Science.gov

Sample records for acids including proline

  1. Vacuolar amino acid transporters upregulated by exogenous proline and involved in cellular localization of proline in Saccharomyces cerevisiae.

    PubMed

    Nishida, Ikuhisa; Watanabe, Daisuke; Tsolmonbaatar, Ariunzaya; Kaino, Tomohiro; Ohtsu, Iwao; Takagi, Hiroshi

    2016-07-14

    In the budding yeast Saccharomyces cerevisiae, the AVT genes (AVT1-7), which encode vacuolar amino acid transporters belonging to the amino acid vacuolar transport (AVT)-family, were significantly upregulated in response to exogenous proline. To reveal a novel role of the Avt proteins in proline homeostasis, we analyzed the effects of deletion or overexpression of the AVT genes on the subcellular distribution of amino acids after the addition of proline to the cells grown in minimal medium. Among seven AVT gene disruptants, avt1Δ and avt7Δ showed the lowest ratios of vacuolar proline. Consistently, overexpression of the AVT1 gene specifically enhanced the vacuolar localization of proline. Since double disruption of the AVT1 and AVT7 genes did not completely abrogate vacuolar accumulation of proline, it is presumed that Avt1 has a dominant role, and Avt7 and other Avt proteins have redundant functions, in the localization of proline into the vacuolar lumen. In contrast, deletion of the AVT3 gene increased vacuolar proline, although the highly expressed AVT3 gene interfered with the accumulation of proline in the vacuole. Based on these results, it appears that Avt3 is the major protein involved in the export of proline from the vacuole. We also observed vacuolar membrane localization of GFP-fused Avt1, Avt3, and Avt7 proteins. Taken together, our data suggest that the AVT genes induced by exogenous proline are involved in the bidirectional transport of proline across the vacuolar membrane. PMID:27246536

  2. Mechanisms independent of abscisic acid (ABA) or proline feedback have a predominant role in transcriptional regulation of proline metabolism during low water potential and stress recovery.

    PubMed

    Sharma, Sandeep; Verslues, Paul E

    2010-11-01

    Proline accumulation in response to abiotic stress is controlled partially by transcriptional regulation of key enzymes including Δ¹-pyrroline-carboxylate synthetase1 (P5CS1), proline dehydrogenase (ProDH), ornithine amino transferase (OAT) and Δ¹-pyrroline-carboxylate dehydrogenase (P5CDH). For these genes, the role of abscisic acid (ABA), role of feedback regulation by high proline and the mechanisms of gene regulation upon stress release remain unclear. An ABA-deficient (aba2-1) mutant, mutants deficient in proline accumulation (p5cs1), as well as double mutants deficient in both, were used to determine the importance of these factors in transcriptional regulation of proline metabolism. Upregulation of P5CS1 by low water potential was less dependent on ABA than that of stress-marker genes used for comparison. ProDH downregulation by low water potential and upregulation by stress release was not impaired in aba2-1, p5cs1 or p5cs1/aba2-1 compared with wild type despite differing ABA and proline levels in these mutants. Thus, ProDH is a model for characterization of novel regulatory mechanisms associated with low water potential and stress recovery. Both OAT and P5CDH were upregulated during low water potential. This contrasts with previous salt stress experiments and raises questions about the flux of metabolites through proline metabolism under low water potential when high levels of proline accumulate.

  3. A Statistical Analysis of the PPII Propensity of Amino Acid Guests in Proline-Rich Peptides

    PubMed Central

    Moradi, Mahmoud; Babin, Volodymyr; Sagui, Celeste; Roland, Christopher

    2011-01-01

    There has been considerable debate about the intrinsic PPII propensity of amino-acid residues in denatured polypeptides. Experimentally, the propensity scale is based on the behavior of guest amino-acid residues placed in the middle of polyproline hosts. We have used classical molecular dynamics simulations, with state-of-the-art force fields to carry out a comprehensive analysis of the conformational equilibria of the proline-based host oligopeptides with single guests. The tracked structural characteristics include the PPII content, the cis/trans isomerization of the prolyl bonds, the puckering of the pyrrolidine rings of the proline residues, and the secondary structural motifs. We find no evidence for an intrinsic PPII propensity in any of the guest amino acids other than proline. Instead, the PPII content as derived from experiments may be explained in terms of: 1), a local correlation between the dihedral angles of the guest amino acid and the proline residue immediately preceding it; and 2), a nonlocal correlation between the cis/trans states of the peptide bonds. In terms of the latter, we find that the presence of a guest (other than proline, tyrosine, or tryptophan) increases the trans content of most of the prolyl bonds, which results in an effective increase of the peptide PPII content. With respect to the local dihedral correlations, we find that these are well described in terms of the so-called odds-ratio statistic. Expressed in terms of free energy language, the PPII content based on the odds-ratio of the relevant residues correlate well with the experimentally measured PPII content. PMID:21320454

  4. Proline might have been the first amino acid in the primitive genetic code.

    PubMed

    Komatsu, Reina; Sawada, Risa; Umehara, Takuya; Tamura, Koji

    2014-06-01

    Stereochemical assignment of amino acids and corresponding codons or anticodons has not been successful so far. Here, we focused on proline and GGG (anticodon of tRNA(Pro)) and investigated their mutual interaction. Circular dichroism spectroscopy revealed that guanosine nucleotides (GG, GGG) formed G-quartet structures. The structures were destroyed by adding high concentrations of proline. We propose that the possibility of the reversible proline/G-quartet interaction could have contributed to the specific assignment of proline on GGG and that this coding could have been the first in the genetic code. PMID:24973301

  5. Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

    NASA Astrophysics Data System (ADS)

    Abutokaikah, Maha T.; Guan, Shanshan; Bythell, Benjamin J.

    2016-10-01

    Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra.

  6. Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2008-04-12

    The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.

  7. Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2006-11-01

    The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.

  8. D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

    PubMed

    Soma, Hiroki; Furuya, Ryuji; Kaneko, Ryo; Tsukamoto, Ayaka; Shirasu, Kazumitsu; Tanigawa, Minoru; Nagata, Yoko

    2013-10-01

    We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

  9. 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.

  10. 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.

  11. 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

  12. Effective prevention of chill-haze in beer using an acid proline-specific endoprotease from Aspergillus niger.

    PubMed

    Lopez, Michel; Edens, Luppo

    2005-10-01

    Chill-haze formation during beer production is known to involve polyphenols that interact with proline-rich proteins. We hypothesized that incubating beer wort with a proline-specific protease would extensively hydrolyze these proline-rich proteins, yielding a peptide fraction that is unable to form a haze. Predigestion of the proline-rich wheat gliadin with different proteases pointed toward a strong haze-suppressing effect by a proline-specific enzyme. This finding was confirmed in small-scale brewing experiments using a recently identified proline-specific protease with an acidic pH optimum. Subsequent pilot plant trials demonstrated that, upon its addition during the fermentation phase of beer brewing, even low levels of this acidic enzyme effectively prevented chill-haze formation in bottled beer. Results of beer foam stability measurements indicated that the enzyme treatment leaves the beer foam almost unaffected. In combination with the enzyme's cost-effectiveness and regulatory status, these preliminary test results seem to favor further industrial development of this enzymatic beer stabilization method.

  13. 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.

  14. The epidermis of scales in gecko lizards contains multiple forms of beta-keratins including basic glycine-proline-serine-rich proteins.

    PubMed

    Toni, M; Dalla Valle, L; Alibardi, L

    2007-05-01

    The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.

  15. Effects of copper on chlorophyll, proline, protein and abscisic acid level of sunflower (Helianthus annuus L.) seedlings.

    PubMed

    Zengin, Fikriye Kirbag; Kirbag, Sevda

    2007-07-01

    The effect of copperchloride (CuCl2) on the level of chlorophyll (a+b), proline, protein and abscisic acid in sunflower (Helianthus annuus L.) seedlings were investigated Control and copper treated (0.4, 0.5 and 0.6 mM) seedlings were grown for ten days in Hoagland solution. Abscisic acid content was determined in root, shoot and leaf tissues of seedlings by HPLC. Copper stress caused significant increase of the abscisic acid contents in roots, shoots and leaves of seedlings. The increase was dependent on the copper salt concentration. Enhanced accumulation of proline in the leaves of seedlings exposed to copper was determined, as well as a decrease of chlorophyll (a+b) and total protein (p < 0.05 or p < 0.01). It was observed that the level of chlorophyll (a+b) and total protein (p < 0.05 or p < 0.01) remarkably decreased as copper concentration increased to 0.6 mM, although the levels of proline and abscisic acid in the leaves of plants were increased--a dose-depended behavior The same trends were also observed with the level of abscisic acid of stems and roots. Copper has dose- depended effects on chlorophyll, proline, protein and abscisic acid level of sunflower (Helianthus annuus L.) seedlings. Thus, we assumed that copper levels increase above some critical points seedling growth get negative effects. This assumption is in line with previous findings.

  16. A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses.

    PubMed

    Inbar, Ehud; Schlisselberg, Doreen; Suter Grotemeyer, Marianne; Rentsch, Doris; Zilberstein, Dan

    2013-01-15

    Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

  17. Salicylic acid alleviates adverse effects of heat stress on photosynthesis through changes in proline production and ethylene formation

    PubMed Central

    Khan, M Iqbal R; Iqbal, Noushina; Masood, Asim; Per, Tasir S; Khan, Nafees A

    2013-01-01

    We investigated the potential of salicylic acid (SA) in alleviating the adverse effects of heat stress on photosynthesis in wheat (Triticum aestivum L.) cv WH 711. Activity of ribulose 1,5-bisphosphate carboxylase (Rubisco), photosynthetic-nitrogen use efficiency (NUE), and net photosynthesis decreased in plants subjected to heat stress (40°C for 6 h), but proline metabolism increased. SA treatment (0.5 mM) alleviated heat stress by increasing proline production through the increase in γ-glutamyl kinase (GK) and decrease in proline oxidase (PROX) activity, resulting in promotion of osmotic potential and water potential necessary for maintaining photosynthetic activity. Together with this, SA treatment restricted the ethylene formation in heat-stressed plants to optimal range by inhibiting activity of 1-aminocyclopropane carboxylic acid (ACC) synthase (ACS). This resulted in improved proline metabolism, N assimilation and photosynthesis. The results suggest that SA interacts with proline metabolism and ethylene formation to alleviate the adverse effects of heat stress on photosynthesis in wheat. PMID:24022274

  18. Salicylic acid alleviates adverse effects of heat stress on photosynthesis through changes in proline production and ethylene formation.

    PubMed

    Khan, M Iqbal R; Iqbal, Noushina; Masood, Asim; Per, Tasir S; Khan, Nafees A

    2013-11-01

    We investigated the potential of salicylic acid (SA) in alleviating the adverse effects of heat stress on photosynthesis in wheat (Triticum aestivum L.) cv WH 711. Activity of ribulose 1,5-bisphosphate carboxylase (Rubisco), photosynthetic-nitrogen use efficiency (NUE), and net photosynthesis decreased in plants subjected to heat stress (40 °C for 6 h), but proline metabolism increased. SA treatment (0.5 mM) alleviated heat stress by increasing proline production through the increase in γ-glutamyl kinase (GK) and decrease in proline oxidase (PROX) activity, resulting in promotion of osmotic potential and water potential necessary for maintaining photosynthetic activity. Together with this, SA treatment restricted the ethylene formation in heat-stressed plants to optimal range by inhibiting activity of 1-aminocyclopropane carboxylic acid (ACC) synthase (ACS). This resulted in improved proline metabolism, N assimilation and photosynthesis. The results suggest that SA interacts with proline metabolism and ethylene formation to alleviate the adverse effects of heat stress on photosynthesis in wheat.

  19. Improving Co-Amorphous Drug Formulations by the Addition of the Highly Water Soluble Amino Acid, Proline

    PubMed Central

    Jensen, Katrine Tarp; Löbmann, Korbinian; Rades, Thomas; Grohganz, Holger

    2014-01-01

    Co-amorphous drug amino acid mixtures were previously shown to be a promising approach to create physically stable amorphous systems with the improved dissolution properties of poorly water-soluble drugs. The aim of this work was to expand the co-amorphous drug amino acid mixture approach by combining the model drug, naproxen (NAP), with an amino acid to physically stabilize the co-amorphous system (tryptophan, TRP, or arginine, ARG) and a second highly soluble amino acid (proline, PRO) for an additional improvement of the dissolution rate. Co-amorphous drug-amino acid blends were prepared by ball milling and investigated for solid state characteristics, stability and the dissolution rate enhancement of NAP. All co-amorphous mixtures were stable at room temperature and 40 °C for a minimum of 84 days. PRO acted as a stabilizer for the co-amorphous system, including NAP–TRP, through enhancing the molecular interactions in the form of hydrogen bonds between all three components in the mixture. A salt formation between the acidic drug, NAP, and the basic amino acid, ARG, was found in co-amorphous NAP–ARG. In comparison to crystalline NAP, binary NAP–TRP and NAP–ARG, it could be shown that the highly soluble amino acid, PRO, improved the dissolution rate of NAP from the ternary co-amorphous systems in combination with either TRP or ARG. In conclusion, both the solubility of the amino acid and potential interactions between the molecules are critical parameters to consider in the development of co-amorphous formulations. PMID:25025400

  20. 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

  1. Proline Coordination with Fatty Acid Synthesis and Redox Metabolism of Chloroplast and Mitochondria1[OPEN

    PubMed Central

    Shinde, Suhas; Villamor, Joji Grace; Lin, Wendar; Verslues, Paul E.

    2016-01-01

    Proline (Pro) accumulation is one of the most prominent changes in plant metabolism during drought and low water potential; however, the regulation and function of Pro metabolism remain unclear. We used a combination of forward genetic screening based on a Proline Dehydrogenase1 (PDH1) promoter-luciferase reporter (PDH1pro:LUC2) and RNA sequencing of the Pro synthesis mutant p5cs1-4 to identify multiple loci affecting Pro accumulation in Arabidopsis (Arabidopsis thaliana). Two mutants having high PDH1pro:LUC2 expression and increased Pro accumulation at low water potential were found to be alleles of Cytochrome P450, Family 86, Subfamily A, Polypeptide2 (CYP86A2) and Long Chain Acyl Synthetase2 (LACS2), which catalyze two successive steps in very-long-chain fatty acid (VLCFA) synthesis. Reverse genetic experiments found additional VLCFA and lipid metabolism-related mutants with increased Pro accumulation. Altered cellular redox status is a key factor in the coordination of Pro and VLCFA metabolism. The NADPH oxidase inhibitor diphenyleneiodonium (DPI) induced high levels of Pro accumulation and strongly repressed PDH1pro:LUC2 expression. cyp86a2 and lacs2 mutants were hypersensitive to diphenyleneiodonium but could be reverted to wild-type Pro and PDH1pro:LUC2 expression by reactive oxygen species scavengers. The coordination of Pro and redox metabolism also was indicated by the altered expression of chloroplast and mitochondria electron transport genes in p5cs1-4. These results show that Pro metabolism is both influenced by and influences cellular redox status via previously unknown coordination with several metabolic pathways. In particular, Pro and VLCFA synthesis share dual roles to help buffer cellular redox status while producing products useful for stress resistance, namely the compatible solute Pro and cuticle lipids. PMID:27512016

  2. 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

  3. Connecting proline and γ-aminobutyric acid in stressed plants through non-enzymatic reactions.

    PubMed

    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.

  4. Transforming growth factor-beta 1 stimulates vascular smooth muscle cell L-proline transport by inducing system A amino acid transporter 2 (SAT2) gene expression.

    PubMed Central

    Ensenat, D; Hassan, S; Reyna, S V; Schafer, A I; Durante, W

    2001-01-01

    Transforming growth factor-beta1 (TGF-beta 1) is a multifunctional cytokine that contributes to arterial remodelling by stimulating vascular smooth muscle cell (SMC) growth and collagen synthesis at sites of vascular injury. Since l-proline is essential for the synthesis of collagen, we examined whether TGF-beta 1 regulates the transcellular transport of l-proline by vascular SMCs. l-Proline uptake by vascular SMCs was primarily sodium-dependent, pH-sensitive, blocked by neutral amino acids and alpha-(methylamino)isobutyric acid, and exhibited trans-inhibition. Treatment of SMCs with TGF-beta 1 stimulated l-proline transport in a concentration- and time-dependent manner. The TGF-beta 1-mediated l-proline uptake was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that TGF-beta 1-induced l-proline transport was mediated by an increase in transport capacity independent of any changes in the affinity for l-proline. TGF-beta 1 stimulated the expression of system A amino acid transporter 2 (SAT2) mRNA in a time-dependent fashion that paralleled the increase in l-proline transport. Reverse transcriptase PCR failed to detect the presence of SAT1 or amino acid transporter 3 (ATA3) in either untreated or TGF-beta 1-treated SMCs. These results demonstrate that l-proline transport by vascular SMCs is mediated predominantly by the SAT and that TGF-beta 1 stimulates SMC l-proline uptake by inducing the expression of the SAT2 gene. The ability of TGF-beta 1 to induce SAT2 expression may function to provide SMCs with the necessary levels of l-proline required for collagen synthesis and cell growth. PMID:11716780

  5. 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."

  6. 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

  7. 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

  8. 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.

  9. Abscisic acid-induced nitric oxide and proline accumulation in independent pathways under water-deficit stress during seedling establishment in Medicago truncatula.

    PubMed

    Planchet, Elisabeth; Verdu, Isabelle; Delahaie, Julien; Cukier, Caroline; Girard, Clément; Morère-Le Paven, Marie-Christine; Limami, Anis M

    2014-05-01

    Nitric oxide (NO) production and amino acid metabolism modulation, in particular abscisic acid (ABA)-dependent proline accumulation, are stimulated in planta by most abiotic stresses. However, the relationship between NO production and proline accumulation under abiotic stress is still poorly understood, especially in the early phases of plant development. To unravel this question, this work investigated the tight relationship between NO production and proline metabolism under water-deficit stress during seedling establishment. Endogenous nitrate reductase-dependent NO production in Medicago truncatula seedlings increased in a time-dependent manner after short-term water-deficit stress. This water-deficit-induced endogenous NO accumulation was mediated through a ABA-dependent pathway and accompanied by an inhibition of seed germination, a loss of water content, and a decrease in elongation of embryo axes. Interestingly, a treatment with a specific NO scavenger (cPTIO) alleviated these water-deficit detrimental effects. However, the content of total amino acids, in particular glutamate and proline, as well as the expression of genes encoding enzymes of synthesis and degradation of proline were not affected by cPTIO treatment under water-deficit stress. Under normal conditions, exogenous NO donor stimulated neither the expression of P5CS2 nor the proline content, as observed after PEG treatment. These results strongly suggest that the modulation of proline metabolism is independent of NO production under short-term water-deficit stress during seedling establishment.

  10. Practical synthesis of enantiomerically pure beta2-amino acids via proline-catalyzed diastereoselective aminomethylation of aldehydes.

    PubMed

    Chi, Yonggui; English, Emily P; Pomerantz, William C; Horne, W Seth; Joyce, Leo A; Alexander, Lane R; Fleming, William S; Hopkins, Elizabeth A; Gellman, Samuel H

    2007-05-01

    Proline-catalyzed diastereoselective aminomethylation of aldehydes using a chiral iminium ion, generated from a readily prepared precursor, provides alpha-substituted-beta-amino aldehydes with 85:15 to 90:10 dr. The alpha-substituted-beta-amino aldehydes can be reduced to beta-substituted-gamma-amino alcohols, the major diastereomer of which can be isolated via crystallization or column chromatography. The amino alcohols are efficiently transformed to protected beta2-amino acids, which are valuable building blocks for beta-peptides, natural products, and other interesting molecules. Because conditions for the aminomethylation and subsequent reactions are mild, beta2-amino acid derivatives with protected functional groups in the side chain, such as beta2-homoglutamic acid, beta2-homotyrosine, and beta2-homolysine, can be prepared in this way. The synthetic route is short, and purifications are simple; therefore, this method enables the preparation of protected beta2-amino acids in useful quantities.

  11. PNRC: a proline-rich nuclear receptor coregulatory protein that modulates transcriptional activation of multiple nuclear receptors including orphan receptors SF1 (steroidogenic factor 1) and ERRalpha1 (estrogen related receptor alpha-1).

    PubMed

    Zhou, D; Quach, K M; Yang, C; Lee, S Y; Pohajdak, B; Chen, S

    2000-07-01

    PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization was demonstrated by immunofluorescence and Western blot analyses. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC was found to function as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. By examining a series of deletion mutants of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence in the carboxy-terminal region, aa 278-300, was shown to be critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. Results from the mutagenesis study demonstrated that the two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23

  12. 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.

  13. 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

  14. Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications.

    PubMed

    Takagi, Hiroshi

    2008-11-01

    Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T (m) of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive gamma-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production. PMID:18802692

  15. 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

  16. Inhibition of corneal neovascularization with a nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Shakiba, Yadollah; Mostafaie, Ali

    2007-10-01

    Corneal neovascularization is a significant, sight-threatening complication of many ocular surface disorders. Various growth factors and proteinases are involved in corneal neovascularization. The data supporting a causal role for vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are extensive. Inhibition of VEGF and MMPs is a main strategy for treating corneal neovascularization. Several findings have shown that corneal neovascularization can be reduced by using anti-VEGF and anti-MMPs agents. Efficacy of a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract has been demonstrated for reducing VEGF and MMPs secretion by various cells. Moreover, NM can inhibit endothelial cell migration and capillary tube formation. We herein note that topical application of NM is potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity. Further investigations in animal models are needed to place NM alongside corneal neovascularization therapeutics.

  17. Synthesis of ω-Oxo Amino Acids and trans-5-Substituted Proline Derivatives Using Cross-Metathesis of Unsaturated Amino Acids.

    PubMed

    Salih, Nabaz; Adams, Harry; Jackson, Richard F W

    2016-09-16

    A range of 7-oxo, 8-oxo, and 9-oxo amino acids, analogues of 8-oxo-2-aminodecanoic acid, one of the key components of the cyclic tetrapeptide apicidin, have been prepared by a three-step process involving copper-catalyzed allylation of serine-, aspartic acid-, and glutamic acid-derived organozinc reagents, followed by cross-metathesis of the resulting terminal alkenes with unsaturated ketones and hydrogenation. The intermediate 7-oxo-5-enones underwent a highly diastereoselective (dr ≥96:4) acid-catalyzed aza-Michael reaction to give trans-2,5-disubstituted pyrrolidines, 5-substituted proline derivatives. The aza-Michael reaction was first observed when the starting enones were allowed to stand in solution in deuterochloroform but can be efficiently promoted by catalytic amounts of dry HCl. PMID:27529354

  18. 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.

  19. 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

  20. Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants.

    PubMed

    Schwan, William R; Wetzel, Keith J; Gomez, Timothy S; Stiles, Melissa A; Beitlich, Brian D; Grunwald, Sandra

    2004-04-01

    Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo. To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1.74 microM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7.5 micromol per spleen and 88.4 micromol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.

  1. Synthesis of a proline-modified acrylic acid copolymer in supercritical CO2 for glass-ionomer dental cement applications.

    PubMed

    Moshaverinia, Alireza; Roohpour, Nima; Darr, Jawwad A; Rehman, Ihtesham U

    2009-06-01

    Supercritical (sc-) fluids (such as sc-CO(2)) represent interesting media for the synthesis of polymers in dental and biomedical applications. Sc-CO(2) has several advantages for polymerization reactions in comparison to conventional organic solvents. It has several advantages in comparison to conventional polymerization solvents, such as enhanced kinetics, being less harmful to the environment and simplified solvent removal process. In our previous work, we synthesized poly(acrylic acid-co-itaconic acid-co-N-vinylpyrrolidone) (PAA-IA-NVP) terpolymers in a supercritical CO(2)/methanol mixture for applications in glass-ionomer dental cements. In this study, proline-containing acrylic acid copolymers were synthesized, in a supercritical CO(2) mixture or in water. Subsequently, the synthesized polymers were used in commercially available glass-ionomer cement formulations (Fuji IX commercial GIC). Mechanical strength (compressive strength (CS), diametral tensile strength (DTS) and biaxial flexural strength (BFS)) and handling properties (working and setting time) of the resulting modified cements were evaluated. It was found that the polymerization reaction in an sc-CO(2)/methanol mixture was significantly faster than the corresponding polymerization reaction in water and the purification procedures were simpler for the former. Furthermore, glass-ionomer cement samples made from the terpolymer prepared in sc-CO(2)/methanol exhibited higher CS and DTS and comparable BFS compared to the same polymer synthesized in water. The working properties of glass-ionomer formulations made in sc-CO(2)/methanol were comparable and better than the values of those for polymers synthesized in water. PMID:19269267

  2. Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

    PubMed Central

    Dierks-Ventling, Christa; Tonelli, Chiara

    1982-01-01

    In excised pro1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [14C]proline, [14C]glutamic acid, [14C]glutamine, and [14C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO2 production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro1-1 mutant proline catabolism prevails over proline synthesis. PMID:16662144

  3. Role of proline under changing environments

    PubMed Central

    Hayat, Shamsul; Hayat, Qaiser; Alyemeni, Mohammed Nasser; Wani, Arif Shafi; Pichtel, John; Ahmad, Aqil

    2012-01-01

    When exposed to stressful conditions, plants accumulate an array of metabolites, particularly amino acids. Amino acids have traditionally been considered as precursors to and constituents of proteins, and play an important role in plant metabolism and development. A large body of data suggests a positive correlation between proline accumulation and plant stress. Proline, an amino acid, plays a highly beneficial role in plants exposed to various stress conditions. Besides acting as an excellent osmolyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signaling molecule. Review of the literature indicates that a stressful environment results in an overproduction of proline in plants which in turn imparts stress tolerance by maintaining cell turgor or osmotic balance; stabilizing membranes thereby preventing electrolyte leakage; and bringing concentrations of reactive oxygen species (ROS) within normal ranges, thus preventing oxidative burst in plants. Reports indicate enhanced stress tolerance when proline is supplied exogenously at low concentrations. However, some reports indicate toxic effects of proline when supplied exogenously at higher concentrations. In this article, we review and discuss the effects of exogenous proline on plants exposed to various abiotic stresses. Numerous examples of successful application of exogenous proline to improve stress tolerance are presented. The roles played by exogenous proline under varying environments have been critically examined and reviewed. PMID:22951402

  4. 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

  5. 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

  6. 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

  7. 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

  8. The folding and stability of rhodanese are influenced by the replacement of glutamic acid 17 in the NH2-terminal helix by proline but not by glutamine.

