrin; Strauss, Joseph

    2016-01-01

    One of the biggest challenges in public health is the rising number of antibiotic resistant pathogens and the lack of novel antibiotics. In recent years there is a rising focus on fungi as sources of antimicrobial compounds due to their ability to produce a large variety of bioactive compounds and the observation that virtually every fungus may still contain yet unknown so called “cryptic,” often silenced, compounds. These putative metabolites could include novel bioactive compounds. Considerable effort is spent on methods to induce production of these “cryptic” metabolites. One approach is the use of small molecule effectors, potentially influencing chromatin landscape in fungi. We observed that the supernatant of the fungus Doratomyces (D.) microsporus treated with valproic acid (VPA) displayed antimicrobial activity against Staphylococcus (S.) aureus and two methicillin resistant clinical S. aureus isolates. VPA treatment resulted in enhanced production of seven antimicrobial compounds: cyclo-(L-proline-L-methionine) (cPM), p-hydroxybenzaldehyde, cyclo-(phenylalanine-proline) (cFP), indole-3-carboxylic acid, phenylacetic acid (PAA) and indole-3-acetic acid. The production of the antimicrobial compound phenyllactic acid was exclusively detectable after VPA treatment. Furthermore three compounds, cPM, cFP, and PAA, were able to boost the antimicrobial activity of other antimicrobial compounds. cPM, for the first time isolated from fungi, and to a lesser extent PAA, are even able to decrease the minimal inhibitory concentration of ampicillin in MRSA strains. In conclusion we could show in this study that VPA treatment is a potent tool for induction of “cryptic” antimicrobial compound production in fungi, and that the induced compounds are not exclusively linked to the secondary metabolism. Furthermore this is the first discovery of the rare diketopiperazine cPM in fungi. Additionally we could demonstrate that cPM and PAA boost antibiotic activity

  12. L-Proline as an efficient and reusable promoter for the synthesis of coumarins in ionic liquid

    PubMed Central

    Liu, Xiu-hong; Fan, Ji-cai; Liu, Yang; Shang, Zhi-cai

    2008-01-01

    The effect of L-proline as a promoter on the condensation reaction of salicylaldehyde or its derivatives with ethyl acetoacetate in neutral ionic liquid [emim]BF4 was studied. All reactions were carried out under mild reaction conditions and achieved high yields. Moreover, the ionic liquid containing L-proline could be recycled and reused for several times without noticeably decreasing in productivity. The results show that the L-proline-[emim]BF4 system has a potential in contribution to the development of environmentally friendly and inexpensive processes in organic syntheses. PMID:19067468

  13. Effects of L-proline on the Growth Performance, and Blood Parameters in Weaned Lipopolysaccharide (LPS)-challenged Pigs

    PubMed Central

    Kang, Ping; Zhang, Lili; Hou, Yongqing; Ding, Binying; Yi, Dan; Wang, Lei; Zhu, Huiling; Liu, Yulan; Yin, Yulong; Wu, Guoyao

    2014-01-01

    This trail was conducted to study the effect of L-proline on the growth performance, and blood parameter in the weaned lipopolysaccharide (LPS)-challenged pigs. Thirty six pigs (9.13±0.85 kg) were assigned randomly to dietary treatments in a 2×3 factorial arrangement in a 20-d growth assay. Factors were intraperitoneal injection with saline or LPS, and three dietary L-proline supplement levels (0%, 0.5%, or 1.0%). On d 10, blood samples were collected at 3 h after LPS (100 μg LPS/kg body weight [BW]) or saline injection. On d 20 of the trial, all pigs were orally administrated D-xylose (0.1 g/kg BW) at 2 h, and blood samples were collected at 3 h after LPS or saline injection. As a result, dietary supplementation with 0.5% proline had a tendency to increase average daily gain (ADG) in piglets during d 10 to 20 (p = 0.088). Without LPS challenge, dietary supplementation with 1.0% proline had no effect on growth hormone (GH) concentrations on d 10 (p>0.05), but decreased it after LPS challenge (p<0.05). There was LPS challenge×proline interaction for GH concentrations on d 10 (p<0.05). Dietary supplementation with 1.0% proline decreased glucagon concentration on d 10 after LPS challenge (p<0.05). In addition, dietary supplementation with proline increased superoxide dismutase (SOD) activity significantly on d 10 and 20 (p<0.05), and 1.0% proline increased heat shock proteins-70 concentration on d 10 (p<0.05). Moreover, proline supplementation increased diamine oxidase (DAO) concentrations after LPS challenge (p<0.05). There was LPS challenge×proline interaction for DAO (p<0.05). Furthermore, dietary supplementation with 1.0% proline increased the D-xylose level when no LPS challenge (p<0.05). These results indicate that proline supplementation could improve growth performance, increase SOD activities, and has a positive effect on the gastrointestinal tract digestibility in early weaned pigs. PMID:25083109

  14. Roles of phytochemicals in amino acid nutrition.

    PubMed

    Kong, Xiangfeng; Wu, Guoyao; Yin, Yinlong

    2011-01-01

    Chinese herbal medicine (CHM) is often used as dietary supplements to maintain good health in animals and humans. Here, we review the current knowledge about effects of CHM (including ultra-fine Chinese herbal powder, Acanthopanax senticosus extracts, Astragalus polysaccharide, and glycyrrhetinic acid) as dietary additives on physiological and biochemical parameters in pigs, chickens and rodents. Additionally, we propose possible mechanisms for the beneficial effects of CHM on the animals. These mechanisms include (a) increased digestion and absorption of dietary amino acids; (b) altered catabolism of amino acids in the small intestine and other tissues; (c) enhanced synthesis of functional amino acids (e.g., arginine, glutamine and proline) and polyamines; and (d) improved metabolic control of nutrient utilization through cell signaling. Notably, some phytochemicals and glucocorticoids share similarities in structure and physiological actions. New research findings provide a scientific and clinical basis for the use of CHM to improve well-being in livestock species and poultry, while enhancing the efficiency of protein accretion. Results obtained from animal studies also have important implications for human nutrition and health. PMID:21196382

  15. Acceleration of the Rate-Limiting Step of Thioredoxin Folding by Replacement of its Conserved cis-Proline with (4 S)-Fluoroproline.

    PubMed

    Roderer, Daniel; Glockshuber, Rudi; Rubini, Marina

    2015-10-12

    The incorporation of the non-natural amino acids (4R)- and (4S)-fluoroproline (Flp) has been successfully used to improve protein stability, but little is known about their effect on protein folding kinetics. Here we analyzed the influence of (4R)- and (4S)-Flp on the rate-limiting trans-to-cis isomerization of the Ile75-Pro76 peptide bond in the folding of Escherichia coli thioredoxin (Trx). While (4R)-Flp at position 76 had essentially no effect on the isomerization rate in the context of the intact tertiary structure, (4S)-Flp accelerated the folding reaction ninefold. Similarly, tenfold faster trans-to-cis isomerization of Ile75-(4S)-Flp76 relative to Ile75-Pro76 was observed in the unfolded state of Trx. Our results show that the replacement of cis prolines by non-natural proline analogues can be used for modulating the folding rates of proteins with cis prolyl-peptide bonds in the native state. PMID:26382254

  16. Structure-Activity Relationships of the Antimicrobial Peptide Arasin 1 — And Mode of Action Studies of the N-Terminal, Proline-Rich Region

    PubMed Central

    Paulsen, Victoria S.; Blencke, Hans-Matti; Benincasa, Monica; Haug, Tor; Eksteen, Jacobus J.; Styrvold, Olaf B.; Scocchi, Marco; Stensvåg, Klara

    2013-01-01

    Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH2 terminus of the peptide and the fragment arasin 1(1–23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1–23) were shown to be non-toxic to human red blood cells and arasin 1(1–23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1–23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1–23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1–23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1–23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC. PMID:23326415

  17. Thermally reversible gels based on acryloyl- L-proline methyl ester as drug delivery systems

    NASA Astrophysics Data System (ADS)

    Martellini, Flavia; Higa, Olga Z.; Takacs, Erzsebet; Safranj, Agneza; Yoshida, Masaru; Katakai, Ryoichi; Carenza, Mario

    1999-06-01

    Thermally reversible hydrogels were synthesized by radiation-induced copolymerization of acryloyl- L-proline methyl ester with hydrophilic or hydrophobic monomers. The swelling behaviour was found to be affected by a proper balance of the latter. In particular, the transition temperature of the different hydrogels shifted to higher or lower values depending on the presence of hydrophilic or hydrophobic moieties in the polymer chain, respectively. Acetaminophen, an analgesic and antipyretic drug, was entrapped into some hydrogels and a wide range of release rates was obtained according to the nature of the comonomers. A novel thermoresponsive hydrogel was also prepared by radiation polymerization of acryloyl- L-proline methyl ester in the presence of 4-acryloyloxy acetanilide, an acrylic derivative of acetaminophen. Again, the swelling curves showed an inverse function of temperature. It was shown that with this hydrogel bearing the drug covalently attached to the polymer backbone, the hydrolysis process was the rate-determining process of the drug release.

  18. Measuring the Elasticity of Poly-l-Proline Helices with Terahertz Spectroscopy.

    PubMed

    Ruggiero, Michael T; Sibik, Juraj; Orlando, Roberto; Zeitler, J Axel; Korter, Timothy M

    2016-06-01

    The rigidity of poly-l-proline is an important contributor to the stability of many protein secondary structures, where it has been shown to strongly influence bulk flexibility. The experimental Young's moduli of two known poly-l-proline helical forms, right-handed all-cis (Form I) and left-handed all-trans (Form II), were determined in the crystalline state by using an approach that combines terahertz time-domain spectroscopy, X-ray diffraction, and solid-state density functional theory. Contrary to expectations, the helices were found to be considerably less rigid than many other natural and synthetic polymers, as well as differing greatly from each other, with Young's moduli of 4.9 and 9.6 GPa for Forms I and II, respectively. PMID:27121300

  19. Menthols as Chiral Auxiliaries for Asymmetric Cycloadditive Oligomerization: Syntheses and Studies of β-Proline Hexamers.

    PubMed

    Kudryavtsev, Konstantin V; Ivantcova, Polina M; Muhle-Goll, Claudia; Churakov, Andrei V; Sokolov, Mikhail N; Dyuba, Artem V; Arutyunyan, Alexander M; Howard, Judith A K; Yu, Chia-Chun; Guh, Jih-Hwa; Zefirov, Nikolay S; Bräse, Stefan

    2015-12-18

    To produce a novel class of structurally ordered poly-β-prolines, an emergent method for synthesizing chiral β-peptide molecular frameworks was developed based on 1,3-dipolar cycloaddition chemistry of azomethine ylides. Functionalized short β-peptides with up to six monomeric residues were efficiently synthesized in homochiral forms using a cycloadditive oligomerization approach. X-ray, NMR, and CD structural analyses of the novel β-peptides revealed secondary structure features that were generated primarily by Z/E-β-peptide bond isomerism. Anticancer in cellulo activity of the new β-peptides toward hormone-refractory prostate cancer cells was observed and was dependent on the absolute configuration of the stereogenic centers and the chain length of the β-proline oligomers. PMID:26624275

  20. Measuring the Elasticity of Poly‐l‐Proline Helices with Terahertz Spectroscopy

    PubMed Central

    Ruggiero, Michael T.; Sibik, Juraj; Orlando, Roberto; Zeitler, J. Axel

    2016-01-01

    Abstract The rigidity of poly‐l‐proline is an important contributor to the stability of many protein secondary structures, where it has been shown to strongly influence bulk flexibility. The experimental Young's moduli of two known poly‐l‐proline helical forms, right‐handed all‐cis (Form I) and left‐handed all‐trans (Form II), were determined in the crystalline state by using an approach that combines terahertz time‐domain spectroscopy, X‐ray diffraction, and solid‐state density functional theory. Contrary to expectations, the helices were found to be considerably less rigid than many other natural and synthetic polymers, as well as differing greatly from each other, with Young's moduli of 4.9 and 9.6 GPa for Forms I and II, respectively. PMID:27121300

  1. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus.

    PubMed

    Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yanjin

    2016-01-01

    Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology. PMID:27548202

  2. Functional analysis of a proline to serine mutation in codon 453 of the thyroid hormone receptor {beta}1 gene

    SciTech Connect

    Ozata, M.; Suzuki, Satoru; Takeda, Teiji

    1995-10-01

    Mutations in the gene encoding human thyroid hormone receptor {beta}(hTR{beta}) have been associated with generalized resistance to thyroid hormone (GRTH). This disorder is associated with significant behavoral abnormalities. We examined the hTR{beta} gene in a family with members who manifest inappropriately normal TSH, elevated free T{sub 4}, and free and total T{sub 3}. Sequence analysis showed a cytosine to thymine transition at nucleotide 1642 in one allele of the index patient`s genomic DNA. This altered proline to serine at codon 453. The resulting mutant receptor when expressed in vitro bound DNA with high affinity, but the T{sub 3} affinity of the receptor was impaired. The mutant TR demonstrated a dominant negative effect when cotransfected with two isoforms of wild-type receptor and also in the presence of TR variant {alpha}2 in COS-1 cells. Mutations of codon 453 occur more frequently than at other sites, and four different amino acid substitutions have been reported. Significant differences in phenotype occur among affected individuals, varying from normality to moderately severe GRTH. There is no clear correlation between K{sub a} or in vitro function of the mutant receptor, and phenotype. This study extends the association between GRTH and illness, and indicates that early diagnosis and counseling are needed in families with TR{beta}1 abnormalities. 34 refs., 5 figs., 2 tabs.

  3. Proline hydration at low temperatures: its role in the protection of cell from freeze-induced stress.

    PubMed

    Fedotova, Marina V; Dmitrieva, Olga A

    2016-07-01

    The natural amino acid L-α-proline (Pro) is a compatible osmolyte which accumulates in the cell cytoplasm to protect structure and function of various proteins and enzymes under abiotic stress, like for instance, freezing. It is assumed that the interactions of Pro with intracellular water play an important role in the protection mechanism. However, until now the details of these interactions are far from being fully understood. We present results of a theoretical study of the hydration of Pro zwitterion (Pro-ZW) in water in the temperature range of 298-248 K. The data were obtained by the integral equation method in the framework of the 1D- and 3D-RISM approaches. The structural data were analyzed in terms of radial and spatial distribution functions. The observed features of Pro-ZW hydration are discussed from the position of the biological role of Pro as a cryoprotectant. In particular, it was found that under cooling conditions this protectant is able to bind a significant amount of water molecules and, thus, is helping to keep water inside the cell. PMID:27085633

  4. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E.

    1996-04-01

    The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

  5. Adaptation to a long term (4 weeks) arginine- and precursor (glutamate, proline and aspartate)-free diet☆

    PubMed Central

    Tharakan, John F.; Yu, Yong M.; Zurakowski, David; Roth, Rachel M.; Young, Vernon R.; Castillo, Leticia

    2008-01-01

    Summary Background & aims It is not known whether arginine homeostasis is negatively affected by a “long term” dietary restriction of arginine and its major precursors in healthy adults. To assess the effects of a 4-week arginine- and precursor-free dietary intake on the regulatory mechanisms of arginine homeostasis in healthy subjects. Methods Ten healthy adults received a complete amino acid diet for 1 week (control diet) and following a break period, six subjects received a 4-week arginine, proline, glutamate and aspartate-free diet (APF diet). The other four subjects continued for 4 weeks with the complete diet. On days 4 and 7 of the first week and days 25 and 28 of the 4-week period, the subjects received 24-h infusions of arginine, citrulline, leucine and urea tracers. Results During the 4-week APF, plasma arginine fluxes for the fed state, were significantly reduced. There were no significant differences for citrulline, leucine or urea fluxes. Arginine de novo synthesis was not affected by the APF intake. However, arginine oxidation was significantly decreased. Conclusions In healthy adults, homeostasis of arginine under a long term arginine- and precursor-free intake is achieved by decreasing catabolic rates, while de novo arginine synthesis is maintained. PMID:18590940

  6. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus

    PubMed Central

    Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yanjin

    2016-01-01

    Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology. PMID:27548202

  7. L-Proline Derived Bifunctional Organocatalysts: Enantioselective Michael Addition of Dithiomalonates to trans-β-Nitroolefins.

    PubMed

    Jin, Hui; Kim, Seung Tae; Hwang, Geum-Sook; Ryu, Do Hyun

    2016-04-15

    A series of novel L-proline derived tertiary amine bifunctional organocatalysts 9 are reported, which were applied to the asymmetric Michael addition of dithiomalonates 2 to trans-β-nitroolefins 1. The reaction proceeded in high yields (up to 99%) with high enantioselectivities (up to 97% ee). The synthetic utility of this methodology was demonstrated in the short synthesis of (R)-phenibut in high yield. PMID:26989804

  8. Organocatalytic enantio- and diastereoselective synthesis of 3,5-disubstituted prolines.

    PubMed

    Riaño, Iker; Díaz, Estibaliz; Uria, Uxue; Reyes, Efraím; Carrillo, Luisa; Vicario, Jose L

    2016-02-01

    The asymmetric synthesis of substituted pyrrolidines has been accomplished using a novel organocatalytic cyclization reaction promoted by a Cinchona alkaloid based primary amine. The reaction proceeds smoothly yielding pyrrolidine-2,2-dicarboxylates after in situ diastereoselective reduction with high levels of enantioselection. Furthermore, these adducts could be easily transformed into N-protected disubstituted prolines through the base-promoted diastereoselective C → N alkoxycarbonyl transfer reaction. PMID:26728548

  9. Changes in antioxidant enzymes activities and proline, total phenol and anthocyanine contents in Hyssopus officinalis L. plants under salt stress.

    PubMed

    Jahantigh, Omolbanin; Najafi, Farzaneh; Badi, Hassanali Naghdi; Khavari-Nejad, Ramazan Ali; Sanjarian, Forough

    2016-06-01

    The relationships between salt stress and antioxidant enzymes activities, proline, phenol and anthocyanine contents in Hyssopus officinalis L. plants in growth stage were investigated. The plants were subjected to five levels of saline irrigation water, 0.37 (tap water as control) with 2, 4, 6, 8 and 10 dSm(-1) of saline water. After two months the uniform plants were harvested for experimental analysis. Antioxidant enzymes activities and proline, phenol and anthocyanine contents of the plants were examinated. Enhanced activities of peroxidase, catalase and superoxide dismutase were determined by increasing salinity that plays an important protective role in the ROS-scavenging process. Proline, phenol and anthocyanine contents increased significantly with increasing salinity. These results suggest that salinity tolerance of Hyssopus officinalis plants might be closely related with the increased capacity of antioxidative system to scavenge reactive oxygen species and with the accumulation of osmoprotectant proline, phenol and anthocyanine contents under salinity conditions. PMID:27165530

  10. Force-dependent isomerization kinetics of a highly conserved proline switch modulates the mechanosensing region of filamin

    PubMed Central

    Rognoni, Lorenz; Möst, Tobias; Žoldák, Gabriel; Rief, Matthias

    2014-01-01

    Proline switches, controlled by cis–trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. In this study, we use single-molecule mechanical measurements to develop a full kinetic and energetic description of a highly conserved proline switch in the force-sensing domain 20 of human filamin and how prolyl isomerization modulates the force-sensing mechanism. Proline isomerization toggles domain 20 between two conformations. A stable cis conformation with slow unfolding, favoring the autoinhibited closed conformation of filamin’s force-sensing domain pair 20–21, and a less stable, uninhibited conformation promoted by the trans form. The data provide detailed insight into the folding mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency in modulating force-sensing, thus combining two previously unconnected regulatory mechanisms, proline switches and mechanosensing. PMID:24706888

  11. Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation.

    PubMed

    Kraus, Allison; Anson, Kelsie J; Raymond, Lynne D; Martens, Craig; Groveman, Bradley R; Dorward, David W; Caughey, Byron

    2015-08-28

    Human prion diseases can have acquired, sporadic, or genetic origins, each of which results in the conversion of prion protein (PrP) to transmissible, pathological forms. The genetic prion disease Gerstmann-Straussler-Scheinker syndrome can arise from point mutations of prolines 102 or 105. However, the structural effects of these two prolines, and mutations thereof, on PrP misfolding are not well understood. Here, we provide evidence that individual mutations of Pro-102 or Pro-105 to noncyclic aliphatic residues such as the Gerstmann-Straussler-Scheinker-linked leucines can promote the in vitro formation of PrP amyloid with extended protease-resistant cores reminiscent of infectious prions. This effect was enhanced by additional charge-neutralizing mutations of four nearby lysine residues comprising the so-called central lysine cluster. Substitution of these proline and lysine residues accelerated PrP conversion such that spontaneous amyloid formation was no longer slower than scrapie-seeded amyloid formation. Thus, Pro-102 and Pro-105, as well as the lysines in the central lysine cluster, impede amyloid formation by PrP, implicating these residues as key structural modulators in the conversion of PrP to disease-associated types of amyloid. PMID:26175152

  12. Three crystal forms of the bifunctional enzyme proline utilization A (PutA) from Bradyrhizobium japonicum.

    PubMed

    Schuermann, Jonathan P; White, Tommi A; Srivastava, Dhiraj; Karr, Dale B; Tanner, John J

    2008-10-01

    Proline utilization A proteins (PutAs) are large (1000-1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains. Here, the first successful crystallization efforts for a PutA protein are described. Three crystal forms of PutA from Bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. The apparent tetragonal and hexagonal crystals were grown in the presence of PEG 3350 and sodium formate near pH 7. The apparent tetragonal form diffracted to 2.7 A resolution and exhibited pseudo-merohedral twinning such that the true space group is P2(1)2(1)2(1) with four molecules in the asymmetric unit. The hexagonal form diffracted to 2.3 A resolution and belonged to space group P6(2)22 with one molecule in the asymmetric unit. Centered monoclinic crystals were grown in ammonium sulfate, diffracted to 2.3 A resolution and had two molecules in the asymmetric unit. Removing the histidine tag was important in order to obtain the C2 crystal form. PMID:18931443

  13. Unique Structural Features and Sequence Motifs of Proline Utilization A (PutA)

    PubMed Central

    Singh, Ranjan K.; Tanner, John J.

    2013-01-01

    Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium japonicum (BjPutA) along with sequence analysis to identify unique structural features of PutAs. This analysis shows that PutAs have secondary structural elements and domains not found in the related monofunctional enzymes. Some of these extra features are predicted to be important for substrate channeling in BjPutA. Multiple sequence alignment analysis shows that some PutAs have a 17-residue conserved motif in the C-terminal 20–30 residues of the polypeptide chain. The BjPutA structure shows that this motif helps seal the internal substrate-channeling cavity from the bulk medium. Finally, it is shown that some PutAs have a 100–200 residue domain of unknown function in the C-terminus that is not found in minimalist PutAs. Remote homology detection suggests that this domain is homologous to the oligomerization beta-hairpin and Rossmann fold domain of BjPutA. PMID:22201760

  14. Three crystal forms of the bifunctional enzyme proline utilization A (PutA) from Bradyrhizobium japonicum

    PubMed Central

    Schuermann, Jonathan P.; White, Tommi A.; Srivastava, Dhiraj; Karr, Dale B.; Tanner, John J.