    PubMed

    Luo, G X; Horowitz, P M

    1993-05-15

    Two site-directed mutants of the enzyme rhodanese which replace glutamic acid 17 with either glutamine (E17Q) or with proline (E17P) were produced and purified. Both mutants displayed specific activities similar to the wild type enzyme. E17Q was equivalent to the wild type enzyme in all assayed characteristics, except that the mutant had slightly more solvent exposure of hydrophobic surfaces. Results with E17Q suggest that the charge on Glu17 is not required for helix stabilization, nor is its titration required for the low pH structural transitions seen previously. In contrast, E17P was significantly different from the wild type enzyme. For example, E17P had (a) higher exposure of hydrophobic surfaces in the unperturbed state; (b) considerably lower stability to perturbation by urea; (c) easier exposure of organized hydrophobic surfaces on initial unfolding, even though denaturation to the final disorganized state was the same as for the wild type; (d) the ability to refold without assistants but with lower yields and somewhat slower folding; and (e) similar susceptibility to trypsin and evidence of a new clip site closer to the NH2 terminus. However, E17P and the wild type enzyme had very similar recoveries with chaperonin-assisted refolding, and the chaperonin protein groEL had a very similar ability to suppress unassisted refolding. These results indicate that changes in the NH2-terminal sequence can have dramatic effects on the stability of rhodanese and on its ability to be refolded in the absence of assistants. They further suggest that interactions with chaperonins do not rely exclusively on the detailed conformation at the NH2 terminus. A model that incorporates observations here includes step(s) in which the NH2-terminal sequence folds onto the NH2-terminal domain late in the folding process after the protein had adopted a near native conformation.

  9. Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Roomi, M W; Ivanov, V; Kalinovsky, T; Niedzwiecki, A; Rath, M

    2006-01-01

    Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P < 0.0001) and 1000 microg/mL NM (P < 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.

  10. Energetics of proline transport in corn mitochondria

    SciTech Connect

    Elthon, T.E.; Stewart, C.R.; Bonner, W.D. Jr.

    1984-08-01

    The mechanism of proline entry into the matrix region of isolated corn mitochondria (Zea mays L. Mo17 x B73) was investigated by measuring osmotically induced changes of mitochondrial size (changes in A/sub 520/) in combination with oxygen uptake measurements. Using NADH oxidation to generate the electrochemical gradient, we have determined that proline transport is stereospecific and that it can be inhibited by the proline analog L-thiazolidine-4-carboxylic acid. The energetics of proline transport was investigated by measuring the effects of FCCP (p-trifluoromethoxycarbonyl cyanide phenylhydrazone) and valinomycin on mitochondrial swelling and substrate oxidation. Proline transport and resulting oxidation were found to be partially dependent upon the energy of the electrochemical gradient. At low proline concentrations, entry was found to be primarily independent of the gradient (based on insensitivity to FCCP), whereas at higher proline concentrations a gradient-dependent mechanism became involved. Results with valimomycin indicated that proline transport and oxidation are dependent upon the pH potential across the membrane rather than the electrical (membrane) potential.

  11. 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.

  12. In vitro release control of ketoprofen from pH-sensitive gels consisting of poly(acryloyl- L-proline methyl ester) and saturated fatty acid sodium salts

    NASA Astrophysics Data System (ADS)

    Negishi, M.; Hiroki, A.; Miyajima, M.; Yoshida, M.; Asano, M.; Katakai, R.

    1999-06-01

    The effect of saturated fatty acid sodium salts (C n), sodium laurate (C 12), sodium myristate (C 14), sodium palmitate (C 16), and sodium stearate (C 18), on the swelling of poly(acryloyl- L-proline methyl ester) (A-ProOMe) gel was investigated in different pH solutions. The C n-loaded gels collapsed in a buffer solution with pH 3.0, while they expanded in a buffer solution with pH 6.5. This effect was strongly influenced by the number of methylene units in C n, as the threshold for causing this sensitivity existed between C 12 and C 14. On the other hand, a pulsatile release of ketoprofen occurred when the gel was cycled in buffer solutions between pH 3.0 and pH 6.5. This behavior may be attributable to the surface-regulated mechanism.

  13. Penultimate proline in neuropeptides.

    PubMed

    Glover, Matthew S; Bellinger, Earl P; Radivojac, Predrag; Clemmer, David E

    2015-08-18

    A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

  14. 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

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

    PubMed

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

    2016-03-15

    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.

  16. 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.

  17. 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

  18. Interactions in L-phenylalanine/L-leucine/L-glutamic Acid/L-proline + 2 M aqueous NaCl/2 M NaNO3 systems at different temperatures

    NASA Astrophysics Data System (ADS)

    Riyazuddeen, Imran Khan; Afrin, Sadaf

    2012-12-01

    Density (ρ) and speed of sound ( u) in 2 M aqueous NaCl and 2 M NaNO3 solutions of amino acids: L-phenylalanine, L-leucine, L-glutamic acid, and L-proline have been measured for several molal concentrations of amino acids at different temperatures. The ρ and u data have been used to calculate the values of isothermal compressibility and internal pressure at different temperatures. The trends of variations of κ T and P i with an increase in molal concentration of amino acid and temperature have been discussed in terms of solute-solvent and solute-solute interactions in the systems.

  19. 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

  20. Mutating a Conserved Proline Residue within the Trimerization Domain Modifies Na+ Binding to Excitatory Amino Acid Transporters and Associated Conformational Changes*

    PubMed Central

    Hotzy, Jasmin; Schneider, Nicole; Kovermann, Peter; Fahlke, Christoph

    2013-01-01

    Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3. PMID:24214974

  1. 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.

  2. Proline-rich Sequence Recognition

    PubMed Central

    Schlundt, Andreas; Sticht, Jana; Piotukh, Kirill; Kosslick, Daniela; Jahnke, Nadin; Keller, Sandro; Schuemann, Michael; Krause, Eberhard; Freund, Christian

    2009-01-01

    The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly “PTAP” interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFκB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461–2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes. PMID:19542561

  3. The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f).

    PubMed Central

    Hay, D I; Bennick, A; Schlesinger, D H; Minaguchi, K; Madapallimattam, G; Schluckebier, S K

    1988-01-01

    Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins. Images Fig. 1. Fig. 4. Fig. 5. PMID:3196309

  4. 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.

  5. 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

  6. Proline metabolism and transport in maize seedlings at low water potential.

    PubMed

    Raymond, Marjorie J; Smirnoff, Nicholas

    2002-06-01

    The growing zone of maize seedling primary roots accumulates proline at low water potential. Endosperm removal and excision of root tips rapidly decreased the proline pool and greatly reduced proline accumulation in root tips at low water potential. Proline accumulation was not restored by exogenous amino acids. Labelling root lips with [14C]glutamate and [14C]proline showed that the rate of proline utilization (oxidation and protein synthesis) exceeded the rate of biosynthesis by five-fold at high and low water potentials. This explains the reduction in the proline pool following root and endosperm excision and the inability to accumulate proline at low water potential. The endosperm is therefore the source of the proline that accumulates in the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [14C]Proline was transported from the scutellum to other parts of the seedling and reached the highest concentration in the root tip. Less [14C]proline was transported at low water potential but because of the lower rate of protein synthesis and oxidation, more accumulated as proline in the root tip. Despite the low biosynthesis capacity of the roots, the extent of proline accumulation in relation to water potential is precisely controlled by transport and utilization rate.

  7. Thermodynamic and conformational study of proline stereoisomers.

    PubMed

    Santos, Ana Filipa L O M; Notario, Rafael; Ribeiro da Silva, Manuel A V

    2014-08-28

    Amino acids play fundamental roles both as building blocks of proteins and as intermediates in metabolism. Proline, one of the 20 natural amino acids, has a primordial function in enzymes, peptide hormones, and proteins. The energetic characterization of these molecules provides information concerning stability and reactivity and has great importance in understanding the activity and behavior of larger molecules containing these structures as fragments. In the present work, parallel experimental and computational studies have been performed. The experimental studies have been based on calorimetric and effusion techniques, from which the enthalpy of formation in the crystalline phase and the enthalpy of sublimation of the sterioisomers L-, D-, and the DL-mixture of proline have been derived. Additionally, vapor pressure measurements have also enabled the determination of the entropies and Gibbs energies of sublimation, at T = 298.15 K. From the former results, the experimental standard (p(o) = 0.1 MPa) molar enthalpies of formation, in the gaseous phase, at T = 298.15 K, of L-proline, D-proline, and DL-proline have been calculated as -388.6 ± 2.3, -391.9 ± 2.0, and -391.5 ± 2.4 kJ·mol(-1), respectively. A computational study at the G3 and G4 levels has been carried out. Conformational analysis has been done and the enthalpy of formation of proline as well as other intrinsic properties such as acidity, basicity, adiabatic ionization enthalpy, electron and proton affinities, and bond dissociation enthalpies have been calculated. There is a very good agreement between calculated and experimental values, when they are available.

  8. 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

  9. Connecting proline metabolism and signaling pathways in plant senescence

    PubMed Central

    Zhang, Lu; Becker, Donald F.

    2015-01-01

    The amino acid proline has a unique biological role in stress adaptation. Proline metabolism is manipulated under stress by multiple and complex regulatory pathways and can profoundly influence cell death and survival in microorganisms, plants, and animals. Though the effects of proline are mediated by diverse signaling pathways, a common theme appears to be the generation of reactive oxygen species (ROS) due to proline oxidation being coupled to the respiratory electron transport chain. Considerable research has been devoted to understand how plants exploit proline metabolism in response to abiotic and biotic stress. Here, we review potential mechanisms by which proline metabolism influences plant senescence, namely in the petal and leaf. Recent studies of petal senescence suggest proline content is manipulated to meet energy demands of senescing cells. In the flower and leaf, proline metabolism may influence ROS signaling pathways that delay senescence progression. Future studies focusing on the mechanisms by which proline metabolic shifts occur during senescence may lead to novel methods to rescue crops under stress and to preserve post-harvest agricultural products. PMID:26347750

  10. Different Type 1 Fimbrial Genes and Tropisms of Commensal and Potentially Pathogenic Actinomyces spp. with Different Salivary Acidic Proline-Rich Protein and Statherin Ligand Specificities

    PubMed Central

    Li, Tong; Khah, Massoud Kheir; Slavnic, Snjezana; Johansson, Ingegerd; Strömberg, Nicklas

    2001-01-01

    Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundii genospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 and fimP) in genospecies 1 and 2 strains, except that orf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oral Actinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C and fimP). Bioinformatics suggested sortase (orfB, orf4, and part of orf5), prepilin peptidase (orfC and orf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1 to -3) functions. Those gene regions corresponding to orf3 and orf5 were divergent, those corresponding to orf2, orf1, and fimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only the orf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both

  11. 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

  12. Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the first step in the racemization of proline catalyzed by proline racemase.

    PubMed

    Williams, Glenn; Maziarz, E Peter; Amyes, Tina L; Wood, Troy D; Richard, John P

    2003-07-15

    Rate constants for the hydrolysis of L-proline methyl ester to form proline and methanol in D(2)O buffered at neutral pD and 25 degrees C and the deuterium enrichment of the proline product determined by electrospray ionization mass spectrometry are reported. The data give k(DO) = 5.3 +/- 0.5 M(-1) s(-1) as the second-order rate constant for carbon deprotonation of N-protonated proline methyl ester by deuterioxide ion in D(2)O at 25 degrees C and I = 1.0 (KCl). The data provide good estimates of carbon acidities of pK(a) = 21 for N-protonated proline methyl ester and pK(a) = 29 for proline zwitterion in water and of the second-order rate constant k(HO) = 4.5 x 10(-5) M(-1) s(-1) for carbon deprotonation of proline zwitterion by hydroxide ion at 25 degrees C. There is no detectable acceleration of the deprotonation of N-protonated proline methyl ester by the Brønsted base 3-quinuclidinone in water, and it is not clear that such Brønsted catalysis would make a significant contribution to the rate acceleration for deprotonation of bound proline at proline racemase. A comparison of the first-order rate constants k(HO)[HO(-)] = 4.5 x 10(-11) s(-1) for deprotonation of free proline zwitterion in water at pH 8 and k(cat) = 2600 s(-1) for deprotonation of proline bound to the active site of proline racemase at pH 8 shows that the enzymatic rate acceleration for proline racemase is ca. 10(13)-fold. This corresponds to a 19 kcal/mol stabilization of the transition state for deprotonation of the enzyme-bound carbon acid substrate by interaction with the protein catalyst. It is suggested that (1) much of the rate acceleration of the enzymatic over the nonenzymatic reaction in water may result from transfer of the substrate proline zwitterion from the polar solvent water to a nonpolar enzyme active site and (2) the use of thiol anions rather than oxygen anions as Brønsted bases at this putative nonpolar enzyme active site may be favored, because of the smaller energetic

  13. 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.

  14. 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

  15. 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

  16. Efficacy of proline in the treatment of menopause

    PubMed Central

    Nam, Sun-Young; Yoou, Myoung-Schook

    2016-01-01

    The amino acids in the placenta have multiple functions; however, the therapeutic effects of proline remain poorly for relief postmenopausal symptoms. The aim of present study was to evaluate the effects of proline in the treatment of menopause using in vitro and in vivo models. We assessed the therapeutic effects and regulatory mechanisms of proline by using MCF-7 estrogen-dependent cells, MG63 osteoblast cells, and ovariectomized mice model. An in vivo study was carried out in eight-week-old sham and ovariectomized group. The ovariectomized mouse was further subdivided into two groups administered orally with 17β-estradiol or proline (10 mg/kg/day) for eight weeks. Proline significantly increased cell proliferation and Ki-67 levels in MCF-7 cells and enhanced cell proliferation, alkaline phosphatase activity, extracellular signal-regulated kinase phosphorylation, and glutamyl-prolyl-tRNA synthetase activation in MG63 cells. The estrogen receptor-β and estrogen-response elements luciferase activity were significantly increased by proline in MCF-7 and MG63 cells. In ovariectomized mice, oral administration of proline (10 mg/kg/day) for eight weeks significantly reduced body and vaginal weights. Proline also significantly increased serum estradiol and alkaline phosphatase levels, whereas serum luteinizing hormone was decreased by proline. In addition, detailed microcomputed tomography analysis showed that the proline notably enhanced bone mineral density, trabecular bone volume, and trabecular number in ovariectomized mice. Those findings implied that proline can be a promising candidate for the treatment of menopause. PMID:26830682

  17. 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

  18. 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).

  19. 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

  20. 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.

  1. Uptake of proline by the scutellum of germinating barley grain

    SciTech Connect

    Vaeisaenen, E.; Sopanen, T.

    1986-04-01

    Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up 1 millimolar L-(/sup 14/C)proline at an initial rate of about 6.5 micromoles gram/sup -1/ fresh weight hour/sup -1/ (pH 5, 30/sup 0/C). The uptake had a pH optimum at 5. The bulk of the uptake (93%) was via carrier-mediated active transport. All of the 19 L-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. D-Proline inhibited this system as strongly as L-proline. Nine of the 16 L-amino tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain.

  2. 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

  3. Antiradical activity of gallic acid included in lipid interphases.

    PubMed

    Salcedo, C L; Frías, M A; Cutro, A C; Nazareno, M A; Disalvo, E A

    2014-10-01

    Polyphenols are well known as antioxidant agents and by their effects on the hydration layers of lipid interphases. Among them, gallic acid and its derivatives are able to decrease the dipole potential and to act in water as a strong antioxidant. In this work we have studied both effects on lipid interphases in monolayers and bilayers of dimyristoylphosphatidylcholine. The results show that gallic acid (GA) increases the negative surface charges of large unilamellar vesicles (LUVs) and decreases the dipole potential of the lipid interphase. As a result, positively charged radical species such as ABTS(+) are able to penetrate the membrane forming an association with GA. These results allow discussing the antiradical activity (ARA) of GA at the membrane phase which may be taking place in water spaces between the lipids.

  4. 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

  5. Comparative aspects of tissue glutamine and proline metabolism.

    PubMed

    Bertolo, Robert F; Burrin, Douglas G

    2008-10-01

    The cellular metabolism of glutamine and proline are closely interrelated, because they can be interconverted with glutamate and ornithine via the mitochondrial pathway involving pyrroline-5-carboxylate (P5C). In adults, glutamine and proline are converted via P5C to citrulline in the gut, then citrulline is converted to arginine in the kidney. In neonates, arginine is a semiindispensable amino acid and is synthesized from proline completely in the gut; because of low P5C synthase activity, glutamine is not an important precursor for neonatal arginine synthesis. Thus, splanchnic metabolism of glutamine and proline is important, because both amino acids serve as key precursors for arginine synthesis with some developmental differences. Studies investigating splanchnic extraction demonstrate that about two-thirds of dietary glutamine and almost all dietary glutamate are extracted on first pass and the vast majority is oxidized in the gut. This capacity to extract glutamine and glutamate appears to be very large, so diets high in glutamine or glutamate probably have little impact on circulating concentrations and consequent potential toxicity. In contrast, it appears that very little proline is extracted by the gut and liver, at least in the neonate, which may result in hyperprolinemia and potential toxicity. Therefore, the upper limits of safe dietary intake for glutamine and proline, and other amino acids, appear to be substantially different depending on the extent of first-pass splanchnic extraction and irreversible catabolism.

  6. Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.

    PubMed

    Moxley, Michael A; Becker, Donald F

    2012-01-10

    The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.

  7. l-Hydroxyproline and d-Proline Catabolism in Sinorhizobium meliloti

    PubMed Central

    Chen, Siyun; White, Catharine E.; diCenzo, George C.; Zhang, Ye; Stogios, Peter J.; Savchenko, Alexei

    2016-01-01

    ABSTRACT Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and several related compounds, such as proline betaine, trans-4-hydroxy-l-proline (trans-4-l-Hyp), and cis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteen hyp genes are induced upon growth of S. meliloti on trans-4-l-Hyp, and of those, hypMNPQ encodes an ABC-type trans-4-l-Hyp transporter and hypRE encodes an epimerase that converts trans-4-l-Hyp to cis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in which cis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as a d-amino acid dehydrogenase, converting cis-4-d-Hyp to Δ1-pyrroline-4-hydroxy-2-carboxylate, which is deaminated by HypD to α-ketoglutarate semialdehyde and then converted to α-ketoglutarate by HypH. The crystal structure of HypD revealed it to be a member of the N-acetylneuraminate lyase subfamily of the (α/β)8 protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown that S. meliloti can catabolize d-proline as both a carbon and a nitrogen source, that d-proline can complement l-proline auxotrophy, and that the catabolism of d-proline is dependent on the hyp cluster. Transport of d-proline involves the HypMNPQ transporter, following which d-proline is converted to Δ1-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted to l-proline through the NADPH-dependent reduction of P2C by the previously uncharacterized HypS protein. Thus, overall, we have now completed detailed genetic and/or biochemical characterization of 9 of the 14 hyp genes. IMPORTANCE Hydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut

  8. Rapid method for proline determination in grape juice and wine.

    PubMed

    Long, Danfeng; Wilkinson, Kerry L; Poole, Kate; Taylor, Dennis K; Warren, Tristan; Astorga, Alejandra M; Jiranek, Vladimir

    2012-05-01

    Proline is typically the most abundant amino acid present in grape juice and wine. The amount present is influenced by viticultural and winemaking factors and can be of diagnostic importance. A method for rapid routine quantitation of proline would therefore be of benefit for wine researchers and the industry in general. Colorimetric determination utilizing isatin as a derivatizing agent has previously been applied to plant extracts, biological fluids, and protein hydrolysates. In the current study, this method has been successfully adapted to grape juice and wine and proved to be sensitive to milligram per liter amounts of proline. At sugar concentrations above 60 g/L, interference from the isatin-proline reaction was observed, such that proline concentrations were considerably underestimated in grape juice and dessert wine. However, the method was robust for the analysis of fermentation samples and table wines. Results were within ±10% agreement with data generated from typical HPLC-based analyses. The isatin method is therefore considered suitable for the routine analysis required to support research into the utilization or release of proline by yeast during fermentation. PMID:22480274

  9. 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.

  10. 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

  11. A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.

    PubMed

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

    2009-02-10

    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 insight 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 analogue l-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 A, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (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 the 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.

  12. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate

    SciTech Connect

    Ostrander, E.L.; Larson, J.D.; Schuermann, J.P.; Tanner, J.J.

    2009-03-02

    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 insight 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 analogue L-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 {angstrom}, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (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 the 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.

  13. Proline biosynthesis is required for endoplasmic reticulum stress tolerance in Saccharomyces cerevisiae.

    PubMed

    Liang, Xinwen; Dickman, Martin B; Becker, Donald F

    2014-10-01

    The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP(+)/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.

  14. 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.

  15. 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)

  16. Proline induced disruption of the structure and dynamics of water.

    PubMed

    Yu, Dehong; Hennig, Marcus; Mole, Richard A; Li, Ji Chen; Wheeler, Cheryl; Strässle, Thierry; Kearley, Gordon J

    2013-12-21

    We use quasi-elastic neutron scattering spectroscopy to study the diffusive motion of water molecules at ambient temperature as a function of the solute molar fraction of the amino acid, proline. We validate molecular dynamics simulations against experimental quasielastic neutron scattering data and then use the simulations to reveal, and understand, a strong dependence of the translational self-diffusion coefficient of water on the distance to the amino acid molecule. An analysis based on the juxtaposition of water molecules in the simulation shows that the rigidity of proline imposes itself on the local water structure, which disrupts the hydrogen-bond network of water leading to an increase in the mean lifetime of hydrogen bonds. The net effect is some distortion of the proline molecule and a slowing down of the water mobility. PMID:24177249

  17. 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.).

  18. Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues.

    PubMed

    Spedaliere, C J; Hamilton, C S; Mueller, E G

    2000-08-01

    On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996) Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families. Contrary to our expectations, the altered enzymes display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.

  19. 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

  20. Na-dependent L-proline transport by eel intestinal brush-border membrane vesicles

    SciTech Connect

    Vilella, S.; Ahearn, G.A.; Cassano, G.; Storelli, C. University of Hawaii at Manoa, Honolulu )

    1988-10-01

    L-({sup 3}H)proline uptake by brush-border membrane vesicles prepared from intestinal mucosa of the European eel, Anguilla anguilla, was stimulated by a transmembrane Na gradient (out > in.) Kinetic analysis of L-proline influx, under short-circuited membrane potential conditions, indicated the presence of an apparent single Na-dependent carrier process and a nonsaturable transfer component with an apparent diffusional permeability (P) of 1.53 {plus minus} 0.35 {mu}l{center dot}mg protein{sup {minus}1}{center dot}min{sup {minus}1}. An imposed transmembrane potential (inside negative) increased apparent L-proline binding affinity (lowered K{sub app}) without appreciably altering maximal amino acid influx (J{sub max}). Hill analysis of L-proline influx over a wide range of external Na concentrations indicated a 1:1 stoichiometry for Na-proline cotransport. Use of amino acid inhibitors of L-proline influx suggested that L-proline transfer may occur by either a classical Na-dependent A System with a wide substrate specificity or by the combination of Na-dependent PHE (phenylalanine preferring) and IMINO (proline, {alpha}-methylaminoisobutyric acid preferring) Systems.

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

    DOEpatents

    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.

  2. 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

  3. Functional specialization in proline biosynthesis of melanoma.

    PubMed

    De Ingeniis, Jessica; Ratnikov, Boris; Richardson, Adam D; Scott, David A; Aza-Blanc, Pedro; De, Surya K; Kazanov, Marat; Pellecchia, Maurizio; Ronai, Ze'ev; Osterman, Andrei L; Smith, Jeffrey W

    2012-01-01

    Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13)C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.

  4. Functional Specialization in Proline Biosynthesis of Melanoma

    PubMed Central

    Richardson, Adam D.; Scott, David A.; Aza-Blanc, Pedro; De, Surya K.; Kazanov, Marat; Pellecchia, Maurizio; Ronai, Ze'ev; Osterman, Andrei L.; Smith, Jeffrey W.

    2012-01-01

    Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis. PMID:23024808

  5. 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

  6. Elimination of self-association as the source of the thermodynamic nonideality in aqueous proline solutions.

    PubMed

    Jones, G P; Paleg, L G; Winzor, D J

    1994-09-28

    The effect of high concentrations of proline on the diffusion coefficient of water has been examined to assess the extent to which the resulting thermodynamic nonideality could be explained on the statistical-mechanical basis of excluded volume. In fact, such a space-filling role not only accounts for the proline concentration-dependence of the diffusion coefficient of water but it also accounts for the nonideality of proline in freezing point depression and isopiestic measurements. These findings refute the conclusion (Schobert, B. and Tschesche, H. (1978) Biochim. Biophys. Acta 541, 270-277) that the stabilization of enzyme structure by high concentrations of proline stems from self-association of the imino acid via intermolecular hydrogen bonding; and thereby support the concept that the protective effect of proline on enzyme stability must reside mainly in its action as an inert, space-filling solute. PMID:7918580

  7. 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.

  8. Pitiprolamide, a proline-rich dolastatin 16 analogue from the marine cyanobacterium Lyngbya majuscula from Guam.

    PubMed

    Montaser, Rana; Abboud, Khalil A; Paul, Valerie J; Luesch, Hendrik

    2011-01-28

    An unusual cyclic depsipeptide, pitiprolamide (1), was isolated from the marine cyanobacterium Lyngbya majuscula collected at Piti Bomb Holes, Guam. The structure was deduced using NMR, MS, X-ray crystallography, and enantioselective HPLC-MS techniques. Remarkably, proline represents half of the residues forming pitiprolamide (1). Other distinctive features include a 4-phenylvaline (dolaphenvaline, Dpv) moiety initially found in dolastatin 16 and the rare 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha) unit condensed in a unique sequence in one single molecule. Pitiprolamide (1) showed weak cytotoxic activity against HCT116 colon and MCF7 breast cancer cell lines, as well as weak antibacterial activities against Mycobacterium tuberculosis and Bacillus cereus.

  9. Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

    PubMed

    Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

    2012-03-01

    Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

  10. 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

  11. Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures 1

    PubMed Central

    Thomas, John C.; De Armond, Richard L.; Bohnert, Hans J.

    1992-01-01

    The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs. ImagesFigure 4Figure 5 PMID:16668687

  12. 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

  13. Actinomyces naeslundii Displays Variant fimP and fimA Fimbrial Subunit Genes Corresponding to Different Types of Acidic Proline-Rich Protein and β-Linked Galactosamine Binding Specificity

    PubMed Central

    Hallberg, K.; Holm, C.; Öhman, U.; Strömberg, N.

    1998-01-01

    Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to β-linked galactosamine (GalNAcβ) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcβ, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) and fimA (type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral Actinomyces isolates. Isolates of genospecies 1 and 2 and A. odontolyticus, but no other Actinomyces species, were positive for hybridization with fimP and fimA full-length probes irrespective of binding to APRPs and statherin, GalNAcβ, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcβ1-3Galα-O-ethyl-inhibitable coaggregation with Streptococcus oralis Ss34 and MPB1, were distinguished by a fimA central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a fimP central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of fimP and fimA central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion, A. naeslundii exhibits variant fimP and fimA genes corresponding to diverse APRP and GalNAcβ specificities, respectively, while A. odontolyticus has a genetically related but distinct adhesin binding specificity. PMID:9712794

  14. Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients.

    PubMed

    An, W S; Lee, S M; Son, Y K; Kim, S E; Kim, K H; Han, J Y; Bae, H R; Park, Y

    2012-01-01

    Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.

  15. 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

  16. Functional genomics and SNP analysis of human genes encoding proline metabolic enzymes

    PubMed Central

    Williams, D. Bart; Zhaorigetu, Siqin; Khalil, Shadi; Wan, Guanghua; Valle, David

    2009-01-01

    Proline metabolism in mammals involves two other amino acids, glutamate and ornithine, and five enzymatic activities, Δ1-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-δ-aminotransferase (OAT). With the exception of OAT, which catalyzes a reversible reaction, the other 4 enzymes are unidirectional, suggesting that proline metabolism is purpose-driven, tightly regulated, and compartmentalized. In addition, this tri-amino-acid system also links with three other pivotal metabolic systems, namely the TCA cycle, urea cycle, and pentose phosphate pathway. Abnormalities in proline metabolism are relevant in several diseases: six monogenic inborn errors involving metabolism and/or transport of proline and its immediate metabolites have been described. Recent advances in the Human Genome Project, in silico database mining techniques, and research in dissecting the molecular basis of proline metabolism prompted us to utilize functional genomic approaches to analyze human genes which encode proline metabolic enzymes in the context of gene structure, regulation of gene expression, mRNA variants, protein isoforms, and single nucleotide polymorphisms. PMID:18506409

  17. Role of abscisic acid (ABA) and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation.

    PubMed

    Verslues, Paul E; Bray, Elizabeth A

    2006-01-01

    The mechanisms by which plants respond to reduced water availability (low water potential) include both ABA-dependent and ABA-independent processes. Pro accumulation and osmotic adjustment are two important traits for which the mechanisms of regulation by low water potential, and the involvement of ABA, is not well understood. The ABA-deficient mutant, aba2-1, was used to investigate the regulatory role of ABA in low water potential-induced Pro accumulation and osmotic adjustment in seedlings of Arabidopsis thaliana. Low water potential-induced Pro accumulation required wild-type levels of ABA, as well as a change in ABA sensitivity or ABA-independent events. Osmotic adjustment, in contrast, occurred independently of ABA accumulation in aba2-1. Quantification of low water potential-induced ABA and Pro accumulation in five ABA-insensitive mutants, abi1-1, abi2-1, abi3, abi4, and abi5, revealed that abi4 had increased Pro accumulation at low water potential, but a reduced response to exogenous ABA. Both of these responses were modified by sucrose treatment, indicating that ABI4 has a role in connecting ABA and sugar in regulating Pro accumulation. Of the other abi mutants, only abi1 had reduced Pro accumulation in response to low water potential and ABA application. It was also observed that abi1-1 and abi2-1 had increased ABA accumulation. The involvement of these loci in feedback regulation of ABA accumulation may occur through an effect on ABA catabolism or conjugation. These data provide new information on the function of ABA in seedlings exposed to low water potential and define new roles for three of the well-studied abi loci.

  18. 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

  19. Diastereoselective Synthesis of 5-Heteroaryl-Substituted Prolines Useful for Controlling Peptide-Bond Geometry.

    PubMed

    Ali, Rafat; Singh, Gajendra; Singh, Shalini; Ampapathi, Ravi Sankar; Haq, Wahajul

    2016-06-17

    A versatile diastereoselective Friedel-Crafts alkylation reaction of heteroaryl systems with a cyclic enecarbamate for the preparation of 5-heteroaryl-substituted proline analogues in good yields has been developed. These heterocyclic tethered cyclic amino acid building blocks constitute important structural motifs in many biologically active molecules. The impact of the substitution on proline cis/trans isomerization was explored by carrying out solution conformational studies by NMR on 5-furanyl-substituted proline-containing peptides. Conformational analysis revealed that the peptide bond is constrained in an exclusively trans conformation. PMID:27228427

  20. 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

  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. The thermodynamic activity of proline in ternary solutions of different water potentials.

    PubMed

    Pahlich, E; Stadermann, T

    1984-06-01

    The particular colligative properties of proline caused us to investigate the thermodynamic activity of this amino acid in detail. The dependence of the activity coefficients γ of proline (γ = thermodynamic activity/molality) on the pH of the solutions, the composition of the solution and the water potential has been measured. The results show that the activity coefficient of proline varies according to the solute milieu. The most pronounced alterations of the activity coefficient could be observed in polyethylene glycol solutions in contrast to KCl- and saccharose solutions where the effect was less distinct. The results described provide a basis for discussing water stress induced metabolic alterations in terms of thermodynamic entities. Changed rates of proline metabolizing sequences and changed ratios of the vacuole/extravacuole distribution of this amino acid in stressed and un-stressed plants may partially be explained by thermodynamic causes.

  3. 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

  4. 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

  5. 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

  6. 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

  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. 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.

  10. Proline inhibits aggregation during protein refolding.

    PubMed Central

    Samuel, D.; Kumar, T. K.; Ganesh, G.; Jayaraman, G.; Yang, P. W.; Chang, M. M.; Trivedi, V. D.; Wang, S. L.; Hwang, K. C.; Chang, D. K.; Yu, C.

    2000-01-01

    The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone. PMID:10716186

  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.

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

    PubMed

    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

  15. An algorithm for computing nucleic acid base-pairing probabilities including pseudoknots.

    PubMed

    Dirks, Robert M; Pierce, Niles A

    2004-07-30

    Given a nucleic acid sequence, a recent algorithm allows the calculation of the partition function over secondary structure space including a class of physically relevant pseudoknots. Here, we present a method for computing base-pairing probabilities starting from the output of this partition function algorithm. The approach relies on the calculation of recursion probabilities that are computed by backtracking through the partition function algorithm, applying a particular transformation at each step. This transformation is applicable to any partition function algorithm that follows the same basic dynamic programming paradigm. Base-pairing probabilities are useful for analyzing the equilibrium ensemble properties of natural and engineered nucleic acids, as demonstrated for a human telomerase RNA and a synthetic DNA nanostructure. PMID:15139042

  16. 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.

  17. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    PubMed

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  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).

  19. Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

    PubMed

    Loomis, R E; Gonzalez, M; Loomis, P M

    1991-11-01

    The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.

  20. 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

  1. 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

  2. Adaptation of an L-Proline Adenylation Domain to Use 4-Propyl-L-Proline in the Evolution of Lincosamide Biosynthesis

    PubMed Central

    Kadlčík, Stanislav; Kučera, Tomáš; Chalupská, Dominika; Gažák, Radek; Koběrská, Markéta; Ulanová, Dana; Kopecký, Jan; Kutejová, Eva; Najmanová, Lucie; Janata, Jiří

    2013-01-01

    Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accomodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin - but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit. PMID:24386435

  3. 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

  4. Colostrinin: a proline-rich polypeptide complex of potential therapeutic interest.

    PubMed

    Janusz, M; Zabłocka, A

    2013-01-01

    A proline-rich polypeptide complex (PRP) subsequently known as ColostrininTM was found for the first time in ovine colostrum as a fraction accompanying colostral IgG2. Subsequently, similar polypeptides were found in human, bovine and caprine colostrum. PRP is a complex of peptides of molecular masses from 500 to 3000 Da. It contains 25% proline residues and 40% hydrophobic amino acids. It is not species specific, and is active both in vivo and in vitro. PRP possesses immunoregulatory properties, including effects on humoral and cellular immune responses, shows regulatory activity in Th1 and Th2 cytokine induction, and has the ability to inhibit the overproduction of reactive oxygen species and nitric oxide. PRP has also shown psychotropic properties. Both immunoregulatory and psychotropic properties suggest potential clinical use of PRP for neurodegenerative disorders. Beneficial effects of PRP/Colostrinin in the case of Alzheimer's disease were shown in double-blind placebo-controlled trials, in long-term open-label studies and in multicenter clinical trials. A very important property of PRP/Colostrinin and one of its components, a nonapeptide (NP), is the prevention of Aβ aggregation and the disruption of aggregates already formed. Moreover, PRP has been found to modulate neurite outgrowth, suppress uncontrolled activation of cells, and reduce 4-HNE-mediated cellular damage. Biological response modifying activity of PRP/Colostrinin can play an important role in its use in the treatment of Alzheimer's disease and suggests its application beyond neurodegenerative disorders. PMID:24200016

  5. 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

  6. 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

  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. 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

  9. 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.

  10. 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

  11. 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

  12. Pitiprolamide, a Proline-Rich Dolastatin 16 Analogue from the Marine Cyanobacterium Lyngbya majuscula from Guam

    PubMed Central

    Montaser, Rana; Abboud, Khalil A.; Paul, Valerie J.; Luesch, Hendrik

    2010-01-01

    An unusual cyclic depsipeptide, pitiprolamide (1), was isolated from the marine cyanobacterium Lyngbya majuscula collected at Piti Bomb Holes, Guam. The structure was deduced using NMR, MS, X-ray crystallography and enantioselective HPLC-MS techniques. Remarkably, proline represents half of the residues forming pitiprolamide (1). Other distinctive features include a 4-phenylvaline (dolaphenvaline, Dpv) moiety initially found in dolastatin 16 and the rare 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha) unit condensed in a unique sequence in one single molecule. Pitiprolamide (1) showed weak cytotoxic activity against HCT116 colon and MCF7 breast cancer cell lines, as well as weak antibacterial activities against Mycobacterium tuberculosis and Bacillus cereus. PMID:21138309

  13. [Properties of post-proline cleaving enzymes from Tenebrio molitor].

    PubMed

    Goptar', I A; Kulemzina, I A; Filippova, I Iu; Lysogorskaia, E N; Oksenoĭt, E S; Zhuzhikov, D P; Dunaevskiĭ, Ia E; Belozerskiĭ, M A; Elpidina, E N

    2008-01-01

    Two post-proline cleaving enzymes PRE1 and PRE2 with molecular masses of 101 and 62 kDa, respectively, capable of hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the midgut of the flour beetle Tenebrio molitor and characterized. PRE1 is active only in acidic media, with a maximum at pH 5.6, whereas PRE2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PRE1 and PRE2 were shown to belong to serine peptidases. Some data indicate that a Cys residue is located close to the PRE2 active site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, inhibits completely PRE2 and partially PRE1. The substrate specificities of the isolated enzymes were studied. It was shown that Z-AlaAla-Pro-pNA was the best substrate for PRE1, and Z-AlaPro-pNA, for PRE2. The combination of the studied properties allowed characterization of PRE2 as a prolyl oligopeptidase.

  14. 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

  15. Influence of fatty acid precursors, including food preservatives, on the growth and fatty acid composition of Listeria monocytogenes at 37 and 10degreesC.

    PubMed

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

    2010-03-01

    Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C(15:0) 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 degrees C and 10 degrees 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, C(4), C(5), and C(6) branched-chain carboxylic acid, and C(3) and C(4) 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.

  16. 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, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  17. 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, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  18. 21 CFR 582.5650 - Proline.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  19. Evidence for a role of proline and hypothalamic astrocytes in the regulation of glucose metabolism in rats.

    PubMed

    Arrieta-Cruz, Isabel; Su, Ya; Knight, Colette M; Lam, Tony K T; Gutiérrez-Juárez, Roger

    2013-04-01

    The metabolism of lactate to pyruvate in the mediobasal hypothalamus (MBH) regulates hepatic glucose production. Because astrocytes and neurons are functionally linked by metabolic coupling through lactate transfer via the astrocyte-neuron lactate shuttle (ANLS), we reasoned that astrocytes might be involved in the hypothalamic regulation of glucose metabolism. To examine this possibility, we used the gluconeogenic amino acid proline, which is metabolized to pyruvate in astrocytes. Our results showed that increasing the availability of proline in rats either centrally (MBH) or systemically acutely lowered blood glucose. Pancreatic clamp studies revealed that this hypoglycemic effect was due to a decrease of hepatic glucose production secondary to an inhibition of glycogenolysis, gluconeogenesis, and glucose-6-phosphatase flux. The effect of proline was mimicked by glutamate, an intermediary of proline metabolism. Interestingly, proline's action was markedly blunted by pharmacological inhibition of hypothalamic lactate dehydrogenase (LDH) suggesting that metabolic flux through LDH was required. Furthermore, short hairpin RNA-mediated knockdown of hypothalamic LDH-A, an astrocytic component of the ANLS, also blunted the glucoregulatory action of proline. Thus our studies suggest not only a new role for proline in the regulation of hepatic glucose production but also indicate that hypothalamic astrocytes are involved in the regulatory mechanism as well. PMID:23274895

  20. Densely Substituted L-Proline Esters as Catalysts for Asymmetric Michael Additions of Ketones to Nitroalkenes.

    PubMed

    Ruiz-Olalla, Andrea; Retamosa, María de Gracia; Cossío, Fernando P

    2015-06-01

    Homochiral methyl 4-aminopyrrolidine-2-carboxylates are readily obtained by means of asymmetric (3 + 2) cycloadditions between azomethine ylides and nitroalkenes, followed by catalytic hydrogenation of the intermediate 4-nitro cycloadducts. These 4-aminopyrrolidine-2-carboxylate esters belong to the L-series of natural amino acids and catalyze asymmetric Michael additions of ketones to nitroalkenes. However, the enantioselectivity observed with these novel unnatural organocatalysts is opposite to that obtained with L-proline. Since both 4-nitro and 4-amino L-proline esters are efficient organocatalysts of aldol reactions, these results permit to modulate asymmetric quimioselective aldol and conjugate addition reactions.

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

    PubMed Central

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

    2016-01-01

    Summary 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

  2. 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.

  3. HR-MAS NMR metabolomics of 'Swingle' citrumelo rootstock genetically modified to overproduce proline.

    PubMed

    de Oliveira, Caroline S; Carlos, Eduardo F; Vieira, Luiz G E; Lião, Luciano M; Alcantara, Glaucia B

    2014-08-01

    The accumulation of proline is a typical physiological response to abiotic stresses in higher plants. 'Swingle' citrumelo, an important rootstock for citrus production, has been modified with a mutated Δ(1)-pyrroline-5-carboxylate synthetase gene (VaP5CSF129A) linked to the cauliflower mosaic virus 35S promoter to induce the overproduction of free proline. This paper presents a comparative metabolomic study of nontransgenic versus transgenic 'Swingle' citrumelo plants with high endogenous proline. (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and multivariate analysis showed significant differences in some metabolites between the nontransgenic and transgenic leaves and roots. The overproduction of proline has reduced the sucrose content in transgenic leaves, revealing a metabolic cost for these plants. In roots, the high level of free proline acts for the adjustment of cation-anion balance, causing the reduction of acetic acid content. The same sucrose level in roots indicates that they can be considered as sucrose sink. Similar behavior may be waited for fruits produced on transgenic rootstock.

  4. 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.

  5. 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.

  6. 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.

  7. Developmental aspects of proline transport in rat renal brush border membranes.

    PubMed

    Medow, M S; Roth, K S; Goldmann, D R; Ginkinger, K; Hsu, B Y; Segal, S

    1986-10-01

    Proline uptake by rat renal brush border membrane vesicles from animals 7 days of age and older has been examined to delineate developmental changes in membrane function that may underlie the physiological hyperprolinuria of young animals. Although the two proline transport systems normally present in adult membranes were found in membranes from young animals, the proline "overshoot" resulting from a sodium ion gradient is minimal and increases with age of the animal from which the membranes were isolated. This is associated with a severalfold faster entry of 22Na into vesicles of the 7-day-old animal compared to entry into membranes prepared from adult kidneys. The very rapid dissipation of the sodium gradient thus diminishing the driving force for transmembrane proline movement may explain the changes in proline overshoot observed in membranes from young animals. The altered sodium permeability is consistent with the fact that young animals have a generalized inability to reabsorb other amino acids whose transport is known to be sodium gradient stimulated. PMID:3463985

  8. Altering the fatty acids in milk fat by including canola seed in dairy cattle diets.

    PubMed

    Chichlowski, M W; Schroeder, J W; Park, C S; Keller, W L; Schimek, D E

    2005-09-01

    The objective was to evaluate the effects of feeding ground canola seed on the fatty acid profile, yield, and composition of milk from dairy cows. Twenty-four multiparous Holstein cows (548.3 +/- 11.9 kg body weight and 28 +/- 9 d in lactation) were randomly assigned to 1 of 2 treatments: Control (CON) or ground canola seed treatment (GCS) with 14% [of diet dry matter (DM)] of the total ration as ground canola seed containing 34% lipid. Diets contained 20% crude protein, but varied in net energy as a result of fat content differences of 2.5% and 6.4% (DM) for CON and GCS, respectively. Diets were composed of corn, corn silage, alfalfa (50:50 ground hay and haylage, DM basis), soybean and blood meal, and vitamins and minerals. Mechanically extruded canola meal was used in the CON diet to adjust for the protein from canola seed in the GCS diet. Cows were housed in tie-stalls and fed and milked twice daily for 10 wk. The inclusion of ground canola seed did not alter DM intake, weight gain, or body condition score of cows. Milk fat from GCS cows had greater proportions of long-chain fatty acids (> or = 18 carbons) and a lower ratio of n-6 to n-3 fatty acids. Feeding GCS reduced the proportion of short- and medium-chain fatty acids. Milk fat from cows fed GCS had a greater proportion of vaccenic acid and tended to have a higher proportion of cis-9,trans-11 conjugated linoleic acid. Actual and 3.5% fat-corrected milk yields were similar between treatments. The milk fat and protein percentages were lower for GCS cows, but total yield of these components was similar between treatments. Milk urea nitrogen was lower and serum urea nitrogen tended to be lower in cows fed canola seed. Serum glucose, insulin, and nonesterified fatty acids were not altered, but serum triglycerides were higher in GCS cows. Ammonia and total volatile fatty acids tended to be lower in ruminal fluid from GCS cows; rumen pH was unchanged. Feeding canola seed to lactating dairy cows resulted in milk

  9. 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…

  10. Antibiosis of some lactic acid bacteria including Lactobacillus acidophilus toward Listeria monocytogenes.

    PubMed

    Raccach, M; McGrath, R; Daftarian, H

    1989-08-01

    Eleven strains of lactic acid bacteria were tested by the 'spot' on the 'lawn' method for their antagonistic activity against four strains of Listeria monocytogenes. Four out of the five strains of lactic acid bacteria most antagonistic toward the pathogen were those cultures known to produce bacteriocins. Four other strains of lactic acid bacteria were not antagonistic against Listeria by this method. Seventeen inhibition zones of the pathogen were obtained at 25 degrees C as compared to 10 at 32 degrees C. Lactobacillus acidophilus strains NU-A and 88, growing in the presence of L. monocytogenes in milk prevented the latter from attaining populations it would have in pure culture (P less than 0.01). 10(1.4)-10(3.5) lower numbers were noted. L. acidophilus in most cases exhibited a bacteriostatic effect toward the pathogen except for strain 88 which appeared to have a bactericidal effect (P less than 0.01) against Listeria strain OH. The lactobacilli reduced the pH of the milk to 4.7 over a 24 h period, showing that acid played a role in the observed antibiosis.

  11. 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

  12. Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus.

    PubMed

    Amin, U S; Lash, T D; Wilkinson, B J

    1995-02-01

    Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. 13C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.

  13. Validation and application of an improved method for the rapid determination of proline in grape berries.

    PubMed

    Rienth, Markus; Romieu, Charles; Gregan, Rebecca; Walsh, Caroline; Torregrosa, Laurent; Kelly, Mary T

    2014-04-16

    A rapid and sensitive method is presented for the determination of proline in grape berries. Following acidification with formic acid, proline is derivatized by heating at 100 °C for 15 min with 3% ninhydrin in dimethyl sulfoxide, and the absorbance, which is stable for at least 60 min, is read at 520 nm. The method was statistically validated in the concentration range from 2.5 to 15 mg/L, giving a repeatability and intermediate precision of generally <3%; linearity was determined using the lack of fit test. Results obtained with this method concurred (r = 0.99) with those obtained for the same samples on an amino acid analyzer. In terms of sample preparation, a simple dilution (5-20-fold) is required, and sugars, primary amino acids, and anthocyanins were demonstrated not to interfere, as the latter are bleached by ninhydrin under the experimental conditions. The method was applied to the study of proline accumulation in the fruits of microvines grown in phytotrons, and it was established that proline accumulation and concentrations closely resemble those of field-grown macrovines. PMID:24617570

  14. 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

  15. 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

  16. Identification of mammalian proline transporter SIT1 (SLC6A20) with characteristics of classical system imino.

    PubMed

    Takanaga, Hitomi; Mackenzie, Bryan; Suzuki, Yoshiro; Hediger, Matthias A

    2005-03-11

    Amino acid homeostasis depends on specific amino acid transport systems, many of which have been characterized at the molecular level. However, the classical System IMINO, defined as the Na+-dependent proline transport activity that escapes inhibition by alanine, had not been identified at the molecular level. We report here the functional characteristics and tissue distribution of Sodium/Imino-acid Transporter 1 (SIT1), which exhibits the properties of classical System IMINO. SIT1, the product of the slc6a20 gene, is a member of the SLC6 Na+- and Cl--dependent neurotransmitter transporter family whose function has remained unknown. When expressed in Xenopus oocytes, rat SIT1 mediated the uptake of imino acids such as proline (K0.5 approximately 0.2 mM) and pipecolate, as well as N-methylated amino acids (e.g. MeAIB, sarcosine). SIT1-mediated proline transport was pH-independent and insensitive to inhibition by alanine or lysine. Proline transport was Na+-dependent, Cl--stimulated, and voltage-dependent. Li+, but not H+, could substitute for Na+. Human SIT1 also functioned as a Na+-dependent proline transporter. Rat SIT1 mRNA was expressed in epithelial cells of duodenum, jejunum, ileum, stomach, cecum, colon, and kidney proximal tubule S 3 segments. SIT1 mRNA was also expressed in the choroid plexus, microglia, and meninges of the brain and in the ovary. Previous reports have documented the marked urinary hyperexcretion of proline in newborn rodents and man. We found that SIT1 was dramatically up-regulated in the kidneys of 3-day-old mice, accounting for the maturation of proline reabsorption in the mouse. The human slc6a20 gene coding SIT1 is an appropriate target for investigation of hereditary forms of iminoaciduria in man.

  17. 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

  18. 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.

  19. Fate of N-nitrosodimethylamine, trihalomethane and haloacetic acid precursors in tertiary treatment including biofiltration.

    PubMed

    Farré, Maria José; Reungoat, Julien; Argaud, Francois Xavier; Rattier, Maxime; Keller, Jürg; Gernjak, Wolfgang

    2011-11-01

    The presence of disinfection by-products (DBPs) such as trihalomethanes (THMs), haloacetic acids (HAAs) and N-nitrosamines in water is of great concern due to their adverse effects on human health. In this work, the removal of N-nitrosodimethylamine (NDMA), total THM and five HAA precursors from secondary effluent by biological activated carbon (BAC) is investigated at full and pilot scale. In the pilot plant two filter media, sand and granular activated carbon, are tested. In addition, we evaluate the influence of ozonation prior to BAC filtration on its performance. Among the bulk of NDMA precursors, the fate of four pharmaceuticals containing a dimethylamino moiety in the chemical structure are individually investigated. Both NDMA formation potential and each of the studied pharmaceuticals are dramatically reduced by the BAC even in the absence of main ozonation prior to the filtration. The low removal of NDMA precursors at the sand filtration in comparison to the removal of NDMA precursors at the BAC suggests that adsorption may play an important role on the removal of NDMA precursors by BAC. Contrary, the precursors for THM and HAA formation are reduced in both sand filtration and BAC indicating that the precursors for the formation of these DBPs are to some extent biodegradable.

  20. Differential regulation of host genes including hepatic fatty acid synthase in HBV-transgenic mice.

    PubMed

    Zhang, Hongmin; Li, Hong; Yang, Yixuan; Li, Sanglin; Ren, Hong; Zhang, Dazhi; Hu, Huaidong

    2013-06-01

    Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans, and it is considered to be a major global health problem. To gain a better understanding of HBV pathogenesis, and identify novel putative targets for anti-HBV therapy, this study was designed to elucidate the differential expression of host proteins in liver tissue from HBV-transgenic mice. Liver samples from two groups, (1) HBV-transgenic (Tg) mice, (2) corresponding background normal mice, wild-type (WT) mice, were collected and subjected to iTRAQ and mass spectrometry analysis. In total, 1950 unique proteins were identified, and 68 proteins were found to be differentially expressed in HBV-Tg mice as compared with that in WT mice. Several differentially expressed proteins were further validated by real-time quantitative RT-PCR, Western blot and immunohistochemical analysis. Furthermore, the association of HBV replication with fatty acid synthase (FASN), one of the highly expressed proteins in HBV-Tg mice, was verified. Silencing of FASN expression in HepG2.2.15 cells suppressed viral replication through the IFN signaling pathway, and some downstream antiviral effectors. The implicated role of FASN in HBV replication provides an opportunity to test existing compounds against FASN for adjuvant therapy and/or treatment of HBV replication. PMID:23675653

  1. 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

  2. L-Proline nutrition and catabolism in Staphylococcus saprophyticus.

    PubMed

    Deutch, Charles E

    2011-05-01

    Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require L-proline but not L-arginine for growth in a defined culture medium. All three strains could utilize L-ornithine as a proline source and contained L-ornithine aminotransferase and Δ(1)-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use L-arginine as a proline source and had L-arginase activity. The proline requirement also could be met by L-prolinamide, L-proline methyl ester, and the dipeptides L-alanyl-L-proline and L-leucyl-L-proline. The bacteria exhibited L-proline degradative activity as measured by the formation of Δ(1)-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of L-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller-Hinton broth. A membrane fraction from this strain had L-proline dehydrogenase activity as detected both by reaction of Δ(1)-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min(-1) mg(-1)) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min(-1) mg(-1)). A soluble fraction from this strain had Δ(1)-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min(-1) mg(-1)) as determined by the NAD(+)-dependent oxidation of DL-Δ(1)-pyrroline-5-carboxylate. Addition of L-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with L: -ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-DL-proline, DL-thiazolidine-2-carboxylate, and L-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.

  3. 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

  4. 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.

  5. 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

  6. Proline-Directed Androgen Receptor Phosphorylation

    PubMed Central

    Gao, Yanfei; Chen, Shaoyong

    2015-01-01

    The androgen receptor (AR) has been identified for decades and mediates essential steroid functions. Like most of biological molecules, AR functional activities are modulated by post-translational modifications. This review is focused on the reported activities and significance of AR phosphorylation, with particular emphasis on proline-directed serine/threonine phosphorylation that occurs predominantly on the receptor. The marked enrichment of AR phosphorylation in the most diverse N-terminal domain suggests that targeting AR phosphorylation can be synergistic to antagonizing the C-terminal domain by clinical antiandrogens. PMID:25866551

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. Proline as chiral auxiliary for the economical asymmetric synthesis of ruthenium(II) polypyridyl complexes.