    2008-01-01

    Proline utilization A proteins (PutAs) are large (1000–1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase domains. Here, the first successful crystallization efforts for a PutA protein are described. Three crystal forms of PutA from Bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. The apparent tetragonal and hexagonal crystals were grown in the presence of PEG 3350 and sodium formate near pH 7. The apparent tetragonal form diffracted to 2.7 Å resolution and exhibited pseudo-merohedral twinning such that the true space group is P212121 with four molecules in the asymmetric unit. The hexagonal form diffracted to 2.3 Å resolution and belonged to space group P6222 with one molecule in the asymmetric unit. Centered monoclinic crystals were grown in ammonium sulfate, diffracted to 2.3 Å resolution and had two molecules in the asymmetric unit. Removing the histidine tag was important in order to obtain the C2 crystal form. PMID:18931443

  15. Unique structural features and sequence motifs of proline utilization A (PutA).

    PubMed

    Singh, Ranjan K; Tanner, John J

    2012-01-01

    Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium japonicum (BjPutA) along with sequence analysis to identify unique structural features of PutAs. This analysis shows that PutAs have secondary structural elements and domains not found in the related monofunctional enzymes. Some of these extra features are predicted to be important for substrate channeling in BjPutA. Multiple sequence alignment analysis shows that some PutAs have a 17-residue conserved motif in the C-terminal 20-30 residues of the polypeptide chain. The BjPutA structure shows that this motif helps seal the internal substrate-channeling cavity from the bulk medium. Finally, it is shown that some PutAs have a 100-200 residue domain of unknown function in the C-terminus that is not found in minimalist PutAs. Remote homology detection suggests that this domain is homologous to the oligomerization beta-hairpin and Rossmann fold domain of BjPutA. PMID:22201760

  16. Use of prolines for improving growth and/or yield

    DOEpatents

    Unkefer, Pat J.; Martinez, Rodolfo A.; Knight, Thomas J.

    2014-08-12

    The present invention describes a composition including a mixture of L- and D-pyroglutamate stereoisomers in a ratio of L to D of from about 80:20 to about 97:3 , and, a carrier medium for application of the L- and D-pyroglutamate stereoisomers to a target plant. The composition can also be used as a germination medium and may be incorporated into a seed coat for assisting in germination. The present invention further describes a method of increasing the agronomic performance of a target plant by treating a target plant with a composition including a mixture of L- and D-pyroglutamate stereoisomers in a ratio of L to D of from about 80:20 to about 97:3 and a carrier medium for said L- and D-pyroglutamate stereoisomers.

  17. Use of prolines for improving growth and/or yield

    DOEpatents

    Unkefer, Pat J; Martinez, Rodolfo A; Knight, Thomas J

    2013-10-08

    The present invention describes a composition including a mixture of L- and D-pyroglutamate stereoisomers in a ratio of L to D of from about 80:20 to about 97:3, and, a carrier medium for application of the L- and D-pyroglutamate stereoisomers to a target plant. The composition can also be used as a germination medium and may be incorporated into a seed coat for assisting in germination. The present invention further describes a method of increasing the agronomic performance of a target plant by treating a target plant with a composition including a mixture of L- and D-pyroglutamate stereoisomers in a ratio of L to D of from about 80:20 to about 97:3 and a carrier medium for said L- and D-pyroglutamate stereoisomers.

  18. Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline-Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays.

    PubMed

    Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

    2016-06-01

    Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular

  19. A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding

    PubMed Central

    Harty, Ronald N.; Paragas, Jason; Sudol, Marius; Palese, Peter

    1999-01-01

    The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the gag-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common. PMID:10074141

  20. Amino Acid Intakes Are Associated With Bone Mineral Density and Prevalence of Low Bone Mass in Women: Evidence From Discordant Monozygotic Twins.

    PubMed

    Jennings, Amy; MacGregor, Alexander; Spector, Tim; Cassidy, Aedín

    2016-02-01

    Although a higher protein intake, particularly from vegetable sources, has been shown to be associated with higher bone mineral density (BMD) the relative impact of specific amino acids on BMD and risk of osteoporosis remains to be determined. Mechanistic research suggests that a number of specific amino acids, including five nonessential amino acids--alanine, arginine, glutamic acid, glycine, and proline--may play a role in bone health, principally through improved production of insulin and insulin-like growth factor 1 and the synthesis of collagen and muscle protein. However to date, no previous studies have examined the associations between habitual intake of amino acids and direct measures of BMD and prevalence of osteoporosis or osteopenia, and no studies have examined this relationship in discordant identical twin-pairs. In these analyses of female monozygotic twin-pairs discordant for amino acid intake (n = 135), twins with higher intakes of alanine and glycine had significantly higher BMD at the spine than their co-twins with within-pair differences in spine-BMD of 0.012 g/cm(2) (SE 0.01; p = 0.039) and 0.014 g/cm(2) (SE 0.01; p = 0.026), respectively. Furthermore, in cross-sectional multivariable analyses of 3160 females aged 18 to 79 years, a higher intake of total protein was significantly associated with higher DXA-measured BMD at the spine (quartile Q4 to quartile Q1: 0.017 g/cm(2), SE 0.01, p = 0.035) and forearm (Q4 to Q1: 0.010 g/cm(2), SE 0.003, p = 0.002). Intake of six amino acids (alanine, arginine, glutamic acid, leucine, lysine, and proline) were associated with higher BMD at the spine and forearm with the strongest association observed for leucine (Q4 to Q1: 0.024 g/cm(2), SE 0.01, p = 0.007). When intakes were stratified by protein source, vegetable or animal, prevalence of osteoporosis or osteopenia was 13% to 19% lower comparing extreme quartiles of vegetable intake for five amino acids (not glutamic acid or proline). These data provide

  1. Amino acid imbalance in cystinuria

    PubMed Central

    Asatoor, A. M.; Freedman, P. S.; Gabriel, J. R. T.; Milne, M. D.; Prosser, D. I.; Roberts, J. T.; Willoughby, C. P.

    1974-01-01

    After oral ingestion of a free amino acid mixture by three cystinuric patients, plasma increments of lysine and arginine were lower and those of many other amino acids were significantly higher than those found in control subjects. Similar results were obtained in control subjects after amino acid imbalance had been artificially induced by the omission of cystine, lysine, and arginine from the amino acid mixture. Especially high increments of alanine and proline provided the best evidence of amino acid imbalance caused by a temporary lysine and, to a lesser extent, arginine and cystine deficit. No such amino acid imbalance was found to occur in the cystinuric patients after ingestion of whole protein, indicating that absorption of oligopeptides produced by protein digestion provided a balanced physiological serum amino acid increment. This is considered to explain the lack of any unequivocal nutritional deficit in cystinuric patients despite poor absorption of the essential free amino acid, lysine. PMID:4411931

  2. Effective atomic numbers and electron densities of bacteriorhodopsin and its comprising amino acids in the energy range 1 keV-100 GeV

    NASA Astrophysics Data System (ADS)

    Ahmadi, Morteza; Lunscher, Nolan; Yeow, John T. W.

    2013-04-01

    Recently, there has been an interest in fabrication of X-ray sensors based on bacteriorhodopsin, a proton pump protein in cell membrane of Halobacterium salinarium. Therefore, a better understanding of interaction of X-ray photons with bacteriorhodopsin is required. We use WinXCom program to calculate the mass attenuation coefficient of bacteriorhodopsin and its comprising amino acids for photon energies from 1 keV to 100 GeV. These amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, Asx1, Asx2, Glx1 and Glx2. We then use that data to calculate effective atomic number and electron densities for the same range of energy. We also emphasize on two ranges of energies (10-200 keV and 1-20 MeV) in which X-ray imaging and radiotherapy machines work.

  3. β-amylase 1 (BAM1) degrades transitory starch to sustain proline biosynthesis during drought stress.

    PubMed

    Zanella, Martina; Borghi, Gian Luca; Pirone, Claudia; Thalmann, Matthias; Pazmino, Diana; Costa, Alex; Santelia, Diana; Trost, Paolo; Sparla, Francesca

    2016-03-01

    During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. β-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly β-amylase-3 (BAM3). A second β-amylase isoform, β-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of β-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops. PMID:26792489

  4. Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.

    PubMed

    Arentson, Benjamin W; Luo, Min; Pemberton, Travis A; Tanner, John J; Becker, Donald F

    2014-08-12

    Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ(1)-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH-P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA. PMID:25046425

  5. Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action

    PubMed Central

    Roy, Ankita; Al-Qusairi, Lama; Donnelly, Bridget F.; Ronzaud, Caroline; Marciszyn, Allison L.; Gong, Fan; Chang, Y.P. Christy; Butterworth, Michael B.; Pastor-Soler, Núria M.; Hallows, Kenneth R.; Staub, Olivier; Subramanya, Arohan R.

    2015-01-01

    The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif–containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2–suppressing antinatriuretic hormones to NCC phosphorylation status. PMID:26241057

  6. Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A

    PubMed Central

    2015-01-01

    Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ1-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH–P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA. PMID:25046425

  7. Enantioselective reduction of ketoimines promoted by easily available (S)-proline derivatives

    PubMed Central

    Bonsignore, Martina; Raimondi, Laura; Orlandi, Manuel; Celentano, Giuseppe

    2013-01-01

    Summary The behavior of readily synthesized and even commercially available (S)-proline derivatives, was studied in the trichlorosilane-mediated reduction of ketoimines. A small library of structurally and electronically modified chiral Lewis bases was considered; such compounds were shown to promote the enantioselective reduction of different substrates in good chemical yields. In the HSiCl3 addition to the model substrate N-phenylacetophenone imine, the organocatalyst of choice led to the formation of the corresponding amine with good stereoselectivity, up to 75% ee. Theoretical studies were also performed in order to elucidate the origin of the stereoselection. PMID:23616807

  8. Steric effect exerted by the proline residue on the antecedent alanine residue.

    PubMed

    Siemión, I Z; Sobczyk, K; Nawrocka, E

    1982-05-01

    Five model tetrapeptides: Ala-Ala-Ala-Ala, Pro-Ala-Ala-Ala, Ala-Pro-Ala-Ala, Ala-Ala-Pro-Ala and Ala-Ala-Ala-Pro, were synthesized and measured in D2O by 13 C-n.m.r. spectroscopy. The spectra analysis led us to the conclusion that for each model (irrespective of pD) in conformational equilibrium, the predominant conformation is the one in which side methyl of alanine preceding proline residue eclipses alanine carbonyl group. The influence of pD changes in cis-trans isomerism of Ala-Pro amide bond was also investigated. PMID:7118413

  9. Prebiotic Synthesis of Hydrophobic and Protein Amino Acids

    PubMed Central

    Ring, David; Wolman, Yecheskel; Friedmann, Nadav; Miller, Stanley L.

    1972-01-01

    The formation of amino acids by the action of electric discharges on a mixture of methane, nitrogen, and water with traces of ammonia was studied in detail. The presence of glycine, alanine, α-amino-n-butyric acid, α-aminoisobutyric acid, valine, norvaline, isovaline, leucine, isoleucine, alloisoleucine, norleucine, proline, aspartic acid, glutamic acid, serine, threonine, allothreonine, α-hydroxy-γ-aminobutyric acid, and α,γ-diaminobutyric acid was confirmed by ion-exchange chromatography and gas chromatography-mass spectrometry. All of the primary α-amino acids found in the Murchison Meteorite have been synthesized by this electric discharge experiment. PMID:4501592

  10. Use of Umbrella Sampling to Calculate the Entrance/Exit Pathway for Z-Pro-Prolinal Inhibitor in Prolyl Oligopeptidase.

    PubMed

    St-Pierre, Jean-François; Karttunen, Mikko; Mousseau, Normand; Róg, Tomasz; Bunker, Alex

    2011-06-14

    Prolyl oligopeptidase (POP), a member of the prolyl endopeptidase family, is known to play a role in several neurological disorders. Its primary function is to cleave a wide range of small oligopeptides, including neuroactive peptides. We have used force biased molecular dynamics simulation to study the binding mechanism of POP. We examined three possible binding pathways using Steered Molecular Dynamics (SMD) and Umbrella Sampling (US) on a crystal structure of porcine POP with bound Z-pro-prolinal (ZPP). Using SMD, an exit pathway between the first and seventh blade of the β-propeller domain of POP was found to be a nonviable route. US on binding pathways through the β-propeller tunnel and the TYR190-GLN208 flexible loop at the interface between both POP domains allowed us to isolate the flexible loop pathway as the most probable. Further analysis of that pathway suggests a long-range covariation of the interdomain H-bond network, which indicates the possibility of large-scale domain reorientation observed in bacterial homologues and hypothesized to also occur in human POP. PMID:26596426

  11. Hypothalamic proline-rich polypeptide enhances bone marrow colony-forming cell proliferation and stromal progenitor cell differentiation.

    PubMed

    Galoyan, A A; Korochkin, L I; Rybalkina, E J; Pavlova, G V; Saburina, I N; Zaraiski, E I; Galoyan, N A; Davtyan, T K; Bezirganyan, K B; Revishchin, A V

    2008-01-01

    The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3-4 days of human BM stromal cell cultivation in the presence of 2-20 microg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3-4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus-bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation. PMID:19177842

  12. Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

    PubMed

    Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

    2006-12-01

    The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD. PMID:17161366

  13. Aspartic acid

    MedlinePlus

    ... also called asparaginic acid. Aspartic acid helps every cell in the body work. It plays a role in: Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as ...

  14. Concordance between isolated cleft palate in mice and alterations within a region including the gene encoding the [beta][sub 3] subunit of the type A [gamma]-aminobutyric acid receptor

    SciTech Connect

    Culiat, C.T.; Stubbs, L.; Nicholls, R.D.; Montgomery, C.S.; Russell, L.B.; Johnson, D.K. ); Rinchik, E.M. Univ. of Florida, Gainesville )

    1993-06-01

    Genetic and molecular analyses of a number of radiation-induced deletion mutations of the pink-eyed dilution (p) locus in mouse chromosome 7 have identified a specific interval on the genetic map associated with a neonatally lethal mutation that results in cleft palate. This interval, closely linked and distal to p, and bracketed by the genes encoding the [alpha][sub 5] and [beta][sub 3] subunits of the type A [gamma]-aminobutyric acid receptor (Gabra5 and Gabrb3, respectively), contains a gene(s) (cp1; cleft palate 1) necessary for normal palate development. The cp1 interval extends from the distal breakpoint of the prenatally lethal p[sup 83FBFo] deletion to the Gabrb3 locus. Among 20 p deletions tested, there was complete concordance between alterations at the Gabrb3 transcription unit and inability to complement the cleft-palate defect. These mapping data, along with previously described in vivo and in vitro teratological effects of [gamma]-aminobutyric acid or its agonists on palate development, suggest the possibility that a particular type A [gamma]-aminobutyric acid receptor that includes the [beta][sub 3] subunit may be necessary for normal palate development. The placement of the cp1 gene within a defined segment of the larger D15S12h (p)-D15S9h-1 interval in the mouse suggests that the highly homologous region of the human genome, 15q11-q13, be evaluated for a role(s) in human fetal facial development. 29 refs., 4 figs., 1 tab.

  15. RhNGF slow unfolding is not due to proline isomerization: possibility of a cystine knot loop-threading mechanism.

    PubMed Central

    De Young, L. R.; Burton, L. E.; Liu, J.; Powell, M. F.; Schmelzer, C. H.; Skelton, N. J.

    1996-01-01

    The unfolding of recombinant human beta-NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 degrees C. Fluorescence spectroscopy, size-exclusion and reversed-phase HPLC, ultra-centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little beta-sheet structure, but retained residual structure in the tryptophan indole and high-field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half-life of 23 h at 25 degrees C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N-terminus. A model for the slow unfolding reaction is proposed where the N-terminus threads through the cystine knot to form M2, a loop-threading reaction, increasing the conformational freedom of the denatured state. PMID:8844846

  16. A putative Δ1-pyrroline-5-carboxylate synthetase involved in the biosynthesis of proline and arginine in Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Fu, Kai-Yun; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2014-12-01

    Delta 1-pyrroline-5-carboxylate synthetase (P5CS) catalyzes the conversion of glutamate (Glu) to Glu semialdehyde (GSA). GSA spontaneously cyclizes to form P5C. P5C is then reduced to proline (Pro) or is converted to ornithine, the intermediate for arginine (Arg) biosynthesis. In the present study, a full-length Ldp5cs complementary DNA was cloned from the Colorado potato beetle Leptinotarsa decemlineata, a notorious insect defoliator of potato in most potato-growing regions of the world. Ldp5cs encodes a 792-amino-acid protein which shares high identity to homologues from other insect species. Quantitative reverse transcription polymerase chain reaction revealed that Ldp5cs was ubiquitously expressed in the eggs, first to fourth-instar larvae, wandering larvae, pupae and sexually mature adults. In the adults, Ldp5cs mRNA levels were higher in the fat body, foregut, midgut and hindgut, moderate in the ventral ganglion, lower in the thorax muscles, epidermis and Malpighian tubules. Two double-stranded RNAs (dsRNAs) (dsLdp5cs1 and dsLdp5cs2) targeting Ldp5cs were constructed and bacterially expressed. Ingestion during 3 consecutive days of dsLdp5cs1 or dsLdp5cs2 successfully silenced Ldp5cs, significantly reduced the contents of Pro and Arg in the hemolymph, decreased flight speed and shortened flight distance of the resulting adults. Furthermore, knocking down Ldp5cs significantly increased adult mortality. Thus, our results suggest that identified Ldp5cs encodes a functional P5CS enzyme that is involved in the biosynthesis of Pro and Arg in L. decemlineata. PMID:25450565

  17. Crosstalk Among Disrupted Glutamatergic and Cholinergic Homeostasis and Inflammatory Response in Mechanisms Elicited by Proline in Astrocytes.

    PubMed

    Loureiro, Samanta Oliveira; Sidegum, Daniele Susana Volkart; Biasibetti, Helena; Pereira, Mery Stefani Leivas; de Oliveira, Diogo Losch; Pessoa-Pureur, Regina; Wyse, Angela T S

    2016-03-01

    Hyperprolinemias are inherited disorder of proline (Pro) metabolism. Patients affected may present neurological manifestations, but the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering that the astrocytes are important players in neurological disorders, the aim of the present work was to study the effects 1 mM Pro on glutamatergic and inflammatory parameters in cultured astrocytes from cerebral cortex of rats, exploring some molecular mechanisms underlying the disrupted homeostasis of astrocytes exposed to this toxic Pro concentration. We showed that cortical astrocytes of rats exposed to 1 mM Pro presented significantly elevated extracellular glutamate and glutamine levels, suggesting glutamate excitotoxicity. The excess of glutamate elicited by Pro together with increased glutamate uptake and upregulated glutamine synthetase (GS) activity supported misregulated glutamate homeostasis in astrocytic cells. High Pro levels also induced production/release of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. We also evidenced misregulation of cholinergic anti-inflammatory system with increased acetylcholinesterase (AChE) activity and decreased acetylcholine (ACh) levels, contributing to the inflammatory status in Pro-treated astrocytes. Our findings highlighted a crosstalk among disrupted glutamate homeostasis, cholinergic mechanisms, and inflammatory cytokines, since ionotropic (DL-AP5 and CNQX) and metabotropic (MCPG and MPEP) glutamate antagonists were able to restore the extracellular glutamate and glutamine levels; downregulate TNFα and IL6 production/release, modulate GS and AChE activities; and restore ACh levels. Otherwise, the non-steroidal anti-inflammatory drugs nimesulide, acetylsalicylic acid, ibuprofen, and diclofenac sodium decreased the extracellular glutamate and glutamine levels, downregulated GS and AChE activities, and restored ACh levels in Pro-treated astrocytes. Altogether, our

  18. Molecular characterization and evolution of the SPRR family of keratinocyte differentiation markers encoding small proline-rich proteins

    SciTech Connect

    Gibbs, S.; Fijneman, R.; Wiegant, J.; Van De Putte, P.; Backendorf, C. ); Van Kessel, A.D. )

    1993-06-01

    SPRR genes (formerly SPR) encode a novel class of polypeptides (small proline rich proteins) that are strongly induced during differentiation of human epidermal keratinocytes in vitro and in vivo. Recently the authors found that the N- and C-terminal domains of these proteins show strong sequence homology to loricrin and involucrin, suggesting that SPRR proteins constitute a new class of cornified envelope precursor proteins. Here they show that SPRR proteins are encoded by closely related members of a gene family, consisting of two genes for SPRR1, approximately seven genes for SPRR2, and a single gene for SPRR3. All SPRR genes are closely linked within a 300-kb DNA segment on human chromosome 1 band q21-q22, a region where the related loricrin and involucrin genes have also been mapped. The most characteristic feature of the SPRR gene family resides in the structure of the central segments of the encoded polypeptides that are built up from tandemly repeated units of either eight (SPRR1 and SPRR3) or nine (SPRR2) amino acids with the general consensus *K*PEP**. Sequencing data of the different members, together with their clustered chromosomal organization, strongly suggest that this gene family has evolved from a single progenitor gene by multiple intra- and intergenic duplications. Analysis of the different SPRR subfamilies reveals a gene-specific bias to either intra- or intergenic duplication. The authors propose that a process of homogenization has acted on the different members of one subfamily, whereas the different subfamilies appear to have diverged from each other, at the levels of both protein structure and gene regulation. 25 refs., 7 figs., 2 tab.

  19. A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes

    PubMed Central

    Lavillette, Dimitri; Maurice, Marielle; Roche, Catherine; Russell, Stephen J.; Sitbon, Marc; Cosset, François-Loïc

    1998-01-01

    The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors. PMID:9811733

  20. Important roles for the arginine family of amino acids in swine nutrition and production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arginine, glutamine, glutamate, proline, aspartate, asparagine, ornithine, and citrulline are interconvertible via complex interorgan metabolism in most mammals (including the pig). The major sites for their metabolism are the small intestine, kidneys, and liver, with cortisol being a key regulatory...

  1. Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

    PubMed Central

    Peter, Heidi; Weil, Brita; Burkovski, Andreas; Krämer, Reinhard; Morbach, Susanne

    1998-01-01

    Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km, 48 μM) to ectoine (Km, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km, 63 μM) to betaine (Km, 333 μM) and proline (Km, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP. PMID:9811661

  2. Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition

    PubMed Central

    Larson, John D.; Jenkins, Jermaine L.; Schuermann, Jonathan P.; Zhou, Yuzhen; Becker, Donald F.; Tanner, John J.