    PubMed

    Fu, Chen; Wenzel, Marianne; Treutlein, Elisabeth; Harms, Klaus; Meggers, Eric

    2012-09-17

    A straightforward method for the synthesis of virtually enantiomerically pure ruthenium(II) polypyridyl complexes [Ru(pp)(pp')(pp")](PF(6))(2), pp = bidentate polypyridyl has been developed. The synthesis draws from the readily available racemic starting material cis-[Ru(pp)(pp')Cl(2)] and the natural amino acids l- or d-proline and relies on a dynamic asymmetric transformation under thermodynamic control.

  14. Selected nutrient contents, fatty acid composition, including conjugated linoleic acid, and retention values in separable lean from lamb rib loins as affected by external fat and cooking method.

    PubMed

    Badiani, Anna; Montellato, Lara; Bochicchio, Davide; Anfossi, Paola; Zanardi, Emanuela; Maranesi, Magda

    2004-08-11

    Proximate composition and fatty acid profile, conjugated linoleic acid (CLA) isomers included, were determined in separable lean of raw and cooked lamb rib loins. The cooking methods compared, which were also investigated for cooking yields and true nutrient retention values, were dry heating of fat-on cuts and moist heating of fat-off cuts; the latter method was tested as a sort of dietetic approach against the more traditional former type. With significantly (P < 0.05) lower cooking losses, dry heating of fat-on rib-loins produced slightly (although only rarely significantly) higher retention values for all of the nutrients considered, including CLA isomers. On the basis of the retention values obtained, both techniques led to a minimum migration of lipids into the separable lean, which was higher (P < 0.05) in dry heating than in moist heating, and was characterized by the prevalence of saturated and monounsaturated fatty acids. On the whole, the response to cooking of the class of CLA isomers (including that of the nutritionally most important isomer cis-9,trans-11) was more similar to that of the monounsaturated than the polyunsaturated fatty acids.

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

    PubMed Central

    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

  16. Metachromatic staining patterns of basic proline-rich proteins from rat and human saliva in sodium dodecyl sulfate-polyacrylamide gels

    SciTech Connect

    Humphreys-Beher, M.G.; Wells, D.J.

    1984-10-01

    A series of basic proteins, rich in proline, were isolated from the salivary secretions of humans and rats. These proteins underwent metachromasia after staining with Coomassie brilliant blue R-250 in sodium dodecyl sulfate-polyacrylamide gels. The technique of destaining gels in several changes of 10% acetic acid after a 30-min staining period is a rapid method of general utility for the identification of proline-rich proteins from total cell lysates from other sources besides saliva.

  17. 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

  18. Simultaneous determination of tetracaine, proline, and enoxacin in human urine by CE with ECL detection.

    PubMed

    Sun, Hanwen; Su, Ming; Li, Liqing

    2010-01-01

    A simple, fast, and sensitive capillary electrophoresis method was developed for the simultaneous determination of tetracaine, proline, and enoxacin in human urine with electrochemiluminescence (ECL) detection. The effects of experimental conditions including the mode of applied voltage signal, the potential of working electrode, pH value, the flow rate of carrier of solution, the concentration of Ru(bpy)(3)(2+), and the ECL intensity of the drugs were investigated in detail. Parameters related to the separation and detection were investigated and optimized. Under optimized conditions, the proposed method displayed a linear range from 0.4 to 100 microg/mL for tetracaine, 0.2 to 80 microg/mL for proline, and 0.1 to 100 microg/mL for enoxacin. Their limits of detection were 0.08, 0.06, and 0.02 microg/mL, respectively. A baseline separation for tetracaine, proline, and enoxacin was achieved within 10 min. Developed method was successfully applied to determine tetracaine, proline, and enoxacin in human urine.

  19. Temporal changes (1997-2012) of perfluoroalkyl acids and selected precursors (including isomers) in Swedish human serum.

    PubMed

    Gebbink, Wouter A; Glynn, Anders; Berger, Urs

    2015-04-01

    Concentrations (including isomer patterns) and temporal changes (1997-2012) of perfluoroalkyl acids (PFAAs) and selected perfluorooctane sulfonate (PFOS) and perfluoroalkyl carboxylic acid (PFCA) precursors were determined in serum samples from Swedish women. Perfluorooctane sulfonamide (FOSA) and perfluorooctane sulfonamidoacetic acid (FOSAA), as well as its N-methyl and N-ethyl derivatives (MeFOSAA and EtFOSAA) were consistently detected. Highest PFOS precursor concentrations were found for EtFOSAA (before year 2000) or MeFOSAA and FOSAA (after 2000). Disappearance half-lives for all PFOS precursors were shorter compared to PFOS. 4:2/6:2 and 6:2/6:2 polyfluoroalkyl phosphate diesters (diPAPs) were detected in <60% of the samples, whereas 6:2/8:2 and 8:2/8:2 diPAPs were detected in >60% of the samples, but showed no significant change in concentrations over time. Linear and sum-branched isomers were quantified separately for three PFAAs and three precursors. Significant changes between 1997 and 2012 in the % linear isomer were observed for PFOA and FOSA (increase) and PFOS (decrease).

  20. SKN-1 and Nrf2 couples proline catabolism with lipid metabolism during nutrient deprivation.

    PubMed

    Pang, Shanshan; Lynn, Dana A; Lo, Jacqueline Y; Paek, Jennifer; Curran, Sean P

    2014-10-06

    Mechanisms that coordinate different metabolic pathways, such as glucose and lipid, have been recognized. However, a potential interaction between amino acid and lipid metabolism remains largely elusive. Here we show that during starvation of Caenorhabditis elegans, proline catabolism is coupled with lipid metabolism by SKN-1. Mutation of alh-6, a conserved proline catabolic enzyme, accelerates fat mobilization, enhances the expression of genes involved in fatty acid oxidation and reduces survival in response to fasting. This metabolic coordination is mediated by the activation of the transcription factor SKN-1/Nrf2, possibly due to the accumulation of the alh-6 substrate P5C, and also requires the transcriptional co-regulator MDT-15. Constitutive activation of SKN-1 induces a similar transcriptional response, which protects animals from fat accumulation when fed a high carbohydrate diet. In human cells, an orthologous alh-6 enzyme, ALDH4A1, is also linked to the activity of Nrf2, the human orthologue of SKN-1, and regulates the expression of lipid metabolic genes. Our findings identify a link between proline catabolism and lipid metabolism, and uncover a physiological role for SKN-1 in metabolism.

  1. 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

  2. New Insight on the Synthesis of Neurotoxins Domoic Acid and Kainic Acid.

    PubMed

    Mollica, Adriano; Costante, Roberto; Stefanucci, Azzurra; Novellino, Ettore

    2015-01-01

    Mono or di-substituted prolines, namely proline chimeras of natural or synthetic origin, carry the side chain of other specific amino acids on the pyrrolidine ring. Thus, proline chimeras are useful tools for a wide range of chemical and biological applications as chiral synthons or building blocks for peptidomimetic design. We focused our attention on domoic acid and kainic acid and we report here a concise and up to date review on their stereoselective and asymmetric syntheses.

  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.

  4. 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

  5. Proline improves copper tolerance in chickpea (Cicer arietinum).

    PubMed

    Singh, Vijeta; Bhatt, Indu; Aggarwal, Anjali; Tripathi, Bhumi Nath; Munjal, Ashok Kumar; Sharma, Vinay

    2010-09-01

    The present study suggests the involvement of proline in copper tolerance of four genotypes of Cicer arietinum (chickpea). Based on the data of tolerance index and lipid peroxidation, the order for copper tolerance was as follows: RSG 888 > CSG 144 > CSG 104 > RSG 44 in the selected genotypes. The basis of differential copper tolerance in chickpea genotypes was characterized by analyzing, antioxidant enzymes (superoxide dismutase, ascorbated peroxidase and catalase), phytochelatins, copper uptake, and proline accumulation. Chickpea genotypes showed stimulated superoxide dismutase activity at all tested concentrations of copper, but H(2)O(2) decomposing enzymes especially; ascorbate peroxidase did not increase with 25 and 50 microM copper treatments. Catalase activity, however, increased at lower copper concentrations but failed to stimulate at 50 microM copper. Such divergence in responses of these enzymes minimizes their importance in protecting chickpea against copper stress. The sensitive genotypes showed greater enhancement of phytochelatins than that of tolerant genotypes. Hence, the possibility of phytochelatins in improving copper tolerance in the test plant is also excluded. Interestingly, the order of proline accumulation in the chickpea genotypes (RSG 888 > CSG 144 > CSG 104 > RSG 44) was exactly similar to the order of copper tolerance. Based on hyperaccumulation of proline in tolerant genotype (RSG 44) and the reduction and improvement of lipid peroxidation and tolerance index, respectively, by proline pretreatment, we conclude that hyperaccumulation of proline improves the copper tolerance in chickpea.

  6. 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.

  7. Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannan.

    PubMed

    Berg, Stefan; Starbuck, James; Torrelles, Jordi B; Vissa, Varalakshmi D; Crick, Dean C; Chatterjee, Delphi; Brennan, Patrick J

    2005-02-18

    D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan, each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. beta-D-Arabinofuranosyl-1-monophosphoryldecaprenol is the only known sugar donor. EmbA, EmbB, and EmbC, products of the paralogous genes embA, embB, and embC, the sites of resistance to the anti-tuberculosis drug ethambutol (EMB), are the only known implicated enzymes. EmbA and -B apparently contribute to the synthesis of arabinogalactan, whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no overall similarity to any known proteins beyond Mycobacterium and related genera. However, functional motifs, equivalent to a proline-rich motif of several bacterial polysaccharide co-polymerases and a superfamily of glycosyltransferases, were found. Site-directed mutagenesis in glycosyltransferase superfamily C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis and accumulation of an unknown intermediate and of the known precursor lipomannan. Yet the pattern of the differently linked d-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis. PMID:15546869

  8. 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

  9. Biosynthesis of prodigiosin by non-proliferating wild-type Serratia marcescens and mutants deficient in catabolism of alanine, histidine, and proline.

    PubMed

    Lim, D V; Qadri, S M; Nichols, C; Williams, R P

    1977-01-01

    Mutants of Serratia marcescens Nima, designated as Aut, Hut, or Put, did not utilize L-alanine, L-histidine, or L-proline, respectively, as a sole carbon source but did utilize other amino acids or glycerol as carbon sources. The bacteria were permeable to alanine, histidine, and proline but lacked the enzymes responsible for degradation of these amino acids. The Aut mutant contained no L-alanine dehydrogenase activity, whereas the Hut and Put mutants contained only 7 and 4% of the histidase and proline oxidase activities, respectively, found in the wild-type strain. Rates of oxygen uptake and protein synthesis were significantly lower when the mutants were incubated in the presence of amino acids they could not degrade. Studies of L-[14C]alanine, L-[14C]histidine, and L-[14C]proline incorporation into prodigiosin synthesized by these mutants and the wild-type strain revealed that proline was incorporated intact, whereas all of alanine except the carboxyl group was incorporated into the pigment molecule. Histidine did not enter prodigiosin directly. These data suggested that the presence of unique biosynthetic pathways, independent of primary metabolism, leads to formation of prodigiosin from specific amino acids.

  10. 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

  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. Stabilization of an α/β-hydrolase by introducing proline residues: salicylic binding protein 2 from tobacco

    PubMed Central

    Huang, Jun; Jones, Bryan J.; Kazlauskas, Romas J.

    2015-01-01

    α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. As a model α/β-hydrolase, we investigated a plant esterase, salicylic acid binding protein 2 (SABP2). SABP2 shows typical stability to urea (unfolding free energy 6.9±1.5 kcal/mol) and to heat inactivation (T1/215 min 49.2±0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homolog or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1) and E215P (+0.9). Introducing proline in the cap domain did not (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/215 min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P-S70P ΔT1/215 min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases. PMID:26110207

  13. Structural characterization of the HIV-1 Vpr N terminus: evidence of cis/trans-proline isomerism.

    PubMed

    Bruns, Karsten; Fossen, Torgils; Wray, Victor; Henklein, Peter; Tessmer, Uwe; Schubert, Ulrich

    2003-10-31

    The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major

  14. The effects of interactions between proline and carbon nanostructures on organocatalysis in the Hajos-Parrish-Eder-Sauer-Wiechert reaction

    NASA Astrophysics Data System (ADS)

    Rance, G. A.; Khlobystov, A. N.

    2014-09-01

    The non-covalent interactions of S-(-)-proline with the surfaces of carbon nanostructures (fullerene, nanotubes and graphite) change the nucleophilic-electrophilic and acid-base properties of the amino acid, thus tuning its activity and selectivity in the organocatalytic Hajos-Parrish-Eder-Sauer-Wiechert (HPESW) reaction. Whilst our spectroscopy and microscopy measurements show no permanent covalent bonding between S-(-)-proline and carbon nanostructures, a systematic investigation of the catalytic activity and selectivity of the organocatalyst in the HPESW reaction demonstrates a clear correlation between the pyramidalisation angle of carbon nanostructures and the catalytic properties of S-(-)-proline. Carbon nanostructures with larger pyramidalisation angles have a stronger interaction with the nitrogen atom lone pair of electrons of the organocatalyst, thereby simultaneously decreasing the nucleophilicity and increasing the acidity of the organocatalyst. These translate into lower conversion rates but higher selectivities towards the dehydrated product of Aldol addition.The non-covalent interactions of S-(-)-proline with the surfaces of carbon nanostructures (fullerene, nanotubes and graphite) change the nucleophilic-electrophilic and acid-base properties of the amino acid, thus tuning its activity and selectivity in the organocatalytic Hajos-Parrish-Eder-Sauer-Wiechert (HPESW) reaction. Whilst our spectroscopy and microscopy measurements show no permanent covalent bonding between S-(-)-proline and carbon nanostructures, a systematic investigation of the catalytic activity and selectivity of the organocatalyst in the HPESW reaction demonstrates a clear correlation between the pyramidalisation angle of carbon nanostructures and the catalytic properties of S-(-)-proline. Carbon nanostructures with larger pyramidalisation angles have a stronger interaction with the nitrogen atom lone pair of electrons of the organocatalyst, thereby simultaneously decreasing the

  15. 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.

  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. 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

  18. 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

  19. Growth inhibition by exogenous proline and its metabolism in saltgrass (Distichlis spicata) suspension cultures.

    PubMed

    Rodriguez, M M; Heyser, J W

    1988-08-01

    The growth of Distichlis spicata suspension cultures in LS medium without NaCl was inhibited 54% by 2 mM proline. In medium containing 260 mM NaCl, 10 mM proline inhibited growth by only 22%. The uptake and metabolism of 10 mM L-[1-(13)C] proline was followed by (13)C NMR and ninhydrin analyses of suspensions cultured in the presence of 0 or 260 mM NaCl. Uptake of 85 to 92% of the exogenous proline occurred within 72 h in all media. In 10 mM proline and no NaCl, cellular proline reached a maximm of 51.5 μmoles/g FW compared to 1.9 μmoles/g FW in suspensions not grown on proline. In medium containing 260 mM NaCl and proline, cellular proline reached 59-65 μmoles/g FW compared to 30-40 μmoles/g FW in controls grown without proline. The (13)C-label in the proline-C1 was either retained in proline or disappeared, presumably released as carbon dioxide, by catabolism through the TCA cycle. Since no metabolite of (13)C-proline was detected by NMR, proline was considered to be the molecule which inhibited the suspension culture growth.

  20. Isolation and identification of microorganisms including lactic acid bacteria and their use in microbial deacidification of wines from domestic vineyards.

    PubMed

    Drozdz, Iwona; Makarewicz, Malgorzata; Tuszyński, Tadeusz

    2013-01-01

    The aim of this study was to identify various bacteria isolated from grapes and their wines. Additionally we investigated the capacity of lactic acid bacteria for microbiological deacidification of wines produced in Poland. We have identified Oenococcus oeni, Lactobacillus acidophilus and Lactobacillus delbrueckii. During the microbial deacidification process, we observed decreases of total acidity and increases of volatile acidity, with statistically significant changes noted for O. oeni in Marechal Foch and Seyval Blanc, and for Lb. acidophilus in Frontenac. On the other hand, a statistically significant increase in pH was observed in Marechal Foch and Seyval Blanc following deacidification by O. oeni.

  1. Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

    PubMed Central

    Medrano, F J; Alonso, J; García, J L; Romero, A; Bode, W; Gomis-Rüth, F X

    1998-01-01

    The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family. PMID:9427736

  2. δ(15) N from soil to wine in bulk samples and proline.

    PubMed

    Paolini, Mauro; Ziller, Luca; Bertoldi, Daniela; Bontempo, Luana; Larcher, Roberto; Nicolini, Giorgio; Camin, Federica

    2016-09-01

    The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright © 2016 John Wiley & Sons, Ltd.

  3. δ(15) N from soil to wine in bulk samples and proline.

    PubMed

    Paolini, Mauro; Ziller, Luca; Bertoldi, Daniela; Bontempo, Luana; Larcher, Roberto; Nicolini, Giorgio; Camin, Federica

    2016-09-01

    The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27479606

  4. Separation of fatty acids or methyl esters including positional and geometric isomers by alumina argentation thin-layer chromatography.

    PubMed

    Breuer, B; Stuhlfauth, T; Fock, H P

    1987-07-01

    This paper describes novel and rapid thin-layer chromatography procedures for the analysis of fatty acids and methyl esters using silver-impregnated alumina sheets. These techniques are known in most laboratories, and the equipment is readily available. The fatty acid method allows a separation of petroselinic (C18:1 delta 6c), oleic (C18:1 delta 9c), elaidic (C18:1 delta 9t), erucic (C22:1 delta 13c), and brassidic acids (C22:1 delta 13t), and the methyl ester method gives an excellent resolution with respect to the number, configuration, and position of the unsaturated centers. Sufficient separation for the subsequent ozonolysis and chromatographic quantification of isomeric C18 and C22 fatty acid methyl esters is obtained with both methods.

  5. 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)...

  6. 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

  7. 21 CFR 172.320 - Amino acids.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...-Leucine L-Lysine DL-Methionine (not for infant foods) L-Methionine L-Phenylalanine L-Proline L-Serine L... Aminoacetic acid (glycine) L-Leucine DL-Methionine L-Methionine L-Tryptophan L-Phenylalanine L-Proline L... DL-Methionine 3.1 L-Phenylalanine 5.8 L-Proline 4.2 L-Serine 8.4 L-Threonine 5.0 L-Tryptophan 1.6...

  8. 21 CFR 172.320 - Amino acids.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...-Leucine L-Lysine DL-Methionine (not for infant foods) L-Methionine L-Phenylalanine L-Proline L-Serine L... Aminoacetic acid (glycine) L-Leucine DL-Methionine L-Methionine L-Tryptophan L-Phenylalanine L-Proline L... DL-Methionine 3.1 L-Phenylalanine 5.8 L-Proline 4.2 L-Serine 8.4 L-Threonine 5.0 L-Tryptophan 1.6...

  9. 21 CFR 172.320 - Amino acids.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...-Leucine L-Lysine DL-Methionine (not for infant foods) L-Methionine L-Phenylalanine L-Proline L-Serine L... Aminoacetic acid (glycine) L-Leucine DL-Methionine L-Methionine L-Tryptophan L-Phenylalanine L-Proline L... DL-Methionine 3.1 L-Phenylalanine 5.8 L-Proline 4.2 L-Serine 8.4 L-Threonine 5.0 L-Tryptophan 1.6...

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut.

    PubMed

    Goptar, Irina A; Filippova, Irina Yu; Lysogorskaya, Elena N; Oksenoit, Elena S; Vinokurov, Konstantin S; Zhuzhikov, Dmitry P; Bulushova, Natalja V; Zalunin, Igor A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N

    2008-03-01

    Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.

  15. Combined supplementation of carbohydrate, alanine, and proline is effective in maintaining blood glucose and increasing endurance performance during long-term exercise in mice.

    PubMed

    Nogusa, Yoshihito; Mizugaki, Ami; Hirabayashi-Osada, Yuri; Furuta, Chie; Ohyama, Kana; Suzuki, Katsuya; Kobayashi, Hisamine

    2014-01-01

    Carbohydrate supplementation is extremely important during prolonged exercise because it maintains blood glucose levels during later stages of exercise. In this study, we examined whether maintaining blood glucose levels by carbohydrate supplementation could be enhanced during long-term exercise by combining this supplementation with alanine and proline, which are gluconeogenic amino acids, and whether such a combination would affect exercise endurance performance. Male C57BL/6J mice were orally administered either maltodextrin (1.25 g/kg) or maltodextrin (1.0 g/kg) with alanine (0.225 g/kg) and proline (0.025 g/kg) 15 min before running for 170 min. Combined supplementation of maltodextrin, alanine, and proline induced higher blood glucose levels than isocaloric maltodextrin alone during the late exercise phase (100-170 min). The hepatic glycogen content of mice administered maltodextrin, alanine, and proline was higher than that of mice ingesting maltodextrin alone 60 min after beginning exercise, but the glycogen content of the gastrocnemius muscle showed no difference. We conducted a treadmill running test to determine the effect of alanine and proline on endurance performance. The test showed that running time to exhaustion of mice that were supplemented with maltodextrin (2.0 g/kg) was longer than that of mice that were supplemented with water alone. Maltodextrin supplementation (1.0 g/kg) with alanine (0.9 g/kg) and proline (0.1 g/kg) further increased running time to exhaustion compared to maltodextrin alone (2.0 g/kg). These results indicate that combined supplementation of carbohydrate, alanine, and proline is effective for maintaining blood glucose and hepatic glycogen levels and increasing endurance performance during long-term exercise in mice.

  16. 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

  17. Proline-induced inhibition of glutamate release in hippocampal area CA1.

    PubMed

    Cohen, S M; Nadler, J V

    1997-09-26

    Concentrations of proline typical of human CSF have been shown to potentiate transmission at Schaffer collateral-commissural synapses on CA1 pyramidal cells of the rat hippocampus. This study tested the hypothesis that proline enhances excitatory synaptic transmission by increasing glutamate release. Two concentrations of proline were used: a concentration typical of normal human CSF (3 microM) and a concentration typical of CSF in persons with the genetic disorder hyperprolinemia type II (30 microM). Continuous exposure of hippocampal slices to either concentration of proline potentiated Schaffer collateral-commissural synaptic transmission. Proline shifted the plot of field EPSP slope against fiber volley amplitude upward. Contrary to the original hypothesis, neither concentration of proline reduced paired-pulse facilitation; 30 microM proline enhanced paired-pulse facilitation, whereas 3 microM proline had no effect. In line with its enhancement of paired-pulse facilitation, 30 microM proline reduced both the K+-evoked release of glutamate and aspartate from CA1 slices and the release of glutamate and aspartate from CA1 synaptosomes evoked by 4-aminopyridine. These results suggest that the proline-induced potentiation of Schaffer collateral-commissural synaptic transmission probably involves a postsynaptic, rather than a presynaptic, mechanism. Concentrations of proline normally found in human CSF little affect glutamate release. However, proline-induced inhibition of glutamate release may contribute to the neuropsychiatric disorders associated with hyperprolinemia type II.

  18. 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

  19. 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

  20. Evaluation of a novel food composition database that includes glutamine and other amino acids derived from gene sequencing data

    PubMed Central

    Lenders, CM; Liu, S; Wilmore, DW; Sampson, L; Dougherty, LW; Spiegelman, D; Willett, WC

    2011-01-01

    Objectives To determine the content of glutamine in major food proteins. Subjects/Methods We used a validated 131-food item food frequency questionnaire (FFQ) to identify the foods that contributed the most to protein intake among 70 356 women in the Nurses’ Health Study (NHS, 1984). The content of glutamine and other amino acids in foods was calculated based on protein fractions generated from gene sequencing methods (Swiss Institute of Bioinformatics) and compared with data from conventional (USDA) and modified biochemical (Khun) methods. Pearson correlation coefficients were used to compare the participants’ dietary intakes of amino acids by sequencing and USDA methods. Results The glutamine content varied from 0.01 to to 9.49 g/100 g of food and contributed from 1 to to 33% of total protein for all FFQ foods with protein. When comparing the sequencing and Kuhn’s methods, the proportion of glutamine in meat was 4.8 vs 4.4%. Among NHS participants, mean glutamine intake was 6.84 (s.d.=2.19) g/day and correlation coefficients for amino acid between intakes assessed by sequencing and USDA methods ranged from 0.94 to 0.99 for absolute intake, −0.08 to 0.90 after adjusting for 100 g of protein, and 0.88 to 0.99 after adjusting for 1000 kcal. The between-person coefficient of variation of energy-adjusted intake of glutamine was 16%. Conclusions These data suggest that (1) glutamine content can be estimated from gene sequencing methods and (2) there is a reasonably wide variation in energy-adjusted glutamine intake, allowing for exploration of glutamine consumption and disease. PMID:19756030

  1. 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

  2. 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.

  3. DMPS-arsenic challenge test. II. Modulation of arsenic species, including monomethylarsonous acid (MMA(III)), excreted in human urine.

    PubMed

    Aposhian, H V; Zheng, B; Aposhian, M M; Le, X C; Cebrian, M E; Cullen, W; Zakharyan, R A; Ma, M; Dart, R C; Cheng, Z; Andrewes, P; Yip, L; O'Malley, G F; Maiorino, R M; Van Voorhies, W; Healy, S M; Titcomb, A

    2000-05-15

    The administration of sodium 2,3-dimercapto-1-propane sulfonate (DMPS) to humans chronically exposed to inorganic arsenic in their drinking water resulted in the increased urinary excretion of arsenic, the appearance and identification of monomethylarsonous acid (MMA(III)) in their urine, and a large decrease in the concentration and percentage of urinary dimethylarsinic acid (DMA). This is the first time that MMA(III) has been detected in the urine. In vitro biochemical experiments were then designed and performed to understand the urinary appearance of MMA(III) and decrease of DMA. The DMPS-MMA(III) complex was not active as a substrate for the MMA(III) methyltransferase. The experimental results support the hypothesis that DMPS competes with endogenous ligands for MMA(III), forming a DMPS-MMA complex that is readily excreted in the urine and points out the need for studying the biochemical toxicology of MMA(III). It should be emphasized that MMA(III) was excreted in the urine only after DMPS administration. The results of these studies raise many questions about the potential central role of MMA(III) in the toxicity of inorganic arsenic and to the potential involvement of MMA(III) in the little-understood etiology of hyperkeratosis, hyperpigmentation, and cancer that can result from chronic inorganic arsenic exposure.

  4. 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

  5. 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

  6. 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.

  7. Developmental and Hormonal Regulation of Genes Coding for Proline-Rich Proteins in Female Inflorescences and Kernels of Maize1

    PubMed Central

    Josè-Estanyol, Matilde; Puigdomènech, Pere

    1998-01-01

    The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments. PMID:9490753

  8. Growth and development of the arborescent cactus Stenocereus queretaroensis in a subtropical semiarid environment, including effects of gibberellic acid.

    PubMed

    Pimienta, Eulogio; Hernandez, Gerardo; Domingues, Alejandro; Nobel, Park S.