    2006-01-01

    PutA (proline utilization A) from Escherichia coli is a 1320-amino-acid residue protein that is both a bifunctional proline catabolic enzyme and an autogenous transcriptional repressor. Here, we report the first crystal structure of a PutA DNA-binding domain along with functional analysis of a mutant PutA defective in DNA binding. Crystals were grown using a polypeptide corresponding to residues 1–52 of E. coli PutA (PutA52). The 2.1 Å resolution structure of PutA52 mutant Lys9Met was determined using Se-Met MAD phasing, and the structure of native PutA52 was solved at 1.9 Å resolution using molecular replacement. Residues 3–46 form a ribbon–helix–helix (RHH) substructure, thus establishing PutA as the largest protein to contain an RHH domain. The PutA RHH domain forms the intertwined dimer with tightly packed hydrophobic core that is characteristic of the RHH family. The structures were used to examine the three-dimensional context of residues conserved in PutA RHH domains. Homology modeling suggests that Lys9 and Thr5 contact DNA bases through the major groove, while Arg15, Thr28, and His30 may interact with the phosphate backbone. Lys9 is shown to be essential for specific recognition of put control DNA using gel shift analysis of the Lys9Met mutant of full-length PutA. Lys9 is disordered in the PutA52 structure, which implies an induced-fit binding mechanism in which the side chain of Lys9 becomes ordered through interaction with DNA. These results provide new insights into the structural basis of DNA recognition by PutA and reveal three-dimensional structural details of the PutA dimer interface. PMID:17001030

  3. Vibrational spectroscopy of bacteriorhodopsin mutants: Evidence for the interaction of proline-186 with the retinylidene chromophore

    SciTech Connect

    Rothschild, K.J.; He, Y.W.; Mogi, T.; Marti, T.; Stern, L.J.; Khorana, H.G. )

    1990-06-26

    Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue by glycine preserves the normal protein structural changes during the photocycle.

  4. Pyrroline-5-carboxylate synthase and proline biosynthesis: From osmotolerance to rare metabolic disease

    PubMed Central

    Pérez-Arellano, Isabel; Carmona-Álvarez, Francisco; Martínez, Ana I; Rodríguez-Díaz, Jesús; Cervera, Javier

    2010-01-01

    Pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that exhibits glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR) activities. The enzyme is highly relevant in humans because it belongs to a combined route for the interconversion of glutamate, ornithine and proline. The deficiency of P5CS activity in humans is associated with a rare, inherited metabolic disease. It is well established that some bacteria and plants accumulate proline in response to osmotic stress. The alignment of P5CSs from different species and analysis of the solved structures of GK and GPR reveal high sequence and structural conservation. The information acquired from different mutant enzymes with increased osmotolerant properties, together with the position of the insertion found in the longer human isoform, permit the delimitation of the regulatory site of GK and P5CS and the proposal of a model of P5CS architecture. Additionally, the GK moiety of the human enzyme has been modeled and the known clinical mutations and polymorphisms have been mapped. PMID:20091669

  5. Proline transport across the intestinal microvillus membrane may be regulated by membrane physical properties.

    PubMed

    Sadowski, D C; Gibbs, D J; Meddings, J B

    1992-03-23

    There is now abundant evidence that integral membrane protein function may be modulated by the physical properties of membrane lipids. The intestinal brush border membrane represents a membrane system highly specialized for nutrient absorption and, thus, provides an opportunity to study the interaction between integral membrane transport proteins and their lipid environment. We have previously demonstrated that alterations in this environment may modulate the function of the sodium-dependent glucose transporter in terms of its affinity for glucose. In this communication we report that membrane lipid-protein interactions are distinctly different for the proline transport proteins. Maximal transport rates for L-proline by either the neutral brush border or imino transport systems are reduced 10-fold when the surrounding membrane environment is made more fluid over the physiological range that exists along the crypt-villus axis. Furthermore, in microvillus membrane vesicles prepared from enterocytes isolated from along the crypt-villus axis a similar gradient exists in the functional activity of these transport systems. This would imply that either the functional activity of these transporters are regulated by membrane physical properties or that the synthesis and insertion of these proteins is coordinated in concert with membrane physical properties as the enterocyte migrates up the crypt-villus axis. PMID:1567897

  6. Co-regulation of mitochondrial respiration by proline dehydrogenase/oxidase and succinate.

    PubMed

    Hancock, Chad N; Liu, Wei; Alvord, W Gregory; Phang, James M

    2016-03-01

    Proline dehydrogenase/oxidase (PRODH/POX) is a mitochondrial protein critical to multiple stress pathways. Because of the roles of PRODH/POX in signaling, and its shared localization to the mitochondrial inner membrane with the electron transport chain (ETC), we investigated whether there was a direct relationship between PRODH/POX and regulation of the ETC. We found that PRODH/POX binds directly to CoQ1 and that CoQ1-dependent PRODH/POX activity required functional Complex III and Complex IV. PRODH/POX supported respiration in living cells during nutrient stress; however, expression of PRODH/POX resulted in an overall decrease in respiratory fitness. Effects on respiratory fitness were inhibited by DHP and NAC, indicating that these effects were mediated by PRODH/POX-dependent reactive oxygen species (ROS) generation. PRODH/POX expression resulted in a dose-dependent down-regulation of Complexes I-IV of the ETC, and this effect was also mitigated by the addition of DHP and NAC. We found that succinate was an uncompetitive inhibitor of PRODH/POX activity, inhibited ROS generation by PRODH/POX, and alleviated PRODH/POX effects on respiratory fitness. The findings demonstrate novel cross-talk between proline and succinate respiration in vivo and provide mechanistic insights into observations from previous animal studies. Our results suggest a potential regulatory loop between PRODH/POX and succinate in regulation of mitochondrial respiration. PMID:26660760

  7. A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

    PubMed

    Fikru, Regassa; Hagos, Ashenafi; Rogé, Stijn; Reyna-Bello, Armando; Gonzatti, Mary Isabel; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe

    2014-01-01

    A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide. PMID:24416292

  8. The effect of salinity on chlorophyll, proline and mineral nutrients in common weeds of coastal rice fields in Malaysia.

    PubMed

    Hakim, M A; Juraimi, Abdul Shukor; Hanafi, M M; Ismail, Mohd Razi; Rafii, M Y; Aslani, Fazad; Selamat, A

    2014-09-01

    Six weed species (Leptochola chinensis, Echinochloa crus-galli, Echinochloa colona, Jussiaea linifolia, Oryza sativa (weedy rice) and Cyperus iria) were tested for their salt tolerant traits in terms of chlorophyll, proline and mineral nutrients accumulation against different salinity levels (0, 4, 8, 16, 24, 32, and 40 dS m(-1)). Chlorophyll a, b and total chlorophyll content, proline and mineral nutrients accumulation were determined. Salt stress showed prominent effect on all the parameters investigated and there were significant variations between the all weed species. Chlorophyll content, K+, Ca(2+) and Mg(2+) ions in both shoots and roots significantly decreased; while proline and Na+ accumulation significantly increased with increasing salinity up to 40 dS m(-1). In terms of overall performance, Cyperus iria and E. crus-galliwere relatively more tolerant; E. colona and J. linifolia were tolerant; L. chinensis and O. sativa L were salt sensitive, respectively. PMID:25204059

  9. Monthly changes of glycogen, lipid and free amino acid of oyster

    NASA Astrophysics Data System (ADS)

    Zhicui, Zhang; Changhu, Xue; Xin, Gao; Zhaojie, Li; Qi, Wang

    2006-07-01

    Monthly difference of the chemical composition of oyster cultured along the eastern coast of Shandong Province was analyzed. The components analyzed included glycogen, fatty acid and free amino acid (FAA). The content of glycogen was high in January and March (2.89 and 2.82 g(100g)-1 on average, respectively) and low in October (2.07 g(100g)-1 on avarage). The low content of neutral lipids in October reflected a relatively poor nutritional value of oyster (1.42 g(100 g)-1 on average). The main fatty acids of oyster were palmitic acid (16:0), oleic acid (18:1), eicosapentaenoic acid (EPA, 20: 5ω-3) and docosahexaenoic acid (DHA, 22:6ω-3). The major FAAs of oyster were Taurine, Glutamicacid, Glycin, Alanine, Arginine and Proline. Taurine was the most abundant FAA with its content ranging from 603 mg (100g)-1 to 1139 mg(100g)-1. The high contents of glycogen, polyunsaturated fatty acid and FAA showed that oyster cultured along the eastern coast of Shandong Province was nutritionally good in January and March.

  10. Effects of hydrostatic pressure and temperature on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium.

    NASA Technical Reports Server (NTRS)

    Paul, K. L.; Morita, R. Y.

    1971-01-01

    Studies of pressure and temperature effects on glutamic acid transport and utilization indicated that hydrostatic pressure and low temperature inhibit glutamate transport more than glutamate respiration. The effects of pressure on transport were reduced at temperatures near the optimum. Similar results were obtained for glycine, phenylalanine, and proline. Pressure effects on the transport systems of all four amino acids were reversible to some degree. Both proline and glutamic acid were able to protect their transport proteins against pressure damage. The data presented indicate that the uptake of amino acids by cells under pressure is inhibited, which is the cause of their inability to grow under pressure.

  11. Metabolomics analysis for hydroxy-L-proline-induced calcium oxalate nephrolithiasis in rats based on ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry.

    PubMed

    Gao, Songyan; Yang, Rui; Peng, Zhongjiang; Lu, Hongtao; Li, Na; Ding, Jiarong; Cui, Xingang; Chen, Wei; Dong, Xin

    2016-01-01

    About 80% of kidney stones are composed of calcium oxalate (CaOx) with variable amounts of calcium phosphate, and hyperoxaluria is considered as an important factor of CaOx nephrolithiasis. However, the underlying metabolic mechanisms of CaOx nephrolithiasis remain undefined. In this study, we successfully developed a rat model with hydroxy-L-proline (HLP) -induced CaOx nephrolithiasis. Rats were continuously orally administrated with HLP for 28 days. Urine and blood samples were collected from the rats treated with or without HLP at four different time points. UPLC-Q-TOF/MS was applied to profile the abundances of metabolites. To obtain more comprehensive analysis of metabolic profiling spectrum, combination of RP-LC and HILIC were applied. We identify 42 significant differential metabolites in the urine, and 13 significant differential metabolites in the blood. Pathway analysis revealed that the pathways involved in amino acid metabolism, taurine metabolism, bile acid synthesis, energy metabolism, TCA cycle, purine metabolism, vitamin metabolism, nicotinic acid and nicotinamide metabolism have been modulated by HLP treatment. This study suggested that a number of metabolic pathways are dysfunctional in the HLP induced crystal kidney injury, and further studies on those pathways are warranted to better understand the metabolic mechanism of CaOx nephrolithiasis. PMID:27443631

  12. Metabolomics analysis for hydroxy-L-proline-induced calcium oxalate nephrolithiasis in rats based on ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry

    PubMed Central

    Gao, Songyan; Yang, Rui; Peng, Zhongjiang; Lu, Hongtao; Li, Na; Ding, Jiarong; Cui, Xingang; Chen, Wei; Dong, Xin

    2016-01-01

    About 80% of kidney stones are composed of calcium oxalate (CaOx) with variable amounts of calcium phosphate, and hyperoxaluria is considered as an important factor of CaOx nephrolithiasis. However, the underlying metabolic mechanisms of CaOx nephrolithiasis remain undefined. In this study, we successfully developed a rat model with hydroxy-L-proline (HLP) -induced CaOx nephrolithiasis. Rats were continuously orally administrated with HLP for 28 days. Urine and blood samples were collected from the rats treated with or without HLP at four different time points. UPLC–Q-TOF/MS was applied to profile the abundances of metabolites. To obtain more comprehensive analysis of metabolic profiling spectrum, combination of RP-LC and HILIC were applied. We identify 42 significant differential metabolites in the urine, and 13 significant differential metabolites in the blood. Pathway analysis revealed that the pathways involved in amino acid metabolism, taurine metabolism, bile acid synthesis, energy metabolism, TCA cycle, purine metabolism, vitamin metabolism, nicotinic acid and nicotinamide metabolism have been modulated by HLP treatment. This study suggested that a number of metabolic pathways are dysfunctional in the HLP induced crystal kidney injury, and further studies on those pathways are warranted to better understand the metabolic mechanism of CaOx nephrolithiasis. PMID:27443631

  13. Unique amino acid signatures that are evolutionarily conserved distinguish simple-type, epidermal and hair keratins

    PubMed Central

    Strnad, Pavel; Usachov, Valentyn; Debes, Cedric; Gräter, Frauke; Parry, David A. D.; Omary, M. Bishr