    1998-01-01

    In Stenocereus queretaroensis (Weber) Buxbaum, an arborescent cactus cultivated in Jalisco, Mexico, for its fruits but studied here in wild populations, stem extension occurred in the autumn at the beginning of the dry season, flowering and fruiting occurred in the spring at the end of the dry season, and new roots grew in the summer during the wet season. The asynchrony of vegetative and reproductive growth reduces competitive sink effects, which may be advantageous for wild populations growing in infertile rocky soils. Seasonal patterns of sugars in the roots and especially the stems of S. queretaroensis were closely related to the main phenological stages, becoming lower in concentration during periods of major stem extension. Cessation of stem extension occurred in 100-year-old plants for which injection of GA(3) reinitiated such growth. Isolated chlorenchyma cylinders had maximum extension in a bathing solution containing 0.1 &mgr;M gibberellic acid.

  9. 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.

  10. 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

  11. Proline iminopeptidase from the outer cell envelope of the human oral spirochete Treponema denticola ATCC 35405.

    PubMed Central

    Mäkinen, K K; Chen, C Y; Mäkinen, P L

    1996-01-01

    Certain periodontopathic organisms have been shown to exhibit high activity of proline iminopeptidase (PIPase). The human oral spirochete Treponema denticola ATCC 35405 was found to contain an easily extractable, novel PIPase (EC 3.4.11.5), which was purified to a sodium dodecyl sulfate- polyacrylamide gel electrophoresis-pure form by means of fast protein liquid chromatographic procedures. The range of the minimum monomeric molecular mass (280 amino acid residues) of the PIPase, based on amino acid analysis, was 30.35 to 30.39 kDa, but the likely in vivo form of the enzyme is a tetramer (minimum mass, 120.2 to 120.4 kDa). The molecular masses based on laser desorption mass spectrometry were 36.058 kDa for the monomer and 72.596 kDa for a dimer. The PIPase cleaves specifically the Pro-Y bond in dipeptides where Y is preferably Arg or Lys. Pro-Gln, Pro-Asn, and Pro-Ala were also good substrates, while Pro-Glu was hydrolyzed slowly and Pro-Asp was not hydrolyzed at all. Tripeptides were poor substrates or were not hydrolyzed (an exception was Pro-Gly-Gly, which cleaved at a moderate rate). Larger molecules, such as poly-L-Pro, were not hydrolyzed. The T. denticola enzyme can be regarded as a true PIPase, since replacing Pro in Pro-Y with other amino acid residues resulted in no hydrolysis. The activity of the PIPase may depend on an active carboxyl group and on an active seryl residue but not on metal cations. Diethylpyrocarbonate inactivated the enzyme in a reaction that was not reversible upon addition of NH2OH. The enzyme contains a relatively large percentage (ca. 15%) of proline residues. The dominance of the PIPase activity among aminopeptidase activities present in T. denticola and the proposed location of the enzyme in the outer cell envelope suggest that it has a vital function in the propagation of the cells within their biological niche (inflamed human periodontal tissues). The biologic role of the PIPase may be envisaged as in the termination of the

  12. 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

  13. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    PubMed

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  14. 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.

  15. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25084341

  17. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE1[OPEN

    PubMed Central

    Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long

    2016-01-01

    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

  18. Proline metabolism in the conceptus: implications for fetal growth and development.

    PubMed

    Wu, G; Bazer, F W; Datta, S; Johnson, G A; Li, P; Satterfield, M C; Spencer, T E

    2008-11-01

    Although there are published studies of proline biochemistry and nutrition in cultured cells and postnatal animals, little is known about proline metabolism and function in the conceptus (embryo/fetus, associated placental membranes, and fetal fluids). Because of the invasive nature of biochemical research on placental and fetal growth, animal models are often used to test hypotheses of biological importance. Recent evidence from studies with pigs and sheep shows that proline is a major substrate for polyamine synthesis via proline oxidase, ornithine aminotransferase, and ornithine decarboxylase in placentae. Both porcine and ovine placentae have a high capacity for proline catabolism and polyamine production. In addition, allantoic and amniotic fluids contain enzymes to convert proline into ornithine, which is delivered through the circulation to placental tissues. There is exquisite metabolic coordination among integrated pathways that support highest rates of polyamine synthesis and concentrations in placentae during early gestation when placental growth is most rapid. Interestingly, reduced placental and fetal growth are associated with reductions in placental proline transport, proline oxidase activity, and concentrations of polyamines in gestating dams with either naturally occurring or malnutrition-induced growth retardation. Conversely, increasing proline availability in maternal plasma through nutritional or pharmacological modulation in pigs and sheep enhances concentrations of proline and polyamines in placentae and fetal fluids, as well as fetal growth. These novel findings suggest an important role for proline in conceptus metabolism, growth and development, as well as a potential treatment for intrauterine growth restriction, which is a significant problem in both human medicine and animal agriculture.

  19. 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

  20. 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

  1. 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

  2. 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

  3. Identification and Disruption of the proBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

    PubMed Central

    Sleator, Roy D.; Gahan, Cormac G. M.; Hill, Colin

    2001-01-01

    Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity. PMID:11375165

  4. Optimal hematologic variables for oxygen transport, including P50, hemoglobin cooperativity, hematocrit, acid-base status, and cardiac function.

    PubMed

    Winslow, R M

    1988-01-01

    The two important blood properties that affect O2 delivery are the O2 equilibrium curve (OEC) and blood viscosity with its subsequent effect on flow (cardiac output). To quantitate these properties blood OEC's were analyzed in terms of the Adair 4-step oxygenation model and the resulting parameters were used to construct a computer nomogram to reproduce the OEC at any combination of effectors that regulate P50 (pH, PCO2, and 2,3-DPG). In this way, the P50 could be changed systematically and the effects on overall O2 transport could be studied. Hematocrit-viscosity-cardiac output relationships were taken from the literature and validated using data from human subjects with various pathological states and high-altitude natives. A model was then developed, using the Bohr integration, to predict the O2 transport function of blood under a variety of conditions including exercise and hypoxia. The results indicate that the optimal hematocrit is about 43-45%, even in hypoxia. The optimal P50, however, depends on the availability of O2: a high P50 is not necessarily beneficial in hypoxia and high cardiac output states. This model and general approach should prove useful in the design of blood substitutes.

  5. 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

  6. Structural and Catalytic Effects of Proline Substitution and Surface Loop Deletion in the Extended Active Site of Human Carbonic Anhydrase II

    PubMed Central

    Boone, Christopher D.; Rasi, Valerio; Tu, Chingkuang; McKenna, Robert

    2015-01-01

    The bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge as several stabilizing elements have been proposed to be the foundation of thermal stability including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions, and aromatic clusters. This study emphasizes the impact of increasing the rigidity of human carbonic anhydrase II (HCA II) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of compactness of HCA II was examined by way of deletion of a surface loop (residues 230 through 240), which had been previously identified as a possible source of thermal stability for the hyperthermophilic CA isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed the electrostatic potential and configuration of the highly acidic loop (residues 230 and 240) plays an important role in its high catalytic activity. Based on these observations and the literature, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements could provide the blueprints for the rational thermal stability engineering of other enzymes. PMID:25683338

  7. Distribution and enantiomeric composition of amino acids in the Murchison meteorite

    NASA Technical Reports Server (NTRS)

    Engel, M. H.; Nagy, B.

    1982-01-01

    Studies of the amino acid contents and enantiomeric compositions of a single stone from the Murchison meteorite are reported. Water-extracted and 6M HCl-extracted samples from the meteorite interior of meteorite fragments were analyzed by gas chromatography and combined gas chromatography-chemical ionization mass spectrometry. Examination of the D/L ratios of glutamic acid, aspartic acid, proline, leucine and alanine reveals those amino acids extractable by water to be partially racemized, whereas the acid-extracted amino acids were less racemized. The amino acid composition of the stone is similar to those previously reported, including the absence of serine, threonine, tyrosine phenylalanine and methionine and the presence of unusual amino acids including such as isovaline, alpha-aminoisobutyric acid and pseudoleucine. It is concluded that the most likely mechanism accounting for the occurrence of nonracemic amino acid mixtures in the Murchison meteorite is by extraterrestrial stereoselective synthesis or decomposition reactions.

  8. 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.

  9. 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.

  10. New force field parameters for metalloproteins I: Divalent copper ion centers including three histidine residues and an oxygen-ligated amino acid residue.

    PubMed

    Wise, Olivia; Coskuner, Orkid

    2014-06-30

    Transition metal ion complexation with proteins is ubiquitous across such diverse fields as neurodegenerative and cardiovascular diseases and cancer. In this study, the structures of divalent copper ion centers including three histidine and one oxygen-ligated amino acid residues and the relative binding affinities of the oxygen-ligated amino acid residues with these metal ion centers, which are debated in the literature, are presented. Furthermore, new force field parameters, which are currently lacking for the full-length metal-ligand moieties, are developed for metalloproteins that have these centers. These new force field parameters enable investigations of metalloproteins possessing these binding sites using molecular simulations. In addition, the impact of using the atom equivalence and inequivalence atomic partial charge calculation procedures on the simulated structures of these metallopeptides, including hydration properties, is described.

  11. Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

    PubMed

    Li, Wenyi; Tailhades, Julien; O'Brien-Simpson, Neil M; Separovic, Frances; Otvos, Laszlo; Hossain, M Akhter; Wade, John D

    2014-10-01

    The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

  12. 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.

  13. 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

  14. Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs.

    PubMed

    Kaino, Tomohiro; Tateiwa, Tetsuya; Mizukami-Murata, Satomi; Shima, Jun; Takagi, Hiroshi

    2008-09-01

    We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.

  15. 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

  16. 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

  17. 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.

  18. Nitrogen availability regulates proline and ethylene production and alleviates salinity stress in mustard (Brassica juncea).

    PubMed

    Iqbal, Noushina; Umar, Shahid; Khan, Nafees A

    2015-04-15

    Proline content and ethylene production have been shown to be involved in salt tolerance mechanisms in plants. To assess the role of nitrogen (N) in the protection of photosynthesis under salt stress, the effect of N (0, 5, 10, 20 mM) on proline and ethylene was studied in mustard (Brassica juncea). Sufficient N (10 mM) optimized proline production under non-saline conditions through an increase in proline-metabolizing enzymes, leading to osmotic balance and protection of photosynthesis through optimal ethylene production. Excess N (20 mM), in the absence of salt stress, inhibited photosynthesis and caused higher ethylene evolution but lower proline production compared to sufficient N. In contrast, under salt stress with an increased demand for N, excess N optimized ethylene production, which regulates the proline content resulting in recovered photosynthesis. The effect of excess N on photosynthesis under salt stress was further substantiated by the application of the ethylene biosynthesis inhibitor, 1-aminoethoxy vinylglycine (AVG), which inhibited proline production and photosynthesis. Without salt stress, AVG promoted photosynthesis in plants receiving excess N by inhibiting stress ethylene production. The results suggest that a regulatory interaction exists between ethylene, proline and N for salt tolerance. Nitrogen differentially regulates proline production and ethylene formation to alleviate the adverse effect of salinity on photosynthesis in mustard.

  19. Amino Acid and Peptide Requirement of Fusiformis necrophorus

    PubMed Central

    Wahren, Ann; Holme, Tord

    1973-01-01

    Uptake of individual amino acids and peptides by Fusiformis necrophorus was studied in growing cultures and resting cell suspensions. The cells were able to incorporate 16 of 17 14C-labeled amino acids into cell protein, the exception being proline. Proline could neither be formed by the cells from any of the other tested amino acids nor be synthesized from glucose or serine when these were used as energy sources. The addition of di- and tripeptides, the octapeptides vasopressin and oxytocin, and the poly (24) peptide ACTH did not stimulate cell growth, but a marked stimulatory effect was noted after the addition of poly-l-proline (mean molecular weight 2,000). It is concluded that cells of F. necrophorus (i) possess transport systems for most amino acids but not for proline, (ii) are dependent on exogenous proline in the form of proline-containing peptides for growth, and (iii) may be cultivated in a defined amino acid medium provided the proline requirement is met by the addition of a proline-containing peptide. PMID:4745417

  20. 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

  1. 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.

  2. Investigations on the origin and metabolism of the carbon skeleton of ornithine, arginine and proline in selected animals.

    PubMed

    Hird, F J; Cianciosi, S C; McLean, R M

    1986-01-01

    The origin and metabolism of the carbon skeletons of the amino acids ornithine and arginine have been investigated in selected animals--an earthworm, an edible mollusc, a starfish, a sea-squirt, a freshwater crustacean and a rat. Only in the rat and microorganisms of sea water was any evidence obtained for the conversion of glutamate (or N-acetylglutamate) to ornithine. Apart from the crustacean, the other animals were able to synthesise the amidine moiety of arginine. All animals were able to hydrolyse (arginase) the amidine moiety from arginine and had the enzymic capacity to convert ornithine to proline. All the animals had some enzymic ability to oxidise proline to pyrroline-5-carboxylic acid. The crustacean (Cherax destructor) was able to conserve the high concentrations of arginine in its tail muscles during fasting. The hypothesis is put forward that, as arginine appears to be an essential amino acid in the diet of this animal, its demonstrated cannibalism is, among other things, a way of supplementing dietary arginine. The results are discussed in relation to the evolution of different phosphagens derived from arginine. PMID:3943303

  3. 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

  4. 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

  5. Proline-rich sequence recognition: II. Proteomics analysis of Tsg101 ubiquitin-E2-like variant (UEV) interactions.

    PubMed

    Schlundt, Andreas; Sticht, Jana; Piotukh, Kirill; Kosslick, Daniela; Jahnke, Nadin; Keller, Sandro; Schuemann, Michael; Krause, Eberhard; Freund, Christian

    2009-11-01

    The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly "PTAP" interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFkappaB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461-2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.

  6. Total lipids of Sarda sheep meat that include the fatty acid and alkenyl composition and the CLA and trans-18:1 isomers.

    PubMed

    Santercole, Viviana; Mazzette, Rina; De Santis, Enrico P L; Banni, Sebastiano; Goonewardene, Laki; Kramer, John K G

    2007-04-01

    The total lipids of the longissimus dorsi muscle were analyzed from commercial adult Sarda sheep in Sardina taken from local abattoirs, and in the subsequent year from three local farms in the Sassari region that provided some information on the amount and type of supplements fed to the pasture-fed sheep. The complete lipid analysis of sheep meat included the fatty acids from O-acyl and N-acyl lipids, including the trans- and conjugated linoleic acid (CLA) isomers and the alk-1-enyl ethers from the plasmalogenic lipids. This analysis required the use of a combination of acid- and base-catalyzed methylation procedures, the former to quantitate the O-acyl, N-acyl and alkenyl ethers, and the latter to determine the content of CLA isomers and their metabolites. A combination of gas chromatographic and silver-ion separation techniques was necessary to quantitate all of the meat lipid constituents, which included a prior separation of the trans-octadecenoic acids (18:1) and a separation of fatty acid methyl esters and the dimethylacetals (DMAs) from the acyl and alk-1-enyl ethers, respectively. The alk-1-enyl moieties of the DMAs were analyzed as their stable cyclic acetals. In general, about half of the meat lipids were triacylglycerols, even though excess fat was trimmed from the meat. The higher fat content in the meat appears to be related to the older age of these animals. The variation in the trans-18:1 and CLA isomer profiles of the Sarda sheep obtained from the abattoirs was much greater than in the profiles from the sheep from the three selected farms. Higher levels of 10t-18:1, 7t9c-18:2, 9t11c-18:2 and 10t12c-18:2 were observed in the commercial sheep meat, which reflected the poorer quality diets of these sheep compared to those from the three farms, which consistently showed higher levels of 11t-18:1, 9c11t-18:2 and 11t13c-18:2. In the second study, sheep were provided with supplements during the spring and summer grazing season, which contributed to higher

  7. Thermodynamic models of aqueous solutions containing inorganic electrolytes and dicarboxylic acids at 298.15 K. 2. Systems including dissociation equilibria.

    PubMed

    Clegg, Simon L; Seinfeld, John H

    2006-05-01

    Atmospheric aerosols contain a significant fraction of water-soluble organic compounds, including dicarboxylic acids. Pitzer activity coefficient models are developed, using a wide range of data at 298.15 K, for the following systems containing succinic acid (H(2)Succ) and/or succinate salts: [H(+), Li(+), Na(+), K(+), Rb(+), Cs(+)]Cl(-)-H(2)Succ-H(2)O, HNO(3)-H(2)Succ-H(2)O, H(+)-NH(4)(+)-HSucc(-)-Succ(2-)-NH(3)-H(2)Succ-H(2)O, NH(4)Cl-(NH(4))(2)Succ-H(2)O, H(+)-Na(+)-HSucc(-)-Succ(2-)-Cl(-)-H(2)Succ-H(2)O, NH(4)NO(3)-H(2)Succ-H(2)O, and H(2)SO(4)-H(2)Succ-H(2)O. The above compositions are given in terms of ions in the cases where acid dissociation was considered. Pitzer models were also developed for the following systems containing malonic acid (H(2)Malo): H(+)-Na(+)-HMalo(-)-Malo(2-)-Cl(-)-H(2)Malo-H(2)O, and H(2)Malo-H(2)SO(4)-H(2)O. The models are used to evaluate the extended Zdanovskii-Stokes-Robinson (ZSR) model proposed by Clegg and Seinfeld (J. Phys. Chem. A 2004, 108, 1008-1017) for calculating water and solute activities in solutions in which dissociation equilibria occur. The ZSR model yields satisfactory results only for systems that contain moderate to high concentrations of (nondissociating) supporting electrolyte. A practical modeling scheme is proposed for aqueous atmospheric aerosols containing both electrolytes and dissociating (organic) nonelectrolytes.

  8. Azetidine-2-carboxylic acid in garden beets (Beta vulgaris).

    PubMed

    Rubenstein, Edward; Zhou, Haihong; Krasinska, Karolina M; Chien, Allis; Becker, Christopher H

    2006-05-01

    Azetidine-2-carboxylic acid (L-Aze) is a toxic and teratogenic non-protein amino acid. In many species, including man, L-Aze is misincorporated into protein in place of proline, altering collagen, keratin, hemoglobin, and protein folding. In animal models of teratogenesis, it causes a wide range of malformations. The role of L-Aze in human disease has been unexplored, probably because the compound has not been associated with foods consumed by humans. Herein we report the presence of L-Aze in the garden or table beet (Beta vulgaris).

  9. [Effects of exogenous nitric oxide, salicylic acid and hydrogen peroxide on free amino acid and soluble protein contents in tobacco leaves].

    PubMed

    Wei, Xiao-Hong; Wang, Li-Min; Long, Rui-Jun; Wang, Gen-Xuan

    2006-04-01

    The work focused on the effects of signal molecules of nitric oxide (NO), salicylic acid (SA) and hydrogen peroxide (H(2)O(2)) on compounds of nitric metabolites of free amino acid, soluble protein and proline in tobacco. The results indicated that NO, SA and H(2)O(2) were able to regulate the proline, free amino acid and soluble protein content in tobacco. Lower concentration of NO and H(2)O(2) raised the level of proline; while higher concentration of NO and H(2)O(2) lowered the proline, free amino acid content in tobacco. The above three signal molecules also showed similar effects on proline, free amino acid and soluble protein content in tobacco.

  10. Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

    PubMed

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

  11. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication.

    PubMed

    Koonin, E V

    1993-06-11

    A new superfamily of (putative) DNA-dependent ATPases is described that includes the ATPase domains of prokaryotic NtrC-related transcription regulators, MCM proteins involved in the initiation of eukaryotic DNA replication, and a group of uncharacterized bacterial and chloroplast proteins. MCM proteins are shown to contain a modified form of the ATP-binding motif and are predicted to mediate ATP-dependent opening of double-stranded DNA in the replication origins. In a second line of investigation, it is demonstrated that the products of unidentified open reading frames from Marchantia mitochondria and from yeast, and a domain of a baculovirus protein involved in viral DNA replication are related to the superfamily III of DNA and RNA helicases that previously has been known to include only proteins of small viruses. Comparison of the multiple alignments showed that the proteins of the NtrC superfamily and the helicases of superfamily III share three related sequence motifs tightly packed in the ATPase domain that consists of 100-150 amino acid residues. A similar array of conserved motifs is found in the family of DnaA-related ATPases. It is hypothesized that the three large groups of nucleic acid-dependent ATPases have similar structure of the core ATPase domain and have evolved from a common ancestor.

  12. 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

  13. Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough.

    PubMed

    Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

    2012-05-01

    Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking. PMID:22280966

  14. 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.

  15. 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

  16. Parameterization of the ReaxFF reactive force field for a proline-catalyzed aldol reaction.

    PubMed

    Hubin, Pierre O; Jacquemin, Denis; Leherte, Laurence; Vercauteren, Daniel P

    2016-11-01

    A parameterization of the ReaxFF reactive FF is performed using a Monte Carlo Simulated Annealing procedure for the modeling of a proline-catalyzed aldol reaction. Emphasis is put on the accurate reproduction of the relative stabilities of several key intermediates of the reaction, as well as, on the description of the reaction path bridging these intermediates based on quantum mechanical calculations. Our training sets include new criteria based on geometry optimizations and short Molecular Dynamics simulations to ensure that the trained ReaxFF potentials adequately predict the structures of all key intermediates. The transferability of the sets of parameters obtained is assessed for various steps of the considered aldol reaction, as well as for different substrates, catalysts, and reagents. This works indeed highlights the challenge of reaching transferable parameters for several reaction steps. © 2016 Wiley Periodicals, Inc.

  17. 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

  18. Evaluation of potential cationic probes for the detection of proline and betaine.

    PubMed

    Kalsoom, Umme; Breadmore, Michael C; Guijt, Rosanne M; Boyce, Mary C

    2014-12-01

    Osmoregulants are the substances that help plants to tolerate environmental extremes such as salinity and drought. Proline and betaine are two of the most commonly studied osmoregulants. An indirect UV CE method has been developed for simultaneous determination of these osmoregulants. A variety of reported probes and compounds were examined as potential probes for the indirect detection of proline and betaine. Mobility and UV-absorption properties highlighted sulfanilamide as a potential probe for indirect analysis of proline and betaine. Using 5 mM sulfanilamide at pH 2.2 with UV detection at 254 nm, proline and betaine were separated in less than 15 min. The LODs for proline and betaine were 11.6 and 28.3 μM, respectively. The developed method was successfully applied to quantification of these two osmoregulants in spinach and beetroot samples.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase.

    PubMed

    Araki, H; Li, Y; Yamamoto, Y; Haneda, M; Nishi, K; Kikkawa, R; Ohkubo, I

    2001-02-01

    We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.

  5. 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

  6. Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

    PubMed

    Mouw, M; Pintel, D J

    1998-11-10

    GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well.

  7. Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: leucine 66 to proline and leucine 91 to proline.

    PubMed

    Gray, T M; Arnoys, E J; Blankespoor, S; Born, T; Jagar, R; Everman, R; Plowman, D; Stair, A; Zhang, D

    1996-04-01

    A class of temperature-sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 degrees C and no activity at 43 degrees C has been selected. These mutants, designated "tight" ts mutants, differ from most other T4 lysozyme mutants that are active at 43 degrees C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T1571, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 degrees C. Two mutants were identified: L91P and L66P. Both insert proline residues into alpha-helical regions of the WT protein structure. The stabilities (delta delta G) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40-ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the alpha-helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain alpha-helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent-filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge.

  8. Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase.

    PubMed

    Mohamed, Saleh A; Mohamed, Tarek M; Fahmy, Afaf S; El-Badry, Mohamed O; Abdel-Gany, Somia S

    2008-01-01

    Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme. PMID:17655846

  9. Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation.

    PubMed

    Perassolo, María; Quevedo, Carla Verónica; Busto, Víctor Daniel; Giulietti, Ana María; Talou, Julián Rodríguez

    2011-07-01

    Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H₂O₂-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.

  10. An update on potato crisps contents of moisture, fat, salt and fatty acids (including trans-fatty acids) with special emphasis on new oils/fats used for frying.

    PubMed

    Gonçalves Albuquerque, Tânia; Sanches-Silva, Ana; Santos, Lèlita; Costa, Helena S

    2012-09-01

    Eighteen brands of potato crisps, frequently consumed, were analyzed to establish their nutritional value in relation to salt, fat and fatty acid (FA) composition. The purpose of the present study was to determine moisture, total fat, salt contents and FA profiles (including trans-FAs), and to identify the oil/fat used for frying of the 18 brands of potato crisps. Our results show that salt content ranged from 0.127 to 2.77 g/100 g and total fat content of potato crisps varied between 20.0 and 42.8 g/100 g. With respect to FAs analysis, palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) were the major FAs found in the analyzed potato crisps. It is clear from our work that nowadays most potato crisps are currently produced using oils with high contents in unsaturated FAs, which can be considered as healthier from a nutritional point of view. Nevertheless, some brands of potato crisps still use palm oil or a blend of palm oil and other fats/oils, which are very rich in saturated FAs.

  11. 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

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

    PubMed Central

    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

  13. Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans.

    PubMed

    Knipp, Gabriele; Honermeier, Bernd

    2006-03-01

    This study aimed to investigate the effect of water stress on the accumulation of proline in the leaves of transgenic potato lines generating fructans. The results of the series of bifactorial experiments in 2002 and 2003 indicated an increase of the proline level in leaves of all potato lines examined under water deficit. In addition, an increase in proline concentration during plant development was observed. The proline content was related to leaf water potential and relative water content (RWC), which indicates that proline could be involved in osmoregulation of potato plants under the experimental conditions. Surprisingly, under water deficit, the proline level was lowest in most of the transgenic SST/FFT-lines, which generate fructan molecules with a high degree of polymerization. Therefore, a pleiotropic effect can not be excluded as the reason for the divergence in behavior of these transgenic lines. The present results suggest that the modification of carbohydrate metabolism, especially the high content of soluble carbohydrates, may affect water stress-induced proline accumulation.

  14. Genetic analysis of proline concentration under osmotic stress in sunflower (Helianthus annuus L.)

    PubMed Central

    Khalil, Farghama; Rauf, Saeed; Monneveux, Philippe; Anwar, Shoaib; Iqbal, Zafar

    2016-01-01

    Proline concentration has been often suggested as an indicator of osmotic stress. A better understanding of the genetics of this trait is however needed. In the present study, proline concentration has been assessed, together with root and stem growth, potassium, calcium and total soluble sugars concentration and stress injury symptoms, in seedlings of sunflower hybrids and their parents grown under control and osmotic conditions. Proline strongly accumulated with osmotic stress. Its concentration exhibited a large variation among genotypes and was higher in hybrids than in parental lines. A positive association was noted between proline concentration and osmotic adjustment that was reflected in a reduction of osmotic stress induced injury, as showed by the reduced number of calli in the hybrids with higher proline concentration. Broad and narrow sense heritability was higher under osmotic stress suggesting applying the selection in osmotic stress condition. In the control treatment, dominance effects explained most of the genetic variation for proline concentration while under osmotic stress both dominance and additive variance were high. The importance of dominance and additive effects suggested that several genomic regions are controlling this trait. Good general combiners, presumably carrying positive additive alleles affecting proline concentration, were identified. PMID:27795671

  15. 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.