    2011-01-01

    Keratins (Ks) consist of central α-helical rod domains that are flanked by non-α-helical head and tail domains. The cellular abundance of keratins, coupled with their selective cell expression patterns, suggests that they diversified to fulfill tissue-specific functions although the primary structure differences between them have not been comprehensively compared. We analyzed keratin sequences from many species: K1, K2, K5, K9, K10, K14 were studied as representatives of epidermal keratins, and compared with K7, K8, K18, K19, K20 and K31, K35, K81, K85, K86, which represent simple-type (single-layered or glandular) epithelial and hair keratins, respectively. We show that keratin domains have striking differences in their amino acids. There are many cysteines in hair keratins but only a small number in epidermal keratins and rare or none in simple-type keratins. The heads and/or tails of epidermal keratins are glycine and phenylalanine rich but alanine poor, whereas parallel domains of hair keratins are abundant in prolines, and those of simple-type epithelial keratins are enriched in acidic and/or basic residues. The observed differences between simple-type, epidermal and hair keratins are highly conserved throughout evolution. Cysteines and histidines, which are infrequent keratin amino acids, are involved in de novo mutations that are markedly overrepresented in keratins. Hence, keratins have evolutionarily conserved and domain-selectively enriched amino acids including glycine and phenylalanine (epidermal), cysteine and proline (hair), and basic and acidic (simple-type epithelial), which reflect unique functions related to structural flexibility, rigidity and solubility, respectively. Our findings also support the importance of human keratin ‘mutation hotspot’ residues and their wild-type counterparts. PMID:22215855

  14. 34 CFR 303.15 - Include; including.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 2 2010-07-01 2010-07-01 false Include; including. 303.15 Section 303.15 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION EARLY INTERVENTION PROGRAM FOR INFANTS AND TODDLERS...

  15. Evaluation of enantiomeric purity of selected amino acids in honey.

    PubMed

    Pawlowska, M; Armstrong, D W

    1994-01-01

    Highly sensitive and accurate HPLC methods were used for the determination of total amounts of proline, leucine and phenylalanine and their enantiomeric ratios in a variety of different honey samples. Significant amounts of D-leucine and D-phenylalanine and relatively low concentrations of D-proline have been found in honeys of different botanical and geographical origins. It is suggested that the enantiomeric ratios of amino acids could be used to test for storage effects, age, and the quality of the processing of the honey. PMID:8068489

  16. Decarboxylative Annulation of α-Amino Acids with γ-Nitroaldehydes

    PubMed Central

    2016-01-01

    Indolizidine and quinolizidine derivatives are readily assembled from proline or pipecolic acid and γ-nitroaldehydes by means of a decarboxylative annulation process. These reactions are promoted by simple acetic acid and involve azomethine ylides as reactive intermediates. The method was applied to the synthesis of an epiquinamide analog. PMID:27509449

  17. The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule.

    PubMed

    Mégroz, Marianne; Kleifeld, Oded; Wright, Amy; Powell, David; Harrison, Paul; Adler, Ben; Harper, Marina; Boyce, John D

    2016-05-01

    The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin. PMID:26883595

  18. Experimental Shock Chemistry of Aqueous Amino Acid Solutions and the Cometary Delivery of Prebiotic Compounds

    NASA Astrophysics Data System (ADS)

    Blank, Jennifer G.; Miller, Gregory H.; Ahrens, Michael J.; Winans, Randall E.

    2001-02-01

    A series of shock experiments were conducted to assess the feasibility of the delivery of organic compounds to the Earth via cometary impacts. Aqueous solutions containing near-saturation levels of amino acids (lysine, norvaline, aminobutyric acid, proline, and phenylalanine) were sealed inside stainless steel capsules and shocked by ballistic impact with a steel projectile plate accelerated along a 12-m-long gun barrel to velocities of 0.5-1.9 km sec^-1. Pressure-temperature-time histories of the shocked fluids were calculated using 1D hydrodynamical simulations. Maximum conditions experienced by the solutions lasted 0.85-2.7 μs and ranged from 5.1-21 GPa and 412-870 K. Recovered sample capsules were milled open and liquid was extracted. Samples were analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). In all experiments, a large fraction of the amino acids survived. We observed differences in kinetic behavior and the degree of survivability among the amino acids. Aminobutyric acid appeared to be the least reactive, and phenylalanine appeared to be the most reactive of the amino acids. The impact process resulted in the formation of peptide bonds; new compounds included amino acid dimers and cyclic diketopiperazines. In our experiments, and in certain naturally occurring impacts, pressure has a greater influence than temperature in determining reaction pathways. Our results support the hypothesis that significant concentrations of organic material could survive a natural impact process.

  19. Experimental shock chemistry of aqueous amino acid solutions and the cometary delivery of prebiotic compounds.

    PubMed

    Blank, J G; Miller, G H; Ahrens, M J; Winans, R E

    2001-01-01

    A series of shock experiments were conducted to assess the feasibility of the delivery of organic compounds to the Earth via cometary impacts. Aqueous solutions containing near-saturation levels of amino acids (lysine, norvaline, aminobutyric acid, proline, and phenylalanine) were sealed inside stainless steel capsules and shocked by ballistic impact with a steel projectile plate accelerated along a 12-m-long gun barrel to velocities of 0.5-1.9 km sec-1. Pressure-temperature-time histories of the shocked fluids were calculated using 1D hydrodynamical simulations. Maximum conditions experienced by the solutions lasted 0.85-2.7 microseconds and ranged from 5.1-21 GPa and 412-870 K. Recovered sample capsules were milled open and liquid was extracted. Samples were analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). In all experiments, a large fraction of the amino acids survived. We observed differences in kinetic behavior and the degree of survivability among the amino acids. Aminobutyric acid appeared to be the least reactive, and phenylalanine appeared to be the most reactive of the amino acids. The impact process resulted in the formation of peptide bonds; new compounds included amino acid dimers and cyclic diketopiperazines. In our experiments, and in certain naturally occurring impacts, pressure has a greater influence than temperature in determining reaction pathways. Our results support the hypothesis that significant concentrations of organic material could survive a natural impact process. PMID:11296518

  20. Intelligent biomedical gels based on pendant l-proline alkyl esters

    NASA Astrophysics Data System (ADS)

    Yoshida, Masaru; Safranj, Agneza; Omichi, Hideki; Katakai, Roichi

    1995-09-01

    Linear poly(acryloyl-L-proline methyl ester, A-ProOMe), obtained by radiation-induced polymerization of its monomer in ethanol, exhibits a lower critical solution temperature (LCST) of 14°C. A minor amount of 2-hydroxypropyl methacrylate (HPMA) was copolymerized with A-ProOMe to obtain an intelligent biomedical gel for application in drug delivery systems. This gel is characterized by an initial shrinkage at the surface in the deswollen state, that results in formation of a rigid membrane barrier devoid of micropores, namely, a surface regulated matrix. Testosterone (T) was incorporated into this gel and, as a result, it was found that the daily dose of T released in vivo from this formulation remained constant at approximately 30 μg/day throughout an experimental period of 54 weeks.

  1. Determination of proline in honey: comparison between official methods, optimization and validation of the analytical methodology.

    PubMed

    Truzzi, Cristina; Annibaldi, Anna; Illuminati, Silvia; Finale, Carolina; Scarponi, Giuseppe

    2014-05-01

    The study compares official spectrophotometric methods for the determination of proline content in honey - those of the International Honey Commission (IHC) and the Association of Official Analytical Chemists (AOAC) - with the original Ough method. Results show that the extra time-consuming treatment stages added by the IHC method with respect to the Ough method are pointless. We demonstrate that the AOACs method proves to be the best in terms of accuracy and time saving. The optimized waiting time for the absorbance recording is set at 35min from the removal of reaction tubes from the boiling bath used in the sample treatment. The optimized method was validated in the matrix: linearity up to 1800mgL(-1), limit of detection 20mgL(-1), limit of quantification 61mgL(-1). The method was applied to 43 unifloral honey samples from the Marche region, Italy. PMID:24360478

  2. Avirulent isolates of Corynebacterium fascians that are unable to utilize agmatine and proline.

    PubMed Central

    Sabart, P R; Gakovich, D; Hanson, R S

    1986-01-01

    Growth of a highly virulent strain of the phytopathogen Corynebacterium fascians on rich media at 37 degrees C resulted in a loss of virulence in a majority of the population within 10 generations. Strains retained virulence during cultivation at 30 degrees C on a minimal medium with ammonia as a nitrogen source. Populations of avirulent strains on the surfaces of pea seedlings decreased, whereas the number of cells of the virulent strain increased 1,000-fold during a 3-week period. All avirulent mutants isolated by growth on rich media at 37 degrees C were unable to grow on media containing agmatine or proline as sole sources of nitrogen. The ability of the mutants to grow on pea seedlings and cause fasciation disease appeared to be related to their ability to utilize nitrogen sources available on plant surfaces. PMID:3729405

  3. The Transmembrane Prolines of the Mitochondrial ADP/ATP Carrier Are Involved in Nucleotide Binding and Transport and Its Biogenesis*

    PubMed Central

    Babot, Marion; Blancard, Corinne; Pelosi, Ludovic; Lauquin, Guy J.-M.; Trézéguet, Véronique

    2012-01-01

    The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy. PMID:22334686

  4. Effect of chronic (4 weeks) ingestion of ethanol on transport of proline into intestinal brush border membrane vesicles

    SciTech Connect

    Beesley, R.C.; Jones, T.D.

    1986-03-01

    Hamsters were separated into two groups and fed liquid diets ad lib. Controls were given a diet similar to that described by DeCarli and Lieber while alcoholics received the same diet containing 5% ethanol isocalorically substituted for sucrose. The volume of diet consumed daily and the gain in body weights of alcoholics were not significantly different from those of controls. After four weeks the animals were sacrificed and the upper third of the small intestine was used to prepare brush border membrane vesicles. In the presence of a Na/sup +/ gradient, uptake of proline into vesicles prepared from both groups was rapid, reaching a maximum accumulation in 1 to 2 min and then decreasing to the equilibrium level. To normalize the results, the amount of proline take up at each time point was divided by the amount present at equilibrium. From the normalized data it was concluded that both the rate of uptake and the maximum accumulation of proline into brush border membrane vesicles isolated from alcoholics were significantly less than those obtained with vesicles from controls. These results suggest that chronic ingestion of ethanol results in a reduction in Na/sup +/-dependent transport of proline across the brush border membrane and, thus, may contribute to the malnutrition which is frequently associated with chronic alcoholism.

  5. Clonal response to cold tolerance in creeping bentgrass and role of proline-associated pentose phosphate pathway.

    PubMed

    Sarkar, Dipayan; Bhowmik, Prasanta C; Kwon, Young-In; Shetty, Kalidas

    2009-11-01

    Single seed origin creeping bentgrass ('Penncross') clonal lines were screened to find genetic heterogeneity, which reflected diversity of phenolic production linked to cold stress within a cross-pollinated cultivar. In this study, total soluble phenolic and antioxidant activity varied among 20 creeping bentgrass clonal lines, confirming wide heterogeneity in this cross-pollinated species. Correlations between phenolic content and proline-associated pentose phosphate pathway were also found among the clonal lines. The active metabolic role of proline in cellular metabolic adjustment to cold stress and its support for likely energy synthesis via mitochondrial oxidative phosphorylation was inferred in creeping bentgrass clonal lines based on the activity of proline dehydrogenase. Results of photochemical efficiency of these clonal lines after cold temperature treatment (4 degrees C) also indicated a close association between stress tolerance and proline-associated pentose phosphate pathway regulation for phenolic biosynthesis and antioxidant response. This study provides a sound metabolic based rationale to screen bentgrass clonal lines for enhanced cold stress tolerance. PMID:19576763

  6. The involvement of proline-rich protein Mus musculus predicted gene 4736 in ocular surface functions

    PubMed Central

    Qi, Xia; Ren, Sheng-Wei; Zhang, Feng; Wang, Yi-Qiang

    2016-01-01

    AIM To research the two homologous predicted proline-rich protein genes, Mus musculus predicted gene 4736 (MP4) and proline-rich protein BstNI subfamily 1 (Prb1) which were significantly upregulated in cultured corneal organs when encountering fungal pathogen preparations. This study was to confirm the expression and potential functions of these two genes in ocular surface. METHODS A Pseudomonas aeruginosa keratitis model was established in Balb/c mice. One day post infection, mRNA level of MP4 was measured using real-time polymerase chain reaction (PCR), and MP4 protein detected by immunohistochemistry (IHC) or Western blot using a customized polyclonal anti-MP4 antibody preparation. Lacrimal glands from normal mice were also subjected to IHC staining for MP4. An online bioinformatics program, BioGPS, was utilized to screen public data to determine other potential locations of MP4. RESULTS One day after keratitis induction, MP4 was upregulated in the corneas at both mRNA level as measured using real-time PCR and protein levels as measured using Western blot and IHC. BioGPS analysis of public data suggested that the MP4 gene was most abundantly expressed in the lacrimal glands, and IHC revealed that normal murine lacrimal glands were positive for MP4 staining. CONCLUSION MP4 and Prb1 are closely related with the physiology and pathological processes of the ocular surface. Considering the significance of ocular surface abnormalities like dry eye, we propose that MP4 and Prb1 contribute to homeostasis of ocular surface, and deserve more extensive functional and disease correlation studies. PMID:27588265

  7. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

    PubMed

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires Júnior, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases. PMID:26661890

  8. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium

    PubMed Central

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases. PMID:26661890

  9. Structural Basis of Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA

    PubMed Central

    Zhou, Yuzhen; Larson, John D.; Bottoms, Christopher A.; Arturo, Emilia C.; Henzl, Michael T.; Jenkins, Jermaine L.; Nix, Jay C.; Becker, Donald F.; Tanner, John J.

    2009-01-01

    Summary The multifunctional Escherichia coli PutA flavoprotein functions as both a membrane-associated proline catabolic enzyme and transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put regulatory region, determined a crystal structure of the PutA ribbon-helix-helix domain (PutA52) complexed with DNA and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25 Å resolution crystal structure of PutA52 bound to one of the operators (operator 2, 21-bp) shows that the protein contacts a 9-bp fragment, corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic with an enthalpy of −1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and 15-fold lower affinity, which shows that base pairs outside of the consensus motif impact binding. The structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators. PMID:18586269

  10. Partial repair of salinity-induced damage to sprouting sugarcane buds by proline and glycinebetaine pretreatment.

    PubMed

    Rasheed, Rizwan; Wahid, Abdul; Hussain, Iqbal; Mahmood, Saqib; Parveen, Abida

    2016-05-01

    Sugarcane shows reduced crop stand under relatively suboptimal conditions; the main reason for this is its sensitivity to ionic stress in the soil solution. This research was performed to explore some physiological and developmental changes in the immature sugarcane buds submitted to salt stress and possible role of glycinebetaine (GB) and proline (Pro) in mitigating the ion toxicity in a time course manner. Salinity stress reduced fresh and dry weight, induced the generation of hydrogen peroxide, increased tissue levels of Na(+) sand Cl(-), reduced K(+) and Ca(2+), and K(+):Na(+) and Ca(2+):Na(+) ratios, while increasing the osmolyte synthesis in expanding sugarcane buds. Salinity stress reduced and delayed the formation of new bud leaves and their expansion, which was mainly because of reduction in the number and area of mesophyll cells and poor development of vascular bundles. The pretreatment of bud chips with 20 mM each of GB and Pro decreased tissue levels of Na(+) and Cl(-), reduced the generation of H2O2, improved K(+) and Ca(2+), K(+):Na(+) and Ca(2+):Na(+) ratios, and further increased the levels of GB, free proline (FP), and soluble sugars in the buds. The pretreatment increased mesophyll cell number and expansion of bud leaves and formation of elaborated vascular tissues, which apparently enabled the sprouting buds to adapt to salinity stress. Of the two osmolytes, GB was a relatively better inducer of salinity tolerance than Pro. In short, salinity-induced oxidative stress was the main cause for altered tissue development, the production of which was offset by pretreatment of bud tissues with Pro and GB. PMID:26043840

  11. A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

    PubMed

    D'Aniello, C; Fico, A; Casalino, L; Guardiola, O; Di Napoli, G; Cermola, F; De Cesare, D; Tatè, R; Cobellis, G; Patriarca, E J; Minchiotti, G

    2015-07-01

    Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity. PMID:25857264

  12. Group A Streptococcus Adheres to Pharyngeal Epithelial Cells with Salivary Proline-rich Proteins via GrpE Chaperone Protein*

    PubMed Central

    Murakami, Jumpei; Terao, Yutaka; Morisaki, Ichijiro; Hamada, Shigeyuki; Kawabata, Shigetada

    2012-01-01

    Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology. PMID:22566698

  13. Neutralization of endotoxin in vitro and in vivo by Bac7(1-35), a proline-rich antibacterial peptide.

    PubMed

    Ghiselli, Roberto; Giacometti, Andrea; Cirioni, Oscar; Circo, Raffaella; Mocchegiani, Federico; Skerlavaj, Barbara; D'Amato, Giuseppina; Scalise, Giorgio; Zanetti, Margherita; Saba, Vittorio

    2003-06-01

    Lipopolysaccharides (LPS), or endotoxins, are structural components of gram-negative bacteria implicated in the pathogenesis of septic shock. In this study the antiendotoxin activity of Bac7(1-35), a synthetic peptide based on the sequence of a proline-rich antibacterial peptide from bovine neutrophils, was investigated in vitro and in an experimental rat model of gram-negative septic shock. The ability of Bac7(1-35) to bind LPS from Escherichia coli O111:B4 was determined using a sensitive Limulus chromogenic assay. In the in vivo study, adult male Wistar rats were given an intraperitoneal injection of 1 x 10(9) colony-forming units of E. coli ATCC 25922. All animals were randomized to receive intraperitoneally 1 mg/kg Bac7(1-35), or isotonic sodium chloride solution (control group C1), 60 mg/kg of piperacillin and 1 mg/kg polymyxin B, 1 mg/kg of polymyxin B plus 60 mg/kg of piperacillin, and 1 mg/kg of Bac7(1-35) plus 60 mg/kg of piperacillin. Each group included 15 animals. Bac7(1-35) was found to completely inhibit the LPS procoagulant activity at approximately 10 microM peptide concentration, as determined by in vitro LAL chromogenic assay. Treatment with Bac7(1-35) resulted in significant decrease in plasma endotoxin levels and lethality rates compared with saline injected control animals. No statistically significant differences were noted between Bac7(1-35) and polymyxin B in reducing all variables measured. These results provide evidence for the ability of Bac7(1-35) to effectively bind LPS and protect animals from lethal effects of this molecule, and point to its potential use for the treatment of endotoxin-induced septic shock. PMID:12785015

  14. Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.

    PubMed Central

    Haffner, C; Jarchau, T; Reinhard, M; Hoppe, J; Lohmann, S M; Walter, U

    1995-01-01

    The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks. Images PMID:7828592

  15. Alterations of amino acid metabolism in osteoarthritis: its implications for nutrition and health.

    PubMed

    Li, Yusheng; Xiao, Wenfeng; Luo, Wei; Zeng, Chao; Deng, Zhenhan; Ren, Wenkai; Wu, Guoyao; Lei, Guanghua

    2016-04-01

    Osteoarthritis (OA) is a common form of arthritis in humans. It has long been regarded as a non-inflammatory disease, but a degree of inflammation is now recognized as being a vital inducer of subpopulation of OA. Besides inflammation, the establishment and development of OA are associated with alterations in metabolism and profiles of amino acids (AA), including glutamate- and arginine-family AA as well as their related metabolites (e.g., creatinine, hydroxyproline, γ-aminobutyrate, dimethylarginines and homoarginine). Functional AA (e.g., glutamine, arginine, glutamate, glycine, proline, and tryptophan) have various benefits (i.e., anti-inflammation and anti-oxidation) in treatment of inflammation-associated diseases, including OA. Thus, these AA have potential as immunomodulatory nutrients for patients with inflammation-induced OA. PMID:26767374

  16. Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

    PubMed

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2015-06-01

    Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of proline (2.5-10 mM) and PEG (2.5-10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control. PMID:25940589

  17. Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit 1

    PubMed Central

    Naidu, Bodapati P.; Aspinall, Donald; Paleg, Leslie G.

    1992-01-01

    This work was undertaken in an effort to reconcile the conflicting proline-accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute-accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings. PMID:16668700

  18. Acid Rain.

    ERIC Educational Resources Information Center

    Openshaw, Peter

    1987-01-01

    Provides some background information on acid deposition. Includes a historical perspective, describes some effects of acid precipitation, and discusses acid rain in the United Kingdom. Contains several experiments that deal with the effects of acid rain on water quality and soil. (TW)

  19. Laser photobioactivation mechanisms: In vitro studies using ascorbic acid uptake and hydroxyproline formation as biochemical markers of irradiation response

    SciTech Connect

    Labbe, R.F.; Skogerboe, K.J.; Davis, H.A.; Rettmer, R.L. )

    1990-01-01

    Clinical investigations of laser photobioactivation, or biostimulation, might be differently designed and more fruitful if knowledge of basic biochemical mechanisms were better understood. In this investigation, biochemical events identified as responses to 904 nm irradiation included increased ascorbic acid uptake by fibroblasts. These cells also showed increased hydroxyproline formation, and this was increased several-fold by the addition of proline to the medium. Maximum biochemical responses were observed at a pulse frequency of 67 Hz and a pulse width of 150 nsec with an energy density of approximately 7 mJ/cm2 per exposure. Elements in the mitochondrial cytochrome system are proposed as the radiation absorbing chromophore(s). Hypothetically, the energy generated is linked to ascorbic acid uptake, which in turn stimulates collagen synthesis.

  20. An apoA-I mimetic peptide containing a proline residue has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide.

    PubMed

    Wool, Geoffrey D; Vaisar, Tomas; Reardon, Catherine A; Getz, Godfrey S

    2009-09-01

    Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated alpha-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE(-/-) mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic alpha-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo. PMID:19433476

  1. An apoA-I mimetic peptide containing a proline residue has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide

    PubMed Central

    Wool, Geoffrey D.; Vaisar, Tomas; Reardon, Catherine A.; Getz, Godfrey S.

    2009-01-01

    Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated α-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE−/− mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic α-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo. PMID:19433476

  2. Nomogram including the total tumoral load in the sentinel nodes assessed by one-step nucleic acid amplification as a new factor for predicting nonsentinel lymph node metastasis in breast cancer patients.