  16. Nutritional assessment of genetically modified rapeseed synthesizing high amounts of mid-chain fatty acids including production responses of growing-finishing pigs.

    PubMed

    Böhme, Hartwig; Rudloff, Eike; Schöne, Friedrich; Schumann, Wolfgang; Hüther, Liane; Flachowsky, Gerhard

    2007-08-01

    The nutritive value of genetically modified myristic acid-rich rapeseed, in which a acyl-thioesterase gene inserted, was studied. Crude nutrients, amino acid and fatty acid profiles as well as mineral and glucosinolate contents were determined and compared with those of the non-transgenic parental cultivar. The concentration of crude nutrients, minerals and amino acids were found to be within the range of natural variance. The myristic and palmitic acid content increased from 0.1 - 11.4% and from 3.6-20%, respectively, at the expense of oleic acid, which decreased from 68.6-42.6% of total fatty acids. The glucosinolate contents increased from 12.4 micromol/g in the parental plant to 19 micromol/g DM in the GM-plant. Full-fat rapeseed of both cultivars was incorporated in pig diets at a level of 15%, and the digestibility and the production efficiency were tested under ad libitum feeding conditions with ten pigs each over the growing finishing period from 32-105 kg BW. The experimental diets did not show significant differences in digestibility and energetic feeding value. However, feed intake and weight gain decreased presumably due to the increasing glucosinolate intake associated with the feeding of transgenic rapeseed. The dietary fatty acids profile influenced the fatty acid profile of body fat. Myristic acid accumulated in back fat and intramuscular fat while the oleic acid content decreased. The increased glucosinolate intake affected the weight of thyroid glands and their iodine concentration.

  17. Response of two mustard (Brassica juncea L.) cultivars differing in photosynthetic capacity subjected to proline.

    PubMed

    Wani, Arif Shafi; Irfan, Mohammad; Hayat, Shamsul; Ahmad, Aqil

    2012-01-01

    The present paper deals with the effect of exogenous application of proline as a shotgun approach on growth, photosynthesis, and antioxidative system in 25-day-old plants of two different cultivars of Brassica juncea L. (Varuna and RH-30) under natural conditions. Exogenous application of proline significantly increased plant growth, photosynthetic rate, and the activities of antioxidant enzymes, compared with untreated seedlings. Pre-sowing seed soaking in 20 mM proline, for 8 h, proved best among all the other concentrations used. PMID:21318454

  18. Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs.

    PubMed

    Kaino, Tomohiro; Tateiwa, Tetsuya; Mizukami-Murata, Satomi; Shima, Jun; Takagi, Hiroshi

    2008-09-01

    We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking. PMID:18641164

  19. The influence of lactation on L-proline absorption from small intestine in the albino rat.

    PubMed

    Datta, U; Sharma, R K

    1985-01-01

    Intestinal absorption of L-proline was studied in control and lactating rats from jejunum and ileum by in vivo method and presented per unit dry weight and per unit length of the respective segment. L-proline absorption was found to be significantly reduced in lactating animals as compared to the virgin controls. The results were discussed in light of serosal to mucosal ratio. By in vitro method also jejunal and ileal uptake of L-proline were found to be significantly reduced in lactating animals as compared to the virgin controls.

  20. 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

  1. The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

    PubMed

    de Caestecker, M P; Yahata, T; Wang, D; Parks, W T; Huang, S; Hill, C S; Shioda, T; Roberts, A B; Lechleider, R J

    2000-01-21

    Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

  2. 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

  3. A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength.

    PubMed Central

    Csonka, L N

    1982-01-01

    Exogenous proline specifically stimulates the growth rate of enteric bacteria in media of inhibitory osmotic strength (J. H. B. Christian, Aust. J. Biol. Sci. 8:490-497, 1955). I observed that Salmonella typhimurium mutants which lack both of the previously known proline permeases (putP proP) are stimulated by proline in media of inhibitory osmolarity. I propose that there is a third proline permease which functions only in media of elevated osmolarity. This conclusion is based on the observations that, in media of elevated osmolarity, (i) the sensitivity of putP proP mutants to toxic proline analogs increases, (ii) proline requirements for maximal growth of proline auxotrophic putP proP mutants decreases, and (iii) the specific rate of incorporation of radioactive proline into protein of growing cells increases. I obtained a Tn10-induced mutation in a gene (proU) required for the functioning of the third proline permease and determined the map location to be at 59 map units of the chromosome, between srlA and tct, 66% linked to nalB in P22 transduction. My results suggest that the function of the third, osmotically stimulated permease might be to accumulate high intracellular proline levels during osmotic stress. Possible mechanisms by which proline might cause growth stimulation are discussed. PMID:7050090

  4. 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

  5. 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

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

    2015-06-01

    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.

  6. Development of glycine-α-methyl-proline-containing tripeptides with neuroprotective properties.

    PubMed

    Cacciatore, Ivana; Fornasari, Erika; Di Stefano, Antonio; Marinelli, Lisa; Cerasa, Laura Serafina; Turkez, Hasan; Aydin, Elanur; Moretto, Alessandro; Ferrone, Alessio; Pesce, Mirko; di Giacomo, Viviana; Reale, Marcella; Costantini, Erica; Di Giovanni, Pamela; Speranza, Lorenza; Felaco, Mario; Patruno, Antonia

    2016-01-27

    Herein is described the synthesis of novel glycine-α-methyl-proline-containing tripeptides (GP(Me)X tripeptides namely GP(Me)R, GP(Me)K, and GP(Me)H) with the aim of obtaining derivatives highly stable in human plasma and able to counteract neuroinflammatory processes that are distinctive of neurodegenerative pathologies. The syntheses of GP(Me)R, GP(Me)K, and GP(Me)H were all achieved both by introducing the Pro(Me) residue into the Gly-Pro-Arg (GPR) sequence in place of the native Pro in P2 position and replacing the basic amino acid Arg in P3 position by Lys or His. Results showed that all novel GP(Me)X tripeptides are stable in human plasma (t1/2 > 51 h) and that GP(Me)H - generating stable intramolecular H-bond in a C11-turn by interaction of His imidazole ring and Gly carbonyl group - restored physiological levels of nitric oxide deriving from neuronal NOS (nNOS) activity, thus preventing the inflammatory response by suppression of the NF-kB activity and, consequently, the expression of inflammatory genes such as inducibile NOS (iNOS). Therefore, GP(Me)H could be a lead compound for further development of peptidomimetics able to contrast neuroinflammatory processes. PMID:26717205

  7. 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

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

    PubMed

    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.

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

    PubMed

    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

  10. Serous tubal intraepithelial carcinoma upregulates markers associated with high-grade serous carcinomas including Rsf-1 (HBXAP), cyclin E and fatty acid synthase.

    PubMed

    Sehdev, Ann Smith; Kurman, Robert J; Kuhn, Elisabetta; Shih, Ie-Ming

    2010-06-01

    Serous tubal intraepithelial carcinoma (STIC) has been proposed as a precursor for many pelvic high-grade serous carcinomas. Our previous analysis of the ovarian cancer genome identified several genes with oncogenic potential that are amplified and/or overexpressed in the majority of high-grade serous carcinomas. Determining whether these genes are upregulated in STICs is important in further elucidating the relationship of STICs to high-grade serous carcinomas and is fundamental in understanding the molecular pathogenesis of high-grade serous carcinomas. In this study, 37 morphologically defined STICs were obtained from 23 patients with stage IIIC/IV high-grade serous carcinomas. Both STICs and the high-grade serous carcinomas were analyzed for expression of Rsf-1 (HBXAP), cyclin E, fatty acid synthase (FASN) and mucin-4. In addition, they were examined for expression of established markers including p53, Ki-67 and p16. We found that diffuse nuclear p53 and p16 immunoreactivity was observed in 27 (75%) of 36 and 18 (55%) of 33 STICs, respectively, whereas an elevated Ki-67 labeling index (>or=10%) was detected in 29 (78%) of 37 STICs. Cyclin E nuclear staining was seen in 24 (77%) of 35 STICs, whereas normal tubal epithelial cells were all negative. Increased Rsf-1 and FASN immunoreactivity occurred in 63%, and 62% of STICs, respectively, compared with adjacent normal-appearing tubal epithelium. Interestingly, only one STIC showed increased mucin-4 immunoreactivity. Carcinomas, when compared with STICs, overexpressed p16, Rsf-1, cyclin E and FASN in a higher proportion of cases. In conclusion, STICs express several markers including Rsf-1, cyclin E and FASN in high-grade serous carcinomas. In contrast, mucin-4 immunoreactivity either did not change or was reduced in most STICs. These results suggest that overexpression of Rsf-1, cyclin E and FASN occurs early in tumor progression.

  11. Origins of the double asymmetric induction on proline-catalyzed aldol reactions.

    PubMed

    Calderón, Félix; Doyagüez, Elisa G; Cheong, Paul Ha-Yeon; Fernández-Mayoralas, Alfonso; Houk, K N

    2008-10-17

    Computational studies to elucidate the origin of the double asymmetric induction on proline-catalyzed aldol reaction have been performed using HF/6-31G(d) calculations. The computed transition structures explain the experimental data obtained. PMID:18811197

  12. 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 ...

  13. Proline accumulation in lemongrass (Cymbopogon flexuosus Stapf.) due to heavy metal stress.

    PubMed

    Handique, G K; Handique, A K

    2009-03-01

    Toxic heavy metals viz. lead, mercury and cadmium induced differential accumulation of proline in lemongrass (Cymbopogon flexuosus Stapf.) grown in soil amended with 50, 100, 200, 350 and 500 mg kg(-1) of the metals have been studied. Proline accumulation was found to be metal specific, organ specific and linear dose dependant. Further, proline accumulation following short term exposure (two months after transplantation) was higher than long term exposure (nine months after transplantation). Proline accumulation following short term exposure was 2.032 to 3.839 micro moles g(-1) for cadmium (50-200 mg kg(-1)); the corresponding range for mercury was 1.968 to 5.670 micro moles g(-1) and 0.830 to 4.567 micro moles g(-1) for lead (50-500 mg kg(-1) for mercury and lead). Proline accumulation was consistently higher in young tender leaf than old leaf, irrespective of the metal or duration of exposure. For cadmium treatment proline level was 2.032 to 3.839 micro moles g(-1) for young leaves while the corresponding value for old leaf was 1.728 to 2.396 micro moles g(-1) following short term exposure. The same trend was observed for the other two metals and duration of exposure. For control set proline accumulation in root was 0.425 micro moles g(-1) as against 0.805 and 0.533 micro moles g(-1) in young and old leaves respectively indicating that proline accumulation in root are lower than leaves, under both normal and stressed condition.

  14. Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells.

    PubMed

    Miltyk, Wojciech; Surazynski, Arkadiusz; Kasprzak, Kazimierz S; Fivash, Matthew J; Buzard, Gregory S; Phang, James M

    2005-04-15

    Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell-matrix and cell-cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation. PMID:15696600

  15. The Composition of Stigmatic Exudate from Lilium longiflorum: Labeling Studies with Myo-inositol, d-Glucose, and l-Proline.

    PubMed

    Labarca, C; Kroh, M; Loewus, F

    1970-07-01

    Stigmatic exudate, a secretion product recovered from the upper surface of Lilium longiflorum pistils, has been examined. Over 99% of the exudate is accounted for as water, carbohydrate, and protein. Exclusive of water, 95% is a high molecular weight, protein-containing polysaccharide composed of galactose, arabinose, rhamnose, glucuronic acid, and galacturonic acid.Detached pistils supplied with myo-inositol-U-(14)C, myo-inositol-2-(3)H, d-glucose-1-(14)C, or l-proline-U-(14)C produce labeled stigmatic exudate. When myo-inositol is supplied, the exudate is rich in labeled arabinose and uronic acids, but some label also recycles through the hexose phosphate pool of secreting cells, causing label to appear in galactose and rhamnose residues. When glucose is provided, galactose is the major constituent labeled but all of the other carbohydrate constituents are also labeled. Proline produces a pattern very similar to that obtained with glucose.Stigmatic exudate also contains a small amount of low molecular weight carbohydrate. If myo-inositol is used to label exudate, free labeled myo-inositol cannot be detected in the low molecular weight fraction until it has been subjected to acid hydrolysis. Similarly, if d-glucose is the source of label, free labeled glucose is found in the low molecular weight fraction only after acid hydrolysis.

  16. 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.

  17. Improvement of crow's feet lines by topical application of 1-carbamimidoyl-L-proline (CLP).

    PubMed

    Kawashima, Makoto; Yokose, Urara; Hachiya, Akira; Fujimura, Tsutomu; Tsukahara, Kazue; Kawada, Hiromitsu; Kitahara, Takashi; Takema, Yoshinori; Terui, Tadashi; Nakagawa, Hidemi

    2013-04-01

    Wrinkles, one of the characteristics of chronic sun-damaged and/or aged skin, are associated with psychological distress. Apart from the deterioration of collagen and elastic fibers in the dermis, which induces the loss of skin elasticity, it has been recently proposed that decreased flexibility or elasticity of the stratum corneum (SC) is also correlated with wrinkle formation. The elasticity of the SC has been shown to be regulated, at least in part, by the amounts and types of amino acids. To evaluate the ability of our newly developed amino acid-derivative (1-carbamimidoyl-L-proline; CLP), which recovers the elastic properties of the SC ex vivo, to improve wrinkles, a clinical test was performed with 126 Japanese female subjects aged 32-50 years who had crow's feet lines on their faces. Three eligible dermatologists evaluated the study according to authorized grades by the Japanese Cosmetic Science Society and scored the subjects who were much improved or improved as 29.7% and 57.8% of all CLP-treated subjects at 4 and 8 weeks, respectively. In contrast, only 1.5% and 8.1% of subjects improved with the placebo lotion at 4 and 8 weeks, respectively. These results suggest a significant efficacy of CLP to improve wrinkles. In parallel with the dermatologists' assessments, skin surface roughness in the CLP-treated group was significantly reduced after treatment with CLP for 4 and 8 weeks compared to the placebo-treated group. The sum of these data suggests that CLP is a promising and useful ingredient for the improvement of wrinkles through its ability to enhance the elasticity of the SC.

  18. 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

  19. 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

  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. 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.

  2. 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

  3. Sinorhizobium meliloti chemoreceptor McpU mediates chemotaxis toward host plant exudates through direct proline sensing.

    PubMed

    Webb, Benjamin A; Hildreth, Sherry; Helm, Richard F; Scharf, Birgit E

    2014-06-01

    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.

  4. 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

  5. CPPU elevates photosynthetic abilities, growth performances and yield traits in salt stressed rice (Oryza sativa L. spp. indica) via free proline and sugar accumulation.

    PubMed

    Gashaw, Arega; Theerawitaya, Cattarin; Samphumphuang, Thapanee; Cha-um, Suriyan; Supaibulwatana, Kanyaratt

    2014-01-01

    Application of N-2-(chloro-4-pyridyl)-N-phenyl urea (CPPU) to salt susceptible cultivar of indica rice (cv. PTT1) effected on free proline and soluble sugar accumulation and regulated the physio-morphological responses when subjected to salt stress condition was firstly demonstrated in this study. Soluble sugars, including sucrose, glucose and fructose, in leaf blade and leaf sheath were enriched in 0.1 mM CPPU pretreated plants subsequently exposed to 16.6 dS m(-1) NaCl for 10 days. In the long period (15 days) salt stress, free proline content in the leaf blade and leaf sheath were evidently peaked to act as osmotic adjustment in the salt-stressed plants. In addition, the photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids, were maintained by the functional regulation of soluble sugar and free proline in the cellular levels, thereby leading to higher net photosynthetic rate. Further, the stomatal closure and transpiration rate in CPPU pretreated plants were retained under salt stress, thereby resulting in alleviation of growth performance and yield traits. This study suggested that exogenous application of CPPU may alternatively play effective role to improve the salt tolerant abilities of salt susceptible rice crop.

  6. 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

  7. Evidence that COMT genotype and proline interact on negative-symptom outcomes in schizophrenia and bipolar disorder

    PubMed Central

    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 Val158Met 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

  8. Temporal trends of perfluoroalkane sulfonic acids and their sulfonamide-based precursors in herring from the Swedish west coast 1991-2011 including isomer-specific considerations.

    PubMed

    Ullah, Shahid; Huber, Sandra; Bignert, Anders; Berger, Urs

    2014-04-01

    A method was developed for simultaneous analysis of perfluoroalkane sulfonic acids (PFSAs) and their sulfonamide-based precursors (perfluoroalkane sulfonamidoacetic acids (FASAAs), sulfonamides (FASAs), and sulfonamidoethanols (FASEs)) in fish muscle. Extraction was performed with acetonitrile followed by a clean-up and fractionation step and instrumental analysis by UPLC/MS/MS and GC/MS. Time trends of PFSAs and their precursors in herring muscle samples originating from the Kattegat at the west coast of Sweden were investigated covering the years 1991-2011. The following analytes were detected, all with decreasing or unchanged trends between 1991 and 2011: Perfluorobutane sulfonic acid (PFBS, below the method detection limit (acid (PFHxS, 9-38pg/g), perfluorooctane sulfonic acid (PFOS, 240-930pg/g), perfluorodecane sulfonic acid (PFDS, acid (MeFOSAA and EtFOSAA, 2-39 and 2-31pg/g, respectively) and perfluorooctane sulfonamide (FOSA, 78-920pg/g). The highest concentrations were found for PFOS and FOSA around the turn of the century. Shorter disappearance half-lives were observed for precursors compared to PFSAs. Assuming that these trend differences are representative for fish consumed by the general Swedish population, this observation suggests that the relative contribution of precursors to total human exposure to PFOS via fish intake has decreased in Sweden over the study period. PFOS precursors in fish may have constituted a significant indirect exposure pathway for PFOS in the 1990s. Isomer-specific analysis of PFOS and FOSA revealed <10% relative contributions of branched isomers to total PFOS and total FOSA. Furthermore, the percentage branched isomers decreased over time for both compounds. These findings are contrary to patterns and temporal trends of PFOS isomers commonly found in human serum. In combination with literature data on isomer patterns

  9. 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.

  10. Grassland species differentially regulate proline concentrations under future climate conditions: an integrated biochemical and modelling approach.

    PubMed

    AbdElgawad, Hamada; De Vos, Dirk; Zinta, Gaurav; Domagalska, Malgorzata A; Beemster, Gerrit T S; Asard, Han

    2015-10-01

    Proline (Pro) is a versatile metabolite playing a role in the protection of plants against environmental stresses. To gain a deeper understanding of the regulation of Pro metabolism under predicted future climate conditions, including drought stress, elevated temperature and CO2 , we combined measurements in contrasting grassland species (two grasses and two legumes) at multiple organisational levels, that is, metabolite concentrations, enzyme activities and gene expression. Drought stress (D) activates Pro biosynthesis and represses its catabolism, and elevated temperature (DT) further elevated its content. Elevated CO2 attenuated the DT effect on Pro accumulation. Computational pathway control analysis allowed a mechanistic understanding of the regulatory changes in Pro metabolism. This analysis indicates that the experimentally observed coregulation of multiple enzymes is more effective in modulating Pro concentrations than regulation of a single step. Pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) play a central role in grasses (Lolium perenne, Poa pratensis), and arginase (ARG), ornithine aminotransferase (OAT) and P5CR play a central role in legumes (Medicago lupulina, Lotus corniculatus). Different strategies in the regulation of Pro concentrations under stress conditions were observed. In grasses the glutamate pathway is activated predominantly, and in the legumes the ornithine pathway, possibly related to differences in N-nutritional status. PMID:26037253

  11. Grassland species differentially regulate proline concentrations under future climate conditions: an integrated biochemical and modelling approach.

    PubMed

    AbdElgawad, Hamada; De Vos, Dirk; Zinta, Gaurav; Domagalska, Malgorzata A; Beemster, Gerrit T S; Asard, Han

    2015-10-01

    Proline (Pro) is a versatile metabolite playing a role in the protection of plants against environmental stresses. To gain a deeper understanding of the regulation of Pro metabolism under predicted future climate conditions, including drought stress, elevated temperature and CO2 , we combined measurements in contrasting grassland species (two grasses and two legumes) at multiple organisational levels, that is, metabolite concentrations, enzyme activities and gene expression. Drought stress (D) activates Pro biosynthesis and represses its catabolism, and elevated temperature (DT) further elevated its content. Elevated CO2 attenuated the DT effect on Pro accumulation. Computational pathway control analysis allowed a mechanistic understanding of the regulatory changes in Pro metabolism. This analysis indicates that the experimentally observed coregulation of multiple enzymes is more effective in modulating Pro concentrations than regulation of a single step. Pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) play a central role in grasses (Lolium perenne, Poa pratensis), and arginase (ARG), ornithine aminotransferase (OAT) and P5CR play a central role in legumes (Medicago lupulina, Lotus corniculatus). Different strategies in the regulation of Pro concentrations under stress conditions were observed. In grasses the glutamate pathway is activated predominantly, and in the legumes the ornithine pathway, possibly related to differences in N-nutritional status.

  12. 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

  13. Improvement of culture conditions for L-proline production by a recombinant strain of Serratia marcescens.

    PubMed

    Masuda, M; Takamatu, S; Nishimura, N; Komatsubara, S; Tosa, T

    1993-12-01

    Serratia marcescens SP511 was previously reported to be an L-proline-producing strain that harbors a recombinant plasmid carrying the mutant type of the proline operon. This strain produced 65 g/L of L-proline in a medium containing 22% sucrose and urea after 5 d of incubation under the conventional culture conditions. We searched for more suitable culture conditions for more abundant L-proline production by SP511. To improve the supply of a nitrogen source to cells, ammonium was used instead of urea and fed to a culture under control of the pH of the medium. The concentrations of MgSO4 and K2HPO4 were increased, and in addition, sucrose was continuously added to the culture at a final concentration of 32%. Under these conditions, the cell amount was increased twofold over that under the previous conditions and L-proline production reached a maximum of more than 100 g/L after 4 d of incubation. PMID:8109960

  14. Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

    PubMed Central

    Moore, Claire E. J.; Mikolajek, Halina; Regufe da Mota, Sergio; Wang, Xuemin; Kenney, Justin W.; Werner, Jörn M.

    2015-01-01

    Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors. PMID:25755286

  15. UV photodissociation of proline-containing peptide ions: insights from molecular dynamics.

    PubMed

    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, S(2) and S(3), lie close to 213 nm. Non-adiabatic MD simulation starting from S(2) and S(3) 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.

  16. 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

  17. Slow tight-binding inhibition of prolyl endopeptidase by benzyloxycarbonyl-prolyl-prolinal.

    PubMed Central

    Bakker, A V; Jung, S; Spencer, R W; Vinick, F J; Faraci, W S

    1990-01-01

    Prolyl endopeptidase is a serine proteinase that specifically cleaves peptides on the carboxy side of proline residues. Wilk & Orlowski [(1983) J. Neurochem. 41, 69-75] have shown that benzyloxycarbonyl-prolyl-prolinal (Z-prolyl-prolinal) is a potent inhibitor of prolyl endopeptidase. We show that Z-prolyl-prolinal is a slow-binding inhibitor of mouse brain prolyl endopeptidase with Ki 0.35 +/- 0.05 nM. Kinetic analysis indicates that the mechanism is a simple, but slow, reversible equilibrium between free and bound enzyme (E + I in equilibrium EI) with rate constants for association (kon) and dissociation (koff) of 1.6 X 10(5) M-1.s-1 and approx. 4 X 10(-5) s-1 respectively. Slow-binding inhibition is dependent on the presence of the aldehyde group since the alcohol (Z-prolyl-prolinol) is a rapid and 50,000-fold poorer inhibitor (Ki 19 microM). Prolyl endopeptidase from human brain is also inhibited by Z-prolyl-prolinal with kinetics similar to those of the mouse brain enzyme. PMID:2241932

  18. Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline.

    PubMed

    Li, Zhong-Guang; Ding, Xiao-Jiao; Du, Pei-Fang

    2013-05-15

    Hydrogen sulfide (H2S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.

  19. 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

  20. 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

  1. Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

    PubMed Central

    Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F.; Dickman, Martin B.

    2006-01-01

    Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants. PMID:16751508

  2. 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

  3. Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing*

    PubMed Central

    Patel, Nikesh; Exell, Jack C.; Jardine, Emma; Ombler, Ben; Finger, L. David; Ciani, Barbara; Grasby, Jane A.

    2013-01-01

    The prototypical 5′-nuclease, flap endonuclease-1 (FEN1), catalyzes the essential removal of single-stranded flaps during DNA replication and repair. FEN1 hydrolyzes a specific phosphodiester bond one nucleotide into double-stranded DNA. This specificity arises from double nucleotide unpairing that places the scissile phosphate diester on active site divalent metal ions. Also related to FEN1 specificity is the helical arch, through which 5′-flaps, but not continuous DNAs, can thread. The arch contains basic residues (Lys-93 and Arg-100 in human FEN1 (hFEN1)) that are conserved by all 5′-nucleases and a cap region only present in enzymes that process DNAs with 5′ termini. Proline mutations (L97P, L111P, L130P) were introduced into the hFEN1 helical arch. Each mutation was severely detrimental to reaction. However, all proteins were at least as stable as wild-type (WT) hFEN1 and bound substrate with comparable affinity. Moreover, all mutants produced complexes with 5′-biotinylated substrate that, when captured with streptavidin, were resistant to challenge with competitor DNA. Removal of both conserved basic residues (K93A/R100A) was no more detrimental to reaction than the single mutation R100A, but much less severe than L97P. The ability of protein-Ca2+ to rearrange 2-aminopurine-containing substrates was monitored by low energy CD. Although L97P and K93A/R100A retained the ability to unpair substrates, the cap mutants L111P and L130P did not. Taken together, these data challenge current assumptions related to 5′-nuclease family mechanism. Conserved basic amino acids are not required for double nucleotide unpairing and appear to act cooperatively, whereas the helical cap plays an unexpected role in hFEN1-substrate rearrangement. PMID:24126913

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. DPPH radical scavenging activity and contents of H2O2, malondialdehyde and proline in determining salinity tolerance in chickpea seedlings.

    PubMed

    Kaur, Narinder; Kumar, Arvind; Kaur, Kamaljit; Gupta, Anil K; Singh, Inderjit

    2014-10-01

    The involvement of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and contents of H2O2, malondialdehyde (MDA) and proline was investigated in determining salinity tolerance among seedlings of thirty chickpea (Cicer arietinum L.) genotypes having different pedigrees. Chickpea genotypes, including cultivars and advanced lines were grown for 7 days under control and salt stress (50 mM NaCl) conditions. The genotypes showed differential response to salt stress in terms of growth, DPPH radical scavenging activity and contents of H2O2, MDA and proline in seedlings. On the basis of seedling growth, the genotypes having better performance under stress conditions had reduced levels of H2O2 and MDA contents, but increased levels of proline and DPPH radical scavenging activity. Stress tolerance index for these parameters was also determined. Agglomerative hierarchal clustering by Pearson correlation coefficient grouped the genotypes into two major clusters--MC I and MC II. MC II and Al-1 sub-cluster of MC-I comprised mainly of genotypes that showed higher stress resistance levels for the respective parameters in comparison to genotypes in other sub-clusters. Thus, it is possible to identify salt-tolerant genotypes on the basis of above parameters without a field trial.

  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. Incorporation of proline into prodigiosin by a Put mutant of Serratia marcesens.

    PubMed

    Lim, D V; Qadri, S M; Williams, R P

    1976-05-01

    A Put mutant of Serratia marcescens, deficient in proline oxidase and therefore unable to degrade proline, was used to assay for an enzymatic reaction responsible for incorporation of proline into prodigiosin. The reaction had a pH optimum of 7.5 and a Km of 1.1 X 10(-4) M at 27 C. At temperatures above 27 C, the velocity of the reaction decreased with increasing temperature and little activity was detected at 42 C. Activity of the enzyme was directly proportional to the quantity of pigment formed and was inhibited by thioproline, a substrate analog. These data suggested the presence of a unique and specific enzyme in the biosynthetic pathway for prodigiosin.

  12. Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat.

    PubMed

    Vendruscolo, Eliane Cristina Gruszka; Schuster, Ivan; Pileggi, Marcos; Scapim, Carlos Alberto; Molinari, Hugo Bruno Correa; Marur, Celso Jamil; Vieira, Luiz Gonzaga Esteves

    2007-10-01

    Water deficit is one of the main abiotic factors that affect spring wheat planted in subtropical regions. Accumulation of proline appears to be a promising approach to maintain the productivity of plants under stress condition. However, morphological alterations and growth reduction are observed in transgenic plants carrying genes coding for osmoprotectants controlled by constitutive promoters. We report here the effects of water deficit on wheat plants transformed with the Vigna aconitifolia Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA that encodes the key regulatory enzyme in proline biosynthesis, under the control of a stress-induced promoter complex-AIPC. Transgenic wheat plants submitted to 15 days of water shortage presented a distinct response. We have found that drought resulted in the accumulation of proline. The tolerance to water deficit observed in transgenic plants was mainly due to protection mechanisms against oxidative stress and not caused by osmotic adjustment.

  13. Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential.

    PubMed

    Sharma, Sandeep; Villamor, Joji Grace; Verslues, Paul E

    2011-09-01

    To better define the still unclear role of proline (Pro) metabolism in drought resistance, we analyzed Arabidopsis (Arabidopsis thaliana) Δ(1)-pyrroline-5-carboxylate synthetase1 (p5cs1) mutants deficient in stress-induced Pro synthesis as well as proline dehydrogenase (pdh1) mutants blocked in Pro catabolism and found that both Pro synthesis and catabolism were required for optimal growth at low water potential (ψ(w)). The abscisic acid (ABA)-deficient mutant aba2-1 had similar reduction in root elongation as p5cs1 and p5cs1/aba2-1 double mutants. However, the reduced growth of aba2-1 but not p5cs1/aba2-1 could be complemented by exogenous ABA, indicating that Pro metabolism was required for ABA-mediated growth protection at low ψ(w). PDH1 maintained high expression in the root apex and shoot meristem at low ψ(w) rather than being repressed, as in the bulk of the shoot tissue. This, plus a reduced oxygen consumption and buildup of Pro in the root apex of pdh1-2, indicated that active Pro catabolism was needed to sustain growth at low ψ(w). Conversely, P5CS1 expression was most highly induced in shoot tissue. Both p5cs1-4 and pdh1-2 had a more reduced NADP/NADPH ratio than the wild type at low ψ(w). These results indicate a new model of Pro metabolism at low ψ(w) whereby Pro synthesis in the photosynthetic tissue regenerates NADP while Pro catabolism in meristematic and expanding cells is needed to sustain growth. Tissue-specific differences in Pro metabolism and function in maintaining a favorable NADP/NADPH ratio are relevant to understanding metabolic adaptations to drought and efforts to enhance drought resistance.

  14. Inhibitory potential of tea polyphenolics and influence of extraction time against Helicobacter pylori and lack of inhibition of beneficial lactic acid bacteria.

    PubMed

    Ankolekar, Chandrakant; Johnson, David; Pinto, Marcia da Silva; Johnson, Kevin; Labbe, Ronald; Shetty, Kalidas

    2011-11-01

    Tea polyphenolics such as catechins are known to have the potential to inhibit many bacterial pathogens. Helicobacter pylori has been identified as an etiologic agent in the development of gastric ulcer, peptic ulcer, gastritis, and many other stomach-related diseases. In this study, we investigated the effect of 9 tea extracts--3 different brands representing 4 different processed types (white, green, oolong, and black)--on the inhibition of H. pylori. Extraction times of 2 and 5 minutes were compared. Most 5-minute extracts showed H. pylori inhibition, whereas 2-minute extracts only of Choice darjeeling black and Tazo white showed inhibition. No recovery was observed after the addition of 0.5 and 5 mM proline, indicating that tea polyphenols do not inhibit H. pylori by inhibition of proline oxidation via proline dehydrogenase. Extracts that showed inhibition were further evaluated for their effect on beneficial lactic acid bacteria. None of the samples showed inhibition, suggesting that tea might be able to inhibit H. pylori without affecting the beneficial lactic acid bacteria. High-performance liquid chromatography indicated the presence of gallic acid, quercetin, caffeine, and tea catechins (including catechin, epicatechin, and epigallocatechin) in all the tea samples. Our study indicates that tea can be potentially used as a low-cost dietary support to combat H. pylori-linked gastric diseases without affecting the beneficial intestinal bacteria.

  15. Mlp24 (McpX) of Vibrio cholerae implicated in pathogenicity functions as a chemoreceptor for multiple amino acids.

    PubMed

    Nishiyama, So-ichiro; Suzuki, Daisuke; Itoh, Yasuaki; Suzuki, Kazuho; Tajima, Hirotaka; Hyakutake, Akihiro; Homma, Michio; Butler-Wu, Susan M; Camilli, Andrew; Kawagishi, Ikuro

    2012-09-01

    The chemotaxis of Vibrio cholerae, the causative agent of cholera, has been implicated in pathogenicity. The bacterium has more than 40 genes for methyl-accepting chemotaxis protein (MCP)-like proteins (MLPs). In this study, we found that glycine and at least 18 L-amino acids, including serine, arginine, asparagine, and proline, serve as attractants to the classical biotype strain O395N1. Based on the sequence comparison with Vibrio parahaemolyticus, we speculated that at least 17 MLPs of V. cholerae may mediate chemotactic responses. Among them, Mlp24 (previously named McpX) is required for the production of cholera toxin upon mouse infection. mlp24 deletion strains of both classical and El Tor biotypes showed defects in taxis toward several amino acids, which were complemented by the expression of Mlp24. These amino acids enhanced methylation of Mlp24. Serine, arginine, asparagine, and proline were shown to bind directly to the periplasmic fragment of Mlp24. The structural information of its closest homolog, Mlp37, predicts that Mlp24 has two potential ligand-binding pockets per subunit, the membrane distal of which was suggested, by mutational analyses, to be involved in sensing of amino acids. These results suggest that Mlp24 is a chemoreceptor for multiple amino acids, including serine, arginine, and asparagine, which were previously shown to stimulate the expression of several virulence factors, implying that taxis toward a set of amino acids plays critical roles in pathogenicity of V. cholerae.

  16. 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

  17. 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

  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.

  19. Suppression of the ELO-2 FA elongation activity results in alterations of the fatty acid composition and multiple physiological defects, including abnormal ultradian rhythms, in Caenorhabditis elegans.

    PubMed Central

    Kniazeva, Marina; Sieber, Matt; McCauley, Scott; Zhang, Kang; Watts, Jennifer L; Han, Min

    2003-01-01

    While the general steps of fatty acid (FA) biosynthesis are well understood, the individual enzymes involved in the elongation of long chain saturated and polyunsaturated FA (PUFA) are largely unknown. Recent research indicates that these enzymes might be of considerable physiological importance for human health. We use Caenorhabditis elegans to study FA elongation activities and associated abnormal phenotypes. In this article we report that the predicted C. elegans F11E6.5/ELO-2 is a functional enzyme with the FA elongation activity. It is responsible for the elongation of palmitic acid and is involved in PUFA biosynthesis. RNAi-mediated suppression of ELO-2 causes an accumulation of palmitate and an associated decrease in the PUFA fraction in triacylglycerides and phospholipid classes. This imbalance in the FA composition results in multiple phenotypic defects such as slow growth, small body size, reproductive defects, and changes in rhythmic behavior. ELO-2 cooperates with the previously reported ELO-1 in 20-carbon PUFA production, and at least one of the enzymes must function to provide normal growth and development in C. elegans. The presented data indicate that suppression of a single enzyme of the FA elongation machinery is enough to affect various organs and systems in worms. This effect resembles syndromic disorders in humans. PMID:12586704

  20. The Saccharomyces cerevisiae poly(A)-binding protein is subject to multiple post-translational modifications, including the methylation of glutamic acid.

    PubMed

    Low, Jason K K; Hart-Smith, Gene; Erce, Melissa A; Wilkins, Marc R

    2014-01-10

    Poly(A)-binding protein in mouse and man was recently found to be highly post-translationally modified. Here we analysed an ortholog of this protein, Pab1 from Saccharomyces cerevisiae, to assess the conservation and thus likely importance of these modifications. Pab1 showed the presence of six sites of methylated glutamate, five sites of lysine acetylation, and one phosphorylation of serine. Many modifications on Pab1 showed either complete conservation with those on human or mouse PABPC1, were present on nearby residues and/or were present in the same domain(s). The conservation of methylated glutamate, an unusual modification, was of particular note and suggests a conserved function. Comparison of methylated glutamate sites in human, mouse and yeast poly(A)-binding protein, along with methylation sites catalysed by CheR L-glutamyl protein methyltransferase from Salmonella typhimurium, revealed that the methylation of glutamate preferentially occurs in EE and DE motifs or other small regions of acidic amino acids. The conservation of methylated glutamate in the same protein between mouse, man and yeast suggests the presence of a eukaryotic l-glutamyl protein methyltransferase and that the modification is of functional significance.

  1. 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

  2. Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana.

    PubMed

    Ben Rejeb, Kilani; Lefebvre-De Vos, Delphine; Le Disquet, Isabel; Leprince, Anne-Sophie; Bordenave, Marianne; Maldiney, Régis; Jdey, Asma; Abdelly, Chedly; Savouré, Arnould

    2015-12-01

    Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2 O2 ) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress-induced proline accumulation and H2 O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2 O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2 O2 , and diphenylene iodonium (DPI), an inhibitor of H2 O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ(1) -pyrroline-5-carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild-type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2 O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant.

  3. 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

  4. 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.

  5. Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough.

    PubMed

    Sasano, Yu; Haitani, Yutaka; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

    2012-01-01

    During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking. PMID:22041027

  6. [Quality of data on folic acid content in vegetables included in several Spanish Food Composition Tables and new data on their folate content].

    PubMed

    Olivares, A B; Bernal, M J; Ros, G; Martínez, C; Periago, M J

    2006-01-01

    The relationship between adequate folate intake, adequate serum levels, and lowering the risk of suffering from cardiovascular diseases, neural tube defects, neural illness and some kind of cancers have been widely studied. Because of the expected health benefits, the consumption of foods with high folate content or enriched foods is increasing. Therefore, an adequate folate intake is important in order to reach acceptable serum levels. Reliable food composition data are necessary in order to evaluate and estimate the populations folate intake, elaborate diets and formulate recommended dietary intakes. For this reason, we revised folic acid data in Spanish Food Composition Tables (FCT). The quality of the data was evaluated and compared with other well-known international Food Composition Tables as well as with a high-resolution liquid chromatographic method (HPLC) validated in our laboratory. We evaluated all data about folate content, as well as all the information given like data origin, analytical method, sampling or original database. For the HPLC method, the food samples were incubated with hog kidney conjugase. After that, the samples were purified and concentrated by strong anion exchange (SAX), then the folate content was quantified by HPLC with a combination of two ultraviolet and fluorescence detectors. The evaluation and comparison of data was established according to some parameters, which define the quality of data, giving punctuation depending on the compliance with these parameters. The study of different sources showed that nutrients were different in definition, analysis method, units and expression of data, and that this fact could have a potential influence on TCA data values. In addition, it has been possible to show a wide variation in food number, name of these foods as well as the analysis of raw or cooked products with different composition. When the quality conditions were tested, the Spanish FCT had the lowest punctuation in folate

  7. 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

  8. 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.

  9. Molecular mechanism for H2 release from BH3NH3, including the catalytic role of the Lewis acid BH3.

    PubMed

    Nguyen, Minh Tho; Nguyen, Vinh Son; Matus, Myrna H; Gopakumar, G; Dixon, David A

    2007-02-01

    Electronic structure calculations using various methods, up to the coupled-cluster CCSD(T) level, in conjunction with the aug-cc-pVnZ basis sets with n = D, T, and Q, extrapolated to the complete basis set limit, show that the borane molecule (BH3) can act as an efficient bifunctional acid-base catalyst in the H2 elimination reactions of XHnYHn systems (X, Y = C, B, N). Such a catalyst is needed as the generation of H2 from isoelectronic ethane and borane amine compounds proceeds with an energy barrier much higher than that of the X-Y bond energy. The asymptotic energy barrier for H2 release is reduced from 36.4 kcal/mol in BH3NH3 to 6.0 kcal/mol with the presence of BH3 relative to the molecular asymptote. The NH3 molecule can also participate in a similar catalytic process but induces a smaller reduction of the energy barrier. The kinetics of these processes was analyzed by both transition-state and RRKM theory. The catalytic effect of BH3 has also been probed by an analysis of the electronic densities of the transition structures using the atom-in-molecule (AIM) and electron localization function (ELF) approaches.

  10. Conserved Proline-Directed Phosphorylation Regulates SR Protein Conformation and Splicing Function

    PubMed Central

    Keshwani, Malik M.; Aubol, Brandon E.; Fattet, Laurent; Ma, Chen-Ting; Qiu, Jinsong; Jennings, Patricia A.; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine-serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine-proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine-serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In this study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1. Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over one hundred human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programs. PMID:25529026

  11. Conserved proline-directed phosphorylation regulates SR protein conformation and splicing function.

    PubMed

    Keshwani, Malik M; Aubol, Brandon E; Fattet, Laurent; Ma, Chen-Ting; Qiu, Jinsong; Jennings, Patricia A; Fu, Xiang-Dong; Adams, Joseph A

    2015-03-01

    The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine-serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine-proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine-serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1 (SR protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programmes.

  12. Collagen labelling with an azide-proline chemical reporter in live cells.

    PubMed

    Amgarten, Beatrice; Rajan, Rakesh; Martínez-Sáez, Nuria; Oliveira, Bruno L; Albuquerque, Inês S; Brooks, Roger A; Reid, David G; Duer, Melinda J; Bernardes, Gonçalo J L

    2015-03-28

    We have developed a strategy for selective imaging of collagen in live foetal ovine osteoblasts. Our approach involves the incorporation of an azide-tagged proline in the biosynthesis of collagen followed by labelling using a strain-promoted [3+2] azide-alkyne cycloaddition reaction.

  13. Genotype differences in the metabolism of proline and polyamines under moderate drought in tomato plants.

    PubMed

    Montesinos-Pereira, D; Barrameda-Medina, Y; Romero, L; Ruiz, J M; Sánchez-Rodríguez, E

    2014-11-01

    Water stress is one of the most important factors limiting the growth and productivity of crops. The implication of compatible osmolytes such as proline and polyamines in osmotic adjustment has been widely described in numerous plants species under stress conditions. In the present study, we investigated the response of five cherry tomato cultivars (Solanum lycopersicum L.) subjected to moderate water stress in order to shed light on the involvement of proline and polyamine metabolism in the mechanisms of tolerance to moderate water stress. Our results indicate that the most water stress-resistant cultivar (Zarina) had increased degradation of proline associated with increased polyamine synthesis, with a higher concentration of spermidine and spermine under stress conditions. In contrast, Josefina, the cultivar most sensitive to water stress, showed a proline accumulation associated with increased synthesis after being subjected to stress. In turn, in this cultivar, no rise in polyamine synthesis was detected. Therefore, all the data appear to indicate that polyamine metabolism is more involved in the tolerance response to moderate water stress. PMID:24750452

  14. 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.

  15. Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

    PubMed

    Muiznieks, Lisa D; Reichheld, Sean E; Sitarz, Eva E; Miao, Ming; Keeley, Fred W

    2015-10-01

    Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties.

  16. 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

  17. 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

  18. Proline-containing cyclopeptides from the marine sponge Phakellia fusca.

    PubMed

    Zhang, Hong-Jun; Yi, Yang-Hua; Yang, Gen-Jin; Hu, Min-Ye; Cao, Gui-Dong; Yang, Fan; Lin, Hou-Wen

    2010-04-23

    Four new cyclopeptides, phakellistatins 15-18 (2-5), together with five known cyclopeptides, phakellistatin 13 (1), hymenistatin 1, and hymenamides G, H, and J, were isolated from the South China Sea sponge Phakellia fusca. Their structures were elucidated by HR-ESIMS, NMR, and MALDI-TOF/TOF sequence analysis. The absolute configurations of the amino acid residues of 2-5 were assigned to be l by enantioselective HPLC analysis.

  19. SELECTIVE INHIBITION OF PROLINE-INDUCED PIGMENTATION IN WASHED CELLS OF SERRATIA MARCESCENS.

    PubMed

    BLIZZARD, J L; PETERSON, G E

    1963-05-01

    Blizzard, John L. (University of Houston, Houston, Texas) and G. E. Peterson. Selective inhibition of proline-induced pigmentation in washed cells of Serratia marcescens. J. Bacteriol. 85:1136-1140. 1963.-Streptomycin, chloramphenicol, and tetracyclines inhibited the synthesis of prodigiosin by Serratia marcescens strain D1. This occurred at concentrations of the antibiotic too low to inhibit the growth of the organism in either agar media or broth cultures. Nonpigmented cells were produced in broth by either adding streptomycin or incubating at 37 C. After being washed and resuspended in aqueous saline containing either casein hydrolysate, l-proline, or a glycine-succinate mixture and incubated at 27 C for 24 hr, these cells formed pigment. The appearance of pigment was preceded by a lag period of 10 hr. Prodigiosin production by these washed suspensions of cells was completely inhibited by either streptomycin or glucose, or by incubation at 37 C instead of 27 C. Even though pigmentation by washed-cell suspensions was induced by proline, the utilization of proline was not affected by streptomycin or glucose, or by incubation at 37 C. To block pigmentation completely, streptomycin had to be added to proline-supplemented cells before they were 10 hr old. Addition of the antibiotic after the end of the induction period caused either partial or no inhibition of pigment production. Streptomycin caused an increase in the endogenous respiration of S. marcescens but failed to affect the constitutive enzymes that oxidize glucose. The possible relationships of these phenomena are discussed. Weil (1952) reported that low concentrations of chloramphenicol and certain tetracyclines inhibit the synthesis of prodigiosin while permitting growth by Serratia marcescens. He noted the potential value to "mode-of-action" studies of an organism having certain functions selectively inhibited by antibiotics. We confirmed Weil's (1952) observations and found that streptomycin at low

  20. 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

  1. Salivary proline-rich proteins in mammals: Roles in oral homeostasis and counteracting dietary tannin.

    PubMed

    McArthur, C; Sanson, G D; Beal, A M

    1995-06-01

    We review information on the structure of proline-rich proteins (PRPs), their various functions related to oral homeostasis and dietary tannin, and the structural basis of these functions. Consideration of the multifunctional nature of these salivary proteins helps explain both the subtle and large variations found in structure and secretion rates both within individuals and between species. We propose that the ancestral function of PRPs is in maintaining oral homeostasis and that counteracting dietary tannins by binding with them is a derived function. PRPs are effective in oral homeostasis at low secretion levels, whereas counteracting tannin depends on high secretion levels. In the dietary habits ranging from carnivores through omnivores to exclusively planteaters, the dietary nitrogen level is progressively reduced, and plant allelochemical intake, including tannins, increases. We suggest that during this evolution from meat-eater to plant-eater, there was some point in omnivory at which selective pressure from nitrogen limitations, arising from a low nitrogen/high tannin diet, became sufficiently great for the evolution of increased secretion level and diversification of PRPs for dealing with tannin. If this hypothesis is correct, carnivorous mammals should secrete low levels of PRPs for oral homeostasis, but should never secrete high levels, unless they are secondarily carnivorous. Omnivores consuming a diet of very little animal tissue but higher levels of tannin-containing foliage or fruit should generally have the capacity to produce high levels of salivary PRPs. Browsers and frugivores should also produce high levels of PRPs, but grazers may have reduced secretion rates depending on the antiquity of the dietary habit. This hypothesis is consistent with the limited information available on the abundance, type, and distribution of PRPs in mammals. Studies are suggested which would test the functional and evolutionary arguments presented.

  2. 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

  3. [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

  4. 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.

  5. 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

  6. 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

  7. Inframolecular acid base studies of the tris and tetrakis myo-inositol phosphates including the 1,2,3-trisphosphate motif

    NASA Astrophysics Data System (ADS)

    Dozol, Hélène; Blum-Held, Corinne; Guédat, Philippe; Maechling, Clarisse; Lanners, Steve; Schlewer, Gilbert; Spiess, Bernard

    2002-12-01

    The intrinsic acid-base properties of the phosphate groups of three myo-inositol derivatives which display the 1,2,3-trisphosphate motif, i.e. (±)- myo-inositol 1,2,3-trisphosphate (Ins(1,2,3)P 3), (±)- myo-inositol 1,2,3,6-tetrakisphosphate (Ins(1,2,3,6)P 4), and (±)- myo-inositol 1,2,3,5-tetrakisphosphate (Ins(1,2,3,5)P 4) are reported. The studies were performed in 0.2 M KCl solution at 37 °C, near physiological ionic strength and temperature. In addition, in order to shed light on the transition metal complexation properties of Ins(1,2,3)P 3, the influence of the Zn 2+ cations on its 31P NMR titration curves was investigated. From the titration curves as well as from the determined protonation microconstants, it appears that for Ins(1,2,3)P 3, the two lateral P1 and P3 phosphates strongly contribute to stabilise a proton on the central P2 phosphate. However, in the fully deprotonated form of Ins(1,2,3)P 3, P1 and P3 repulse each other so that they establish hydrogen bonds with, respectively, their neighbouring OH6 and OH4 hydroxyls. The 1,2,3-trisphosphate motif of Ins(1,2,3,5)P 4 behaves very similarly to that of Ins(1,2,3)P 3 indicating a poor interaction with the distant P5 phosphate. By contrast, moving a phosphate group from position 5 to position 6 on the myo-inositol ring as in Ins(1,2,3,6)P 4, leads to major changes in the basicity and cooperativity of the phosphate groups. Finally, the presence of Zn 2+ cations has a marked influence on the 31P NMR titration curves of Ins(1,2,3)P 3, leading to the conclusion that two equatorial phosphates, assisted by a middle axial one, afford an optimal chelating moiety that is able to occupy all sites of the metal coordination polyhedron which could be the reason for its antioxidant properties.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. The effects of ursodeoxycholic acid treatment for intrahepatic cholestasis of pregnancy on maternal and fetal outcomes: a meta-analysis including non-randomized studies.

    PubMed

    Grand'Maison, Sophie; Durand, Madeleine; Mahone, Michèle

    2014-07-01

    Objectif : Les avantages de l’utilisation d’acide ursodésoxycholique (AUDC) pour la prise en charge de la cholestase intrahépatique de la grossesse (CIG) demeurent incertains. Une analyse Cochrane de 2010 ayant porté sur des essais comparatifs randomisés n’a pas été en mesure de se prononcer pour ou contre l’utilisation d’AUDC pour la prise en charge de la CIG. Nous avons mené une méta-analyse de la littérature, en englobant tant les études non randomisées (ENR) que les ECR. Nous avions pour objectif de déterminer si les patientes ayant participé aux ENR étaient comparables à celles qui avaient participé aux ECR; nous avions également pour objectif de déterminer si l’inclusion des ENR pouvait renforcer les données probantes disponibles et orienter la pratique clinique quant à l’utilisation d’AUDC chez les femmes qui présentent une CIG. Sources de données : Nous avons mené des recherches dans Medline (Ovid), Embase (Ovid), EMB Reviews, Cinahl (Ebsco) et Web of Knowledge (Thomson Reuters) en vue d’en tirer les articles publiés entre 1966 et juin 2012. Sélection des études : Nous avons inclus tous les ECR admissibles ayant comparé l’AUDC à un placebo ou à d’autres traitements et toutes les ENR ayant comparé l’AUDC à tout autre traitement chez des femmes présentant une CIG. Synthèse des données : Nous avons inclus 11 ECR (n = 625 grossesses) et six ENR (n = 211 grossesses). Bien que les femmes ayant participé aux ECR et aux ENR aient été comparables, la qualité des études était plus faible dans le cas des ENR. De façon générale, les femmes traitées à l’AUDC ont connu une atténuation du prurit dans 73 % des ECR et dans 100 % des ENR disposant de données disponibles. Les épreuves de fonction hépatique ont présenté une amélioration dans 82 % des ECR et dans 100 % des ENR disposant de données disponibles. Bien que l’utilisation d’AUDC n’ait pas affecté le taux de c

  14. Lewis acid promoted highly diastereoselective Petasis Borono-Mannich reaction: efficient synthesis of optically active β,γ-unsaturated α-amino acids.

    PubMed

    Li, Yi; Xu, Ming-Hua

    2012-04-20

    An efficient and straightforward method for the preparation of highly enantiomerically enriched β,γ-unsaturated α-amino acid derivatives by a Lewis acid promoted diastereoselective Petasis reaction of vinylboronic acid, N-tert-butanesulfinamide, and glyoxylic acid has been developed. The synthetic utilities of the approach were demonstrated by the rapid and convenient construction of challenging cyclopenta[c]proline derivatives.

  15. Conformational analysis and intramolecular interactions of L-proline methyl ester and its N-acetylated derivative through spectroscopic and theoretical studies.

    PubMed

    Braga, Carolyne B; Ducati, Lucas C; Tormena, Cláudio F; Rittner, Roberto

    2014-03-01

    This work reports a detailed study regarding the conformational preferences of L-proline methyl ester (ProOMe) and its N-acetylated derivative (AcProOMe) to elucidate the effects that rule their behaviors, through nuclear magnetic resonance (NMR) and infrared (IR) spectroscopies combined with theoretical calculations. These compounds do not present a zwitterionic form in solution, simulating properly amino acid residues in biological media, in a way closer than amino acids in the gas phase. Experimental (3)JHH coupling constants and infrared data showed excellent agreement with theoretical calculations, indicating no variations in conformer populations on changing solvents. Natural bond orbital (NBO) results showed that hyperconjugative interactions are responsible for the higher stability of the most populated conformer of ProOMe, whereas for AcProOMe both hyperconjugative and steric effects rule its conformational equilibrium.

  16. Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

    PubMed Central

    Michalkova, Veronika; Weiss, Brian L.; Attardo, Geoffrey M.; Aksoy, Serap

    2014-01-01

    The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production. PMID:25038091

  17. Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7).

    PubMed

    Maes, Marie-Berthe; Lambeir, Anne-Marie; Gilany, Kambiz; Senten, Kristel; Van der Veken, Pieter; Leiting, Barbara; Augustyns, Koen; Scharpé, Simon; De Meester, Ingrid

    2005-03-01

    The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell proline dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).

  18. Effect of heat stress on polyamine metabolism in proline-over-producing tobacco plants.

    PubMed

    Cvikrová, Milena; Gemperlová, Lenka; Dobrá, Jana; Martincová, Olga; Prásil, Ilja T; Gubis, Jozef; Vanková, Radomira

    2012-01-01

    The effect of heat stress on the accumulation of proline and on the level of polyamines (PAs) in tobacco plants was investigated. Responses to heat stress were compared in the upper and lower leaves and roots of tobacco plants that constitutively over-express a modified gene for the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CSF129A) and in the corresponding wild-type. In the initial phases of heat stress (after 2h at 40°C), the accumulation of proline increased in the wild type but slightly decreased in the transformants. The response to heat stress in proline-over-producing tobacco plants involved a transient increase in the levels of free and conjugated putrescine (Put) and in the levels of free spermidine (Spd), norspermidine (N-Spd) and spermine (Spm) after a 2-h lag phase, which correlated with stimulation of the activity of the corresponding biosynthetic enzymes. Diamine oxidase (DAO) activity increased in both plant genotypes, most significantly in the leaves of WT plants. Polyamine oxidase (PAO) activity increased in the roots of WT plants and decreased in the leaves and roots of the transformants. After 6h of heat stress, proline accumulation was observed in the transformants, especially in the lower leaves; much more modest increase was observed in the WT plants. A decrease in the levels of free and conjugated Put coincided with down-regulation of the activity of ornithine decarboxylase and marked stimulation of DAO activity in the leaves and roots of the transformants. PAO activity increased in the roots of the transformants but decreased in the leaves. Conversely, in WT tobacco subjected to 6h of heat stress, slight increases in free and conjugated PA levels were observed and the activity of DAO only increased in the roots; PAO activity did not change from the value observed during the initial phase of heat stress. 6 Hours' heat stress had no effect on the level of malondialdehyde (MDA; a product of lipid peroxidation), in

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Synthesis of amino acids by the heating of formaldehyde and ammonia.

    PubMed

    Fox, S W; Windsor, C R

    1970-11-27

    The heating of formaldehyde and ammonia yields a product that, on hydrolysis, is converted into seven amino acids: aspartic acid, glutamic acid, serine, proline, valine, glycine, and alanine. Glycine is the predominant amino acid. Inasmuch as formaldehyde and ammonia have been identified as compounds in galactic clouds, these experimental results are interpreted in a cosmochemical and geochemical context.

  5. Use of prolines for improving growth and/or yield

    SciTech Connect

    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.

  6. 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.

  7. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

    PubMed Central

    Siddiqui, A H; Brandriss, M C

    1989-01-01

    The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction. Images PMID:2689862

  8. Elucidating proline dynamics in spider dragline silk fibre using 2H-13C HETCOR MAS NMR.

    PubMed

    Shi, Xiangyan; Yarger, Jeffery L; Holland, Gregory P

    2014-05-14

    (2)H-(13)C HETCOR MAS NMR is performed on (2)H/(13)C/(15)N-Pro enriched A. aurantia dragline silk. Proline dynamics are extracted from (2)H NMR line shapes and T1 in a site-specific manner to elucidate the backbone and side chain molecular dynamics for the MaSp2 GPGXX β-turn regions for spider dragline silk in the dry and wet, supercontracted states.

  9. 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

  10. Over-expression of a proline specific aminopeptidase from Aspergillus oryzae JN-412 and its application in collagen degradation.

    PubMed

    Ding, Guo-Wei; Zhou, Nan-Di; Tian, Ya-Ping

    2014-08-01

    A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg(-1) at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7% from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn2+ and Cu2+. DTT, β-mercaptoethanol, EDTA, and ions like Co2+, Mg2+, Mn2+, and Ca2+ had no influence on enzyme activity, whereas Ni2+ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.

  11. Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it.

    PubMed

    Bacon, J R; Rhodes, M J

    2000-03-01

    Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin.

  12. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.

    PubMed

    Igoh, Akihisa; Tomotake, Sho; Doi, Yusuke

    2015-05-01

    Saliva is one of the most common body fluids found at a crime scene. Therefore, identifying saliva is important in forensic science. However, the current protein marker assays used to identify saliva are not sufficiently specific. Although proline-rich proteins (PRPs) are highly specific for saliva, their forensic potential has not yet been investigated. In this study, we developed enzyme-linked immunosorbent assays (ELISAs) to detect acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2 (PRB2). The specificity, sensitivity, and efficiency of the ELISAs for PRH1/2 and PRB2 were compared with those of the ELISA for statherin (STATH), a known protein marker for saliva. The levels of PRH1/2 were significantly higher in saliva and saliva stains than in other body fluids (nasal secretions, urine, semen, vaginal fluid, blood, and sweat). PRB2 and STATH were detected in both nasal secretions and saliva. The PRH1/2 ELISA showed sensitivity similar to that of STATH ELISA. The detection rate of PRH1/2 ELISA was almost similar to that of STATH ELISA, followed by the ELISA for PRB2. The PRH1/2 ELISA had higher specificity for saliva than STATH ELISA. Therefore, the PRH1/2 ELISA has potential as a method to identify saliva for forensic investigation.

  13. 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

  14. Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it.

    PubMed

    Bacon, J R; Rhodes, M J

    2000-03-01

    Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin. PMID:10725160

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

    PubMed Central

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

    1973-01-01

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

  16. A hydrophobic proline-rich motif is involved in the intracellular targeting of temperature-induced lipocalin.

    PubMed

    Hernández-Gras, Francesc; Boronat, Albert

    2015-06-01

    Temperature-induced lipocalins (TILs) play an essential role in the response of plants to different abiotic stresses. In agreement with their proposed role in protecting membrane lipids, TILs have been reported to be associated to cell membranes. However, TILs show an overall hydrophilic character and do not contain any signal for membrane targeting nor hydrophobic sequences that could represent transmembrane domains. Arabidopsis TIL (AtTIL) is considered the ortholog of human ApoD, a protein known to associate to membranes through a short hydrophobic loop protruding from strands 5 and 6 of the lipocalin β-barrel. An equivalent loop (referred to as HPR motif) is also present between β-strands 5 and 6 of TILs. The HPR motif, which is highly conserved among TIL proteins, extends over as short stretch of eight amino acids and contains four invariant proline residues. Subcellular localization studies have shown that TILs are targeted to a variety of cell membranes and organelles. We have also found that the HPR motif is necessary and sufficient for the intracellular targeting of TILs. Modeling studies suggest that the HPR motif may directly anchor TILs to cell membranes, favoring in this way further contact with the polar group of membrane lipids. However, some particular features of the HPR motif open the possibility that targeting of TILs to cell membranes could be mediated by interaction with other proteins. The functional analysis of the HPR motif unveils the existence of novel mechanisms involved in the intracellular targeting of proteins in plants.

  17. 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.

  18. 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.

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

    PubMed

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

    1973-07-01

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

  20. Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII.

    PubMed Central

    Leiting, Barbara; Pryor, KellyAnn D; Wu, Joseph K; Marsilio, Frank; Patel, Reshma A; Craik, Charles S; Ellman, Jonathan A; Cummings, Richard T; Thornberry, Nancy A

    2003-01-01

    There is currently intense interest in the emerging group of proline-specific dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other proline-specific dipeptidases, DPP-VII (also known as quiescent cell proline dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279-288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283-290]. To facilitate the identification of selective substrates and inhibitors for these enzymes, a complete biochemical profile of these enzymes was obtained. The pH profiles, substrate specificities as determined by positional scanning, Michaelis-Menten constants and inhibition profiles for DPP-VII and DPP-II were shown to be virtually identical, strongly supporting the hypothesis that they are the same protease. In addition, substrate specificities, catalytic constants and IC(50) values were shown to be markedly different from those of DPP-IV. Selective DPP-IV and DPP-VII substrates were identified and they can be used to design selective inhibitors and probe further into the biology of these enzymes. PMID:12529175

  1. 4(R/S)-Amino/guanidino-substituted proline peptides: design, synthesis and DNA transfection properties.

    PubMed

    Umashankara, M; Nanda, Manaswini; Sonar, Mahesh; Ganesh, Krishna N

    2012-01-01

    Collagen is a major structural protein found in the connective tissues of higher organisms and mammals and its biomechanical properties are related to the high thermal stability of its triple helical structure. The primary structure of collagen is composed of the repeating tripeptide motif of Pro-Hyp-Gly, where Hyp is 4 R -hydroxy proline. Cationic collagen mimetics consisting of [Pro(X)-Pro(Y)-Gly](6) where Pro(X) and Pro(Y) are 4(R/S)-amino/guanidine proline have been synthesized and shown to form triplexes more stable than the unmodified collagen peptide [Pro-Hyp-Gly](6). The origin of hyperstability is due to conformational pre-organization of proline pucker arising from the electronegativity of the cationic group. These cationic collagen peptides are shown to be effective cell penetrating and plasmid DNA transfecting agents. The results have potential for design of new collagen mimetics for biomaterial applications and efficient cell penetrating agents for drug delivery applications.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores.

    PubMed

    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 against antibiotic

  9. 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

  10. Radioautography of rat incisor dentin as a continuous record of the incorporation of a single dose of /sup 3/H-labeled proline and tyrosine

    SciTech Connect

    Josephsen, K.; Warshawsky, H.

    1982-05-01

    After injection of labeled precursors such as /sup 3/H-proline or /sup 3/H-tyrosine into rats, the incisor dentin contains a continuous and stable record of precursor incorporation into labeled proteins. This record was visualized and quantitated with radioautography in order to evaluate the quantitative changes in enamel where newly secreted proteins randomize with older proteins and both are eventually lost. Up to 4 hours after injection, the pulse-dose was incorporated as a highly labeled band of predentin. The band was entirely within calcified dentin at 2 days and was further removed from new predentin by 4 and 8 days. Dentin which formed proximal to the heavily labeled band contained an amount of radioactivity reflecting the level of labeled precursor available at that time. A standardizing factor for experimental error was obtained by quantitating the reaction in the heavily labeled band, and a post-pulse incorporation factor was determined from the amount of radioactivity added per day as weakly labeled dentin. The variation within the heavily labeled band was assumed to reflect experimental error. The number of grains in the bands were averaged from 4 hours to 8 days to give the standardizing factor. This was multiplied by the ratio of enamel to dentin counts in the same section to obtain a corrected enamel count. With proline it amounted to 5% increase per day from 1 to 4 days and 2.5% per day from 4 to 8 days after injection. In addition, with /sup 3/H-proline the incorporation into predentin increased from 30 minutes to 4 hours. With tyrosine, the counts increased from 30 minutes to 1 hour, but decreased by nearly one third from 1 to 4 hours. This was interpreted as a loss of short-lived matrix proteins including procollagen peptides produced during conversion from procollagen to tropocollagen in the predentin.

  11. Frequent amplification of ORAOV1 gene in esophageal squamous cell cancer promotes an aggressive phenotype via proline metabolism and ROS production

    PubMed Central

    Togashi, Yosuke; Arao, Tokuzo; Kato, Hiroaki; Matsumoto, Kazuko; Terashima, Masato; Hayashi, Hidetoshi; de Velasco, Marco A; Fujita, Yoshihiko; Kimura, Hideharu; Yasuda, Takushi; Shiozaki, Hitoshi; Nishio, Kazuto

    2014-01-01

    Chromosomal band 11q13 seems to be one of the most frequently amplified lesions in human cancer, including esophageal squamous cell cancer (ESCC). The oral cancer overexpressed 1 (ORAOV1) gene has been identified within this region, but its detailed biological function in human ESCC remains largely unclear. In our clinical samples of stage III ESCC, ORAOV1 amplification was observed in 49 of 94 cases (53%). ORAOV1 amplification was significantly associated with a poorly differentiated histology and tumors located in the upper or middle esophagus. Patients with ORAOV1 amplification tended to have a shorter survival period, although the difference was not significant. To investigate the function of ORAOV1, we created ORAOV1-overexpressed ESCC cell lines that exhibited increased cellular proliferation and colony formation, compared with in vitro controls. In vivo, ORAOV1-overexpressed cells exhibited a significantly increased tumorigenicity and a significantly larger tumor volume and poorer differentiation than controls. The peptide mass fingerprinting technique demonstrated that ORAOV1 bound to pyrroline-5-carboxylate reductase (PYCR), which is associated with proline metabolism and reactive oxygen species (ROS) production. Then, ORAOV1-overexpressed cell lines were resistant to stress treatment, which was cancelled by PYCR-knockdown. In addition, the ORAOV1-overexpressed cell line had a higher intracellular proline concentration and a lower ROS level. Our findings indicate that the ORAOV1 gene is frequently amplified in ESCC, enhances tumorigenicity and tumor growth, and is associated with a poorly differentiated tumor histology via proline metabolism and ROS production. ORAOV1 could be a novel target for the treatment of ESCC. PMID:24930674

  12. A novel proline-rich glycoprotein associated with the extracellular matrix of vascular bundles of Brassica petioles.

    PubMed

    Davies, H A; Findlay, K; Daniels, M J; Dow, J M

    1997-01-01

    A panel of monoclonal antibodies (MAC204, MAC236, MAC265) which recognise extracellular matrix glycoproteins implicated in plant-microbe interactions has been used to study glycoprotein antigens in petioles of turnip (Brassica campestris L.). While MAC204 recognised two glycoproteins (gp120 and gp45) with apparent M(r) 120,000 and 45,000 in petiole extracts made with 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer containing sodium dodecyl sulfate, MAC236 recognised gp120 but not gp45, and MAC265 gave no or only weak reactivity. Tissue dissection studies established that gp120 was predominantly associated with the vascular bundle whereas gp45 was largely associated with the pith. This was consistent with results from tissue prints probed with MAC204 and MAC236 which also suggested a vascular localisation for gp120. Immunoelectronmicroscopy showed that MAC204 and MAC236 both labelled three-way junctions between cells of the phloem and sclerid fibres. Both gp120 and gp45 were shown to carry epitopes in common with known hydroxyproline-rich glycoproteins. Unlike gp45, gp120 could be extracted from petioles with Tris buffer alone and then isolated from this extract by trichloroacetic acid treatment (which left gp120 soluble), followed by size-exclusion and ion-exchange chromatography. Amino acid analysis revealed gp120 to be a novel glycoprotein, particularly rich in proline, lysine, valine and threonine but relatively poor in hydroxyproline. The most abundant sugars were arabinose and galactose. The potential role of this very basic cell surface glycoprotein in plant defence against microbes is discussed.

  13. 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

  14. 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

  15. [Free amino acids of hemolymph during pubertal molting and senescence in Spaeroma serratum (Isopoda, Flabellifera].

    PubMed

    Charmantier, G; Voss-Foucart, M F; Trilles, J P; Jeuniaux, Ch

    1975-08-01

    In Sphaeroma serratum, the amino-acidemia is high (about 120 mg/100 ml) during the intermolt stages. The relative proportions of the different free amino acids show only slight differences in young males and senescent ones. At the time of the puberty molt, the total amino-acids concentration (including taurine) of the hemolymph increases sharply during premolt (up to 267 mg/100 ml), falls after ecdysis (down to 97 mg/100 ml and then rises up to the level of the intermolt stage. These variations are due not only to concentration or dilution of the whole hemolymph constituents, but also to specific modifications of the concentration of some of the free amino-acids with respect to the others. These variations of the amino-acidemia, mainly those of serine, proline, glycine, alanine and of the amine taurine, could play a definite role in increasing the osmotic pressure of the hemolymph during premolt, as a preparation to ecdysis.

  16. Enhanced bio-decolorization of 1-amino-4-bromoanthraquinone-2-sulfonic acid by Sphingomonas xenophaga with nutrient amendment.

    PubMed

    Lu, Hong; Guan, Xiaofan; Wang, Jing; Zhou, Jiti; Zhang, Haikun

    2015-01-01

    Bacterial decolorization of anthraquinone dye intermediates is a slow process under aerobic conditions. To speed up the process, in the present study, effects of various nutrients on 1-amino-4-bromoanthraquinone-2-sulfonic acid (ABAS) decolorization by Sphingomonas xenophaga QYY were investigated. The results showed that peptone, yeast extract and casamino acid amendments promoted ABAS bio-decolorization. In particular, the addition of peptone and casamino acids could improve the decolorization activity of strain QYY. Further experiments showed that l-proline had a more significant accelerating effect on ABAS decolorization compared with other amino acids. l-Proline not only supported cell growth, but also significantly increased the decolorization activity of strain QYY. Membrane proteins of strain QYY exhibited ABAS decolorization activities in the presence of l-proline or reduced nicotinamide adenine dinucleotide, while this behavior was not observed in the presence of other amino acids. Moreover, the positive correlation between l-proline concentration and the decolorization activity of membrane proteins was observed, indicating that l-proline plays an important role in ABAS decolorization. The above findings provide us not only a novel insight into bacterial ABAS decolorization, but also an l-proline-supplemented bioaugmentation strategy for enhancing ABAS bio-decolorization.

  17. A rapid method for the determination of perfluoroalkyl substances including structural isomers of perfluorooctane sulfonic acid in human serum using 96-well plates and column-switching ultra-high performance liquid chromatography tandem mass spectrometry.

    PubMed

    Salihovic, Samira; Kärrman, Anna; Lindström, Gunilla; Lind, P Monica; Lind, Lars; van Bavel, Bert

    2013-08-30

    To facilitate high-throughput analysis suitable for large epidemiological studies we developed an automated column-switching ultra-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for determination of perfluorocarboxylic acids (PFCAs; C5, C6, C7, C8, C9, C10, C11, C12, and C13), perfluoroalkyl sulfonic acids (PFSAs; C4, C6, C8, and C10), perfluorooctane sulfonamide (PFOSA), and five groups of structural perfluorooctane sulfonic acid (PFOS) isomers in human serum or plasma. The analytical procedure involves rapid protein precipitation using 96-well plates followed by an automated sample clean-up using an on-line trap column removing many potentially interfering sample components while through the mobile phase gradient the target analytes are eluted onto the analytical column for further separation and subsequent mass detection. The method was linear (R(2)<0.995) at concentrations ranging from 0.01 to 60ngmL(-1) with method detection limits ranging between 0.01 and 0.17ngmL(-1) depending on the analyte. The developed method was precise, with repeatability (n=7) and reproducibility (n=103) coefficients of variation between 2% and 20% for most compounds including PFOS (2% and 8%) and its structural isomers (2-6% and 4-8%). The method was in conformity with a standard reference material. The column-switching HPLC-MS/MS method has been successfully applied for the determination of perfluoroalkyl substances including structural PFOS isomers in human plasma from an epidemiological study.

  18. 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.

  19. Physiologically-Relevant Oxidative Degradation of Oligo(proline)-Crosslinked Polymeric Scaffolds

    PubMed Central

    Yu, Shann S.; Koblin, Rachel L.; Zachman, Angela L.; Perrien, Daniel S.; Hofmeister, Lucas H.; Giorgio, Todd D.; Sung, Hak-Joon

    2011-01-01

    Chronic inflammation-mediated oxidative stress is a common mechanism of implant rejection and failure. Therefore, polymer scaffolds that can degrade slowly in response to this environment may provide a viable platform for implant site-specific, sustained release of immunomodulatory agents over a long time period. In this work, proline oligomers of varying lengths (Pn) were synthesized and exposed to oxidative environments, and their accelerated degradation under oxidative conditions was verified via high performance liquid chromatography and gel permeation chromatography. Next, diblock copolymers of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) were carboxylated to form 100 kDa terpolymers of 4%PEG-86%PCL-10%cPCL (cPCL = poly(carboxyl-ε-caprolactone); i% indicates molar ratio). The polymers were then crosslinked with bi-aminated PEG-Pn-PEG chains—where Pn indicates the length of the proline oligomer flanked by PEG chains. Salt-leaching of the polymeric matrices created scaffolds of macroporous and microporous architecture as observed by scanning electron microscopy. The degradation of scaffolds was accelerated under oxidative conditions, as evidenced by mass loss and differential scanning calorimetry measurements. Immortalized murine bone marrow-derived macrophages were then seeded on the scaffolds, and activated through the addition of γ-interferon and lipopolysaccharide throughout the 9-day study period. This treatment promoted the release of H2O2 by the macrophages, and the degradation of proline-containing scaffolds compared to the control scaffolds. The accelerated degradation was evidenced by increased scaffold porosity, as visualized through scanning electron microscoopy and X-ray microtomography imaging. The current study provides insight into the development of scaffolds that respond to oxidative environments through gradual degradation, for the controlled release of therapeutics targeted to diseases that feature chronic inflammation and

  20. Physiologically relevant oxidative degradation of oligo(proline) cross-linked polymeric scaffolds.

    PubMed

    Yu, Shann S; Koblin, Rachel L; Zachman, Angela L; Perrien, Daniel S; Hofmeister, Lucas H; Giorgio, Todd D; Sung, Hak-Joon

    2011-12-12

    Chronic inflammation-mediated oxidative stress is a common mechanism of implant rejection and failure. Therefore, polymer scaffolds that can degrade slowly in response to this environment may provide a viable platform for implant site-specific, sustained release of immunomodulatory agents over a long time period. In this work, proline oligomers of varying lengths (P(n)) were synthesized and exposed to oxidative environments, and their accelerated degradation under oxidative conditions was verified via high performance liquid chromatography and gel permeation chromatography. Next, diblock copolymers of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) were carboxylated to form 100 kDa terpolymers of 4%PEG-86%PCL-10%cPCL (cPCL = poly(carboxyl-ε-caprolactone); i% indicates molar ratio). The polymers were then cross-linked with biaminated PEG-P(n)-PEG chains, where P(n) indicates the length of the proline oligomer flanked by PEG chains. Salt-leaching of the polymeric matrices created scaffolds of macroporous and microporous architecture, as observed by scanning electron microscopy. The degradation of scaffolds was accelerated under oxidative conditions, as evidenced by mass loss and differential scanning calorimetry measurements. Immortalized murine bone-marrow-derived macrophages were then seeded on the scaffolds and activated through the addition of γ-interferon and lipopolysaccharide throughout the 9-day study period. This treatment promoted the release of H(2)O(2) by the macrophages and the degradation of proline-containing scaffolds compared to the control scaffolds. The accelerated degradation was evidenced by increased scaffold porosity, as visualized through scanning electron microscopy and X-ray microtomography imaging. The current study provides insight into the development of scaffolds that respond to oxidative environments through gradual degradation for the controlled release of therapeutics targeted to diseases that feature chronic

  1. 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

  2. Effect of salt stress on growth, Na+ accumulation and proline metabolism in potato (Solanum tuberosum) cultivars.

    PubMed

    Jaarsma, Rinse; de Vries, Rozemarijn S M; de Boer, Albertus H

    2013-01-01

    Potato (Solanum tuberosum) is a major crop world-wide and the productivity of currently used cultivars is strongly reduced at high soil salt levels. We compared the response of six potato cultivars to increased root NaCl concentrations. Cuttings were grown hydroponically and treated with 0 mM, 60 mM and 180 mM NaCl for one week. Growth reduction on salt was strongest for the cultivars Mozart and Mona Lisa with a severe senescence response at 180 mM NaCl and Mozart barely survived the treatment. The cultivars Desiree and Russett Burbank were more tolerant showing no senescence after salt treatment. A clear difference in Na(+) homeostasis was observed between sensitive and tolerant cultivars. The salt sensitive cultivar Mozart combined low Na(+) levels in root and stem with the highest leaf Na(+) concentration of all cultivars, resulting in a high Na(+) shoot distribution index (SDI) for Mozart as compared to Desiree. Overall, a positive correlation between salt tolerance and stem Na(+) accumulation was found and the SDI for Na(+) points to a role of stem Na(+) accumulation in tolerance. In stem tissue, Mozart accumulated more H2O2 and less proline compared to the tolerant cultivars. Analysis of the expression of proline biosynthesis genes in Mozart and Desiree showed a clear reduction in proline dehydrogenase (PDH) expression in both cultivars and an increase in pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Desiree, but not in Mozart. Taken together, current day commercial cultivars show promising differences in salt tolerance and the results suggest that mechanisms of tolerance reside in the capacity of Na(+) accumulation in stem tissue, resulting in reduced Na(+) transport to the leaves.

  3. 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

  4. 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

  5. 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

  6. Folding of helical membrane proteins: the role of polar, GxxxG-like and proline motifs.

    PubMed

    Senes, Alessandro; Engel, Donald E; DeGrado, William F

    2004-08-01

    Helical integral membrane proteins share several structural determinants that are widely conserved across their universe. The discovery of common motifs has furthered our understanding of the features that are important to stability in the membrane environment, while simultaneously providing clues about proteins that lack high-resolution structures. Motif analysis also helps to target mutagenesis studies, and other experimental and computational work. Three types of transmembrane motifs have recently seen interesting developments: the GxxxG motif and its like; polar and hydrogen bonding motifs; and proline motifs.

  7. Deficiency in plastidic glutamine synthetase alters proline metabolism and transcriptomic response in Lotus japonicus under drought stress.

    PubMed

    Díaz, Pedro; Betti, Marco; Sánchez, Diego H; Udvardi, Michael K; Monza, Jorge; Márquez, Antonio J

    2010-12-01

    The role of plastidic glutamine synthetase (GS2) in proline biosynthesis and drought stress responses in Lotus japonicus was investigated using the GS2 mutant, Ljgln2-2. Wild-type (WT) and mutant plants were submitted to different lengths of time of water and nutrient solution deprivation. Several biochemical markers were measured and the transcriptional response to drought was determined by both quantitative real-time polymerase chain reaction and transcriptomics. The Ljgln2-2 mutant exhibited normal sensitivity to mild water deprivation, but physiological, biochemical and massive transcriptional differences were detected in the mutant, which compromised recovery (rehydration) following re-watering after severe drought stress. Proline accumulation during drought was substantially lower in mutant than in WT plants, and significant differences in the pattern of expression of the genes involved in proline metabolism were observed. Transcriptomic analysis revealed that about three times as many genes were regulated in response to drought in Ljgln2-2 plants compared with WT. The transcriptomic and accompanying biochemical data indicate that the Ljgln2-2 mutant is subject to more intense cellular stress than WT during drought. The results presented here implicate plastidic GS2 in proline production during stress and provide interesting insights into the function of proline in response to drought.

  8. Amino Acid Intakes Are Associated With Bone Mineral Density and Prevalence of Low Bone Mass in Women: Evidence From Discordant Monozygotic Twins.

    PubMed

    Jennin