    PubMed

    Rubio, Isabel T; Espinosa-Bravo, Martin; Rodrigo, Maxi; Amparo Viguri Diaz, Maria; Hardisson, David; Sagasta, Amaia; Dueñas, Basilio; Peg, Vicente

    2014-09-01

    Several models have been developed to predict non-sentinel nodes (NSLN) metastasis in patients with a positive sentinel node (SLN) that incorporates a standard pathology examination of the SLN. It has been reported that total tumoral load (TTL) in the SLNs assessed by one-step nucleic acid amplification (OSNA) is a predictive factor for additional NSLN metastasis in the axillary lymph node dissection (ALND). The objective was to develop a nomogram that predicts patient´s risk of additional NSLN metastasis incorporating TTL in the SLNs assessed by OSNA. Six hundred and ninety-seven consecutive patients with positive SLN evaluation by OSNA and a completion ALND were recruited. Pathologic features of the primary tumor and SLN metastases, including TTL were collected. Multivariate logistic regression identified factors predictive of non-SLN metastasis. A nomogram was developed with these variables and validated in an external cohort. On multivariate logistic regression analysis, tumor size, number of affected SLN, Her2 overexpression, lymphovascular invasion, and TTL were each associated with the likelihood of additional NSLN metastasis (p < 0.05). The overall predictive accuracy of the nomogram, as measured by the AUC was 0.7552 (95 %CI 0.7159-0.7945). When applied to the external cohort the nomogram was accurate with an AUC = 0.678 (95 %CI 0.621-0.736). This novel nomogram that incorporates TTL assessed by OSNA performs well and may help clinicians to make decisions about ALND for individual patients. Moreover, the standardization of pathologic assessment by OSNA may help to achieve interinstitutional reproducibility among nomograms. PMID:25164972

  3. Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

    PubMed

    Milner, J L; Grothe, S; Wood, J M

    1988-10-15

    Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift. PMID:3049595

  4. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition

    PubMed Central

    2014-01-01

    Amino acids are building blocks for proteins in all animals. Based on growth or nitrogen balance, amino acids were traditionally classified as nutritionally essential or nonessential for mammals, birds and fish. It was assumed that all the “nutritionally nonessential amino acids (NEAA)” were synthesized sufficiently in the body to meet the needs for maximal growth and optimal health. However, careful analysis of the scientific literature reveals that over the past century there has not been compelling experimental evidence to support this assumption. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, fertility, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and to protect the integrity of the intestinal mucosa. Thus, diets for animals must contain all NEAA to optimize their survival, growth, development, reproduction, and health. Furthermore, NEAA should be taken into consideration in revising the “ideal protein” concept that is currently used to formulate swine and poultry diets. Adequate provision of all amino acids (including NEAA) in diets enhances the efficiency of animal production. In this regard, amino acids should not be classified as nutritionally essential or nonessential in animal or human nutrition. The new Texas A&M University’s optimal ratios of dietary amino acids for swine and chickens are expected to beneficially reduce dietary protein content and improve the efficiency of their nutrient utilization, growth, and production performance. PMID:24999386

  5. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition.

    PubMed

    Wu, Guoyao

    2014-01-01

    Amino acids are building blocks for proteins in all animals. Based on growth or nitrogen balance, amino acids were traditionally classified as nutritionally essential or nonessential for mammals, birds and fish. It was assumed that all the "nutritionally nonessential amino acids (NEAA)" were synthesized sufficiently in the body to meet the needs for maximal growth and optimal health. However, careful analysis of the scientific literature reveals that over the past century there has not been compelling experimental evidence to support this assumption. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, fertility, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and to protect the integrity of the intestinal mucosa. Thus, diets for animals must contain all NEAA to optimize their survival, growth, development, reproduction, and health. Furthermore, NEAA should be taken into consideration in revising the "ideal protein" concept that is currently used to formulate swine and poultry diets. Adequate provision of all amino acids (including NEAA) in diets enhances the efficiency of animal production. In this regard, amino acids should not be classified as nutritionally essential or nonessential in animal or human nutrition. The new Texas A&M University's optimal ratios of dietary amino acids for swine and chickens are expected to beneficially reduce dietary protein content and improve the efficiency of their nutrient utilization, growth, and production performance. PMID:24999386

  6. Proline Substitutions and Threonine Pseudophosphorylation of the SH3 Ligand of 18.5-kDa Myelin Basic Protein Decrease Its Affinity for the Fyn-SH3 Domain and Alter Process Development and Protein Localization in Oligodendrocytes

    PubMed Central

    Smith, Graham S.T.; De Avila, Miguel; Paez, Pablo M.; Spreuer, Vilma; Wills, Melanie K.B.; Jones, Nina; Boggs, Joan M.; Harauz, George

    2012-01-01

    The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92–99 (murine sequence –T92PRTPPPS99–) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP’s SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca2+ influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein–protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP’s SH3 ligand domain. These results suggest that MBP’s SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that

  7. Mefenamic Acid

    MedlinePlus

    Mefenamic acid is used to relieve mild to moderate pain, including menstrual pain (pain that happens before or during a menstrual period). Mefenamic acid is in a class of medications called NSAIDs. ...

  8. Evaluating placental inter-ordinal phylogenies with novel sequences including RAG1, gamma-fibrinogen, ND6, and mt-tRNA, plus MCMC-driven nucleotide, amino acid, and codon models.

    PubMed

    Waddell, Peter J; Shelley, Shawn

    2003-08-01

    It is essential to test a priori scientific hypotheses with independent data, not least to partly negate factors such as gene-specific base composition biases misleading our models. Seven new gene segments and sequences plus Bayesian likelihood phylogenetic methods were used to compare and test five recent placental phylogenies. These five phylogenies are similar to each other, yet quite different from Fthose of previously proposed trees, and span Waddell et al. [Syst. Biol. 48 (1999) 1] to Murphy et al. [Science 294 (2001b) 2348]. Trees for RAG1, gamma-fibrinogen, ND6, mt-tRNA, mt-RNA, c-MYC, epsilon -globin, and GHR are significantly congruent with the four main groups of mammals common to the five phylogenies, i.e., Afrotheria, Laurasiatheria, Euarchontoglires, Xenarthra plus Boreoeutheria (Laurasiatheria plus Euarchontoglires). Where these five a priori phylogenies differ, remain areas generally hard to resolve with the new sequences. The root remains ambiguous and does not reject a basal Afrotheria (the Exafroplacentalia hypothesis), Afrotheria plus Xenarthra together with basal (Atlantogenata), or Epitheria (Xenarthra basal) convincingly. Good evidence is found that Eulipotyphla is monophyletic and is located at the base of Laurasiatheria. The shrew mole, Uropsilus, is found to cluster consistently with other moles, while Solenodon may be the sister taxa to all other eulipotyphlans. Support is found for a probable sister pairing of just hedgehogs/gymnures and shrews. Relationships within Afrotheria, except the Paenungulata clade, remain hard to resolve, although there is congruent support for Afroinsectiphillia (aardvark, elephant shrews, golden moles, and tenrecs). A first-time use is made of MCMC enacted general time-reversible (GTR) amino acid and codon-based models for general tree selection. Even with ND6, a GTR amino acid model provided resolution of fine features, such as the sister group relationship of walrus to Otatriidae, and with BRCA a more

  9. Effect of ultraviolet radiation on chlorophyll, carotenoid, protein and proline contents of some annual desert plants.

    PubMed

    Salama, Hediat M H; Al Watban, Ahlam A; Al-Fughom, Anoud T

    2011-01-01

    Investigation was carried out to find whether enhanced ultraviolet radiation influences the Malva parviflora L., Plantago major L., Rumex vesicarius L. and Sismbrium erysimoids Desf. of some annual desert plants. The seeds were grown in plastic pots equally filled with a pre-sieved normal sandy soil for 1 month. The planted pots from each species were randomly divided into equal groups (three groups). Plants of the first group exposed to white-light tubes (400-700 nm) 60 w and UV (365 nm) 8 w tubes. The second group was exposed to white-light tubes (400-700 nm) 60 w and UV (302 nm) 8 w tubes. The third group was exposed to white-light tubes (400-700 nm) 60 w and UV (254 nm) 8 w tubes, respectively, for six days. The results indicated that the chlorophyll contents were affected by enhanced UV radiation. The chlorophyll a, b, and total contents were decreased compared with the control values and reduced with the enhanced UV radiation, but the carotenoid was increased compared with the control and also reduced with the enhanced UV radiation. So, the contents of chlorophylls varied considerably. M. parviflora showed the highest constitutive levels of accumulated chlorophyll a, b, and total chlorophyll (0.463, 0.307 and 0.774 mg g(-1) f w) among the investigated plant species. P. major showed the lowest constitutive levels of the chloroplast pigments, 0.0036, 0.0038 and 0.0075 mg g(-1) f w for chlorophyll a, b, and total chlorophyll at UV-365 nm, respectively. The protein content was decreased significantly in both root and shoot systems compared with the control values but, it was increased with increasing wave lengths of UV-radiation of all tested plants. R. vesicarius showed the highest protein contents among the investigated plants; its content was 3.8 mg g(-1) f w at UV-365 nm in shoot system. On the other hand, decreasing ultraviolet wave length induced a highly significant increase in the level of proline in both root and shoot of all

  10. Chemiluminescence detection of amino acids, peptides, and proteins using tris-2,2 prime -bipyridine ruthenium(III)

    SciTech Connect

    Li He; Cox, K.A.; Danielson, N.D. )

    1990-01-01

    The feasibility of using the tris-2-2{prime}-bipyridine ruthenium(III) chemiluminescent (CL) reaction for the detection of amino acids, peptides, and proteins has been studied. Detection limits of the amino acids as determined by flow injection analysis (FIA) ranged from 20 pmol of proline to 50 nmol of asparagine. In general, amino acids containing secondary amine groups yielded the strongest responses. A reaction mechanism for Ru(bpy){sub 3}{sup 3+} chemiluminescence of aliphatic amines has been proposed. Studies of peptide molecules and poly-prolines showed that the peptide bond barely contributes to the detection signals. The separation of hydroxyproline and proline in synthetic collagen by HPLC with Ru(bpy){sub 3}{sup 3+} chemiluminescence detection has been shown to be possible.

  11. Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α

    PubMed Central

    Jambunathan, Kalyani; Watson, Douglas S.; Endsley, Aaron N.; Kodukula, Krishna; Galande, Amit K.

    2012-01-01

    Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery. PMID:22750443

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

    SciTech Connect

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

    1993-09-01

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

  13. Cyclopropyl glycine and proline-containing preparation noopept evoke two types of membrane potential responses in synaptoneurosomes.

    PubMed

    Lutsenko, V K; Vukolova, M N; Gudasheva, T A

    2003-06-01

    Proline, cyclo(Pro-Gly), and acyl-prolyl-containing dipeptide GVS-111 decreased synaptoneurosome membrane potential in a Ca2+-free medium. The efficiency of these preparations decreased in the following order: GVS>cyclo(Pro-Gly)>proline. Depolarization responses induced by endogenous nootropic agent cyclo(Pro-Gly) was dose-dependent and saturable; the threshold concentration of cyclo(Pro-Gly) was 10(-9) M. In a Ca2+-containing medium GVS and cyclo(Pro-Gly) induced both hyperpolarizing and depolarizing membrane responses of synaptoneurosomes. Possible mechanisms underlying changes in the membrane potential of synaptoneurosomes induced by nootropic agents are discussed. It was interesting whether modulation of electrogenesis can improve memory and potentiate the neuroprotective effect of the test nootropic agents. PMID:12937673

  14. Modulation of physicochemical and spectroscopic properties of l-serine and l-proline by propionate based food preservatives.

    PubMed

    Banipal, Tarlok S; Kaur, Navalpreet; Kaur, Jaspreet; Komal; Banipal, Parampaul K

    2016-10-15

    To have an insight into the effect of preservatives on various ingredients of processed items, it is important to study their thermodynamic, transport and spectroscopic properties in aqueous solutions to elucidate various solute-co-solute interactions. The densities, viscosities and enthalpies of dilution of l-serine and l-proline have been determined in water and in aqueous solutions of sodium propionate and calcium propionate at different temperatures. The derived parameters elucidate the changes in taste quality and hydration number of l-serine and l-proline in the presence of the studied preservatives. Predominance of dehydration effect has been observed from calorimetry and changes in chemical shifts from nuclear magnetic resonance spectroscopy also support the above results. PMID:271